Tissue engineering applied to ophthalmology today faces a global shortage of corneas available for transplantation. While corneal pathologies represent one of the leading causes of blindness worldwide, with millions of patients awaiting transplantation, research has oriented itself toward innovative solutions capable of overcoming the limitations of conventional techniques. It is within this scenario that membranes derived from silkworm cocoons establish themselves as an important alternative for ocular tissue reconstruction. These biomaterials, far from being a mere scientific curiosity, represent a genuine revolution in the field of reconstructive ophthalmic surgery, offering unique characteristics that make them particularly suited to the extremely delicate and complex environment of the human eye.
Biomimetic properties and optical transparency
Fibroin membranes demonstrate light transmission exceeding ninety percent across the entire visible spectrum, independent of fabrication method and post-production treatments, a fundamental property considering that transparency represents the primary requirement for any material destined for corneal reconstruction. This exceptional optical clarity derives from the particular organization of protein chains, in which highly ordered crystalline regions alternate with more flexible amorphous domains, creating a structure that permits light passage without causing significant scattering.
Fibroin films prove suitable for corneal tissue engineering thanks to their optical transparency, mechanical resistance, and biocompatibility, characteristics that are rarely found combined in other biomaterials. The material's versatility furthermore permits fine modulation of mechanical properties through various treatment processes, such as annealing with methanol at different concentrations, allowing the achievement of rigidity in the kilopascal range, similar to that of native corneal tissue. This customization capability represents a determining advantage, since corneal epithelial cells are extremely sensitive to the rigidity of the substrate upon which they grow, and inadequate rigidity can compromise crucial processes such as cellular adhesion, migration, and expression of stem cell markers.
Biocompatibility and minimal immune response
It has been demonstrated that fibroin can generate minimal immune and inflammatory responses when implanted in the organism, and the material can be completely degraded by naturally present proteolytic enzymes. This characteristic is particularly relevant in the ocular context, where excessive inflammatory reactions can compromise corneal transparency and lead to scar formation that prevents visual function recovery.
The controlled degradation of fibroin represents a further strategic advantage for ophthalmic applications. The degradation rate follows this order: films prepared in aqueous solution show the most rapid degradation, followed by those prepared with hexafluoroisopropanol, while those prepared with formic acid show the slowest degradation. This modulability permits surgeons and researchers to design scaffolds with specific degradation kinetics, adapted to the regenerative necessities of the individual patient. During the degradation process, the material is gradually replaced by regenerating native tissue, eliminating the necessity for a second intervention for implant removal and reducing the risks of long-term foreign body reactions.
When implanted in animal models with corneal endothelial dysfunction, fibroin membranes adhere tightly to the corneal stroma, effectively preventing aqueous humor penetration while permitting nutrient diffusion, with minimal inflammatory and foreign body reactions during a three-month observation period. This capacity to function as a selective barrier, blocking the passage of excess fluids but permitting that of essential nutrients, represents exactly the type of functionality required to support tissue regeneration while maintaining corneal homeostasis.
Applications in corneal epithelial layer reconstruction
The corneal surface, constituted by the epithelium, represents the eye's first line of defense against external agents and trauma. Corneal epithelial cells can adhere and proliferate on fibroin maintaining a cobblestone pavement appearance, abundant microvilli on the surface, and extensive connections with adjacent cells, morphological characteristics that indicate a healthy and functional cellular phenotype. This capacity of fibroin to support epithelial growth while maintaining the native characteristics of cells represents a fundamental requirement for any biomaterial destined for ocular surface reconstruction.
Transplants of polyethylene glycol-modified fibroin membranes containing limbal epithelial stem cells have repaired corneal epithelial defects and reversed limbal stem cell deficiency, with restoration of neovascularization and corneal clarity scores in animal models. The corneal limbus, the transition zone between cornea and conjunctiva, contains stem cells essential for corneal epithelium maintenance. When this cell population is damaged by chemical burns, trauma, or autoimmune diseases, a condition known as limbal stem cell deficiency occurs, which leads to corneal opacification, neovascularization, and vision loss. Polyethylene glycol-modified fibroin offers an optimal substrate for cultivating these precious stem cells ex vivo and subsequently transplanting them into the patient, restoring the regenerative capacity of the corneal epithelium.
Compared to human amniotic membrane, the unique characteristics of fibroin include transparency, ease of manipulation and transfer, and intrinsic absence of disease transmission risk, rendering it a promising substrate for corneal wound repair and for tissue engineering purposes. Amniotic membrane, currently the clinical standard for ocular surface reconstruction, in fact presents significant limitations: its semi-transparent nature can obstruct light transmission, poor mechanical resistance creates difficulties in surgical manipulation, and above all there exists the risk, albeit minimal, of transmitting infectious pathologies such as HIV, viral hepatitis, or syphilis. The utilization of fibroin permits complete circumvention of these problems while maintaining, and in some cases surpassing, the therapeutic efficacy of amniotic membrane.
Biomimetic scaffolds for stromal regeneration
The corneal stroma, which constitutes approximately ninety percent of total corneal thickness, represents the most complex challenge in corneal tissue engineering. This specialized connective tissue is characterized by a highly ordered organization of collagen fibrils arranged in superimposed lamellae with specific orientations, a sophisticated structure that underlies both the mechanical resistance and optical transparency of the cornea. Human corneal stromal stem cells cultivated on fibroin substrates with microgrooved patterns have successfully differentiated into keratocytes, secreting multilayer lamellae with orthogonally oriented collagen fibrils, in a pattern that mimics human corneal stromal tissue.
The capacity of fibroin membranes to guide cellular organization and collagen fibril orientation represents a result of extraordinary importance. While other substrates generically support cellular growth, fibroin with specific surface patterns is capable of instructing cells to deposit extracellular matrix according to the highly ordered architecture typical of native stroma. This level of control over tissue morphogenesis is essential to obtain not merely a filling of the defect, but a true functional reconstruction of tissue with appropriate biomechanical and optical properties.
Aligned poly-ε-caprolactone-fibroin scaffolds with weight ratios of sixty-forty and fifty-fifty show greater transparency, hydrophilicity, water absorption, and in vitro degradation rate compared to other scaffolds, with overall results that recommend these scaffolds as having great potential for human corneal stromal regeneration. The integration of fibroin with synthetic polymers such as poly-ε-caprolactone permits combining the advantages of both materials: the biocompatibility and biological properties of fibroin with the mechanical resistance and processability of synthetic polymers. This hybrid strategy represents a particularly promising approach for creating scaffolds that can resist the mechanical stresses of the intraocular environment while maintaining optimal biological characteristics for tissue regeneration.
Treatment of corneal ulcers and wounds
The wound healing effect of fibroin-derived protein was evaluated in a rabbit corneal injury model, where corneas treated with fibroin-derived protein in eye drop formulation showed over ninety-five percent corneal surface repair within the first forty-eight hours post-treatment, with the highest repair rate observed in the fibroin-treated group. This significant acceleration of reparative processes represents a potential paradigm shift in the treatment of corneal injuries, where every hour of delay in healing increases the risk of secondary infections, deep ulcerations, and permanent vision loss.
The mechanism through which fibroin accelerates wound healing is complex and multifactorial. Fibroin activates various molecular pathways that promote wound healing, including the phosphoinositide three-kinase pathway (PI3K/AKT), the mitogen-activated protein kinase kinase pathway (MEK1), and the c-Jun N-terminal kinase pathway (JNK), resulting in activation of c-Jun expression induced through downstream mechanisms and cellular migration. These intracellular signaling cascades are fundamental for coordinating the processes of cellular proliferation, migration, and extracellular matrix deposition that characterize tissue healing. Fibroin's capacity to simultaneously stimulate multiple pro-regenerative pathways makes it particularly effective in promoting rapid and complete healing.
In the dry eye model, after a ten-day treatment with fibroin, both fibroin-treated groups showed a fourfold increase in tear production, while the saline-treated group showed only a threefold increase, with epithelial cell detachment showing a ninety-four percent decrease in fibroin-treated groups compared to a thirty-one percent decrease in the PBS group. These data demonstrate how fibroin does not merely passively support tissue regeneration, but actively intervenes in restoring ocular surface homeostasis, improving tear production and reducing epithelial damage. This capacity to positively modulate the ocular surface environment is particularly relevant for patients with chronic pathologies such as dry eye syndrome, where compromise of the tear film creates a vicious cycle of recurrent epithelial damage.
Innovations in endothelial reconstruction
The corneal endothelium, the monolayer of cells lining the cornea's internal surface, plays a critical role in maintaining corneal deturgescence by actively pumping fluids out of the stroma. Endothelial dysfunction is one of the principal indications for corneal transplantation, but endothelial cells have extremely limited regenerative capacity in adults. Artificial fibroin endothelial transplants have shown characteristic endothelial markers zonula occludens (ZO-1) and Na+/K+ ATPase, and in a rabbit endothelial keratoplasty model have restored corneal transparency and thickness at six weeks of follow-up.
Zonula occludens-1 is a tight junction protein essential for maintaining endothelial barrier integrity, while Na+/K+ ATPase represents the ionic pump that generates the osmotic gradient necessary for stromal hydration control. The fact that endothelial cells cultivated on fibroin membranes maintain expression of these functional markers indicates that the biomaterial not only supports cellular survival, but also preserves the differentiation and specialized functionality of these critical cells. This is fundamental because an endothelium that loses its functionality, even if vital, cannot prevent stromal edema and consequent loss of transparency.
Fibroin-plasticizer membranes developed through spin coating and water-annealing techniques exhibit excellent transparency and flexibility, with water-annealing further improving mechanical resistance for intraocular surgical implantation, demonstrating significant clinical potential as temporary treatment for corneal endothelial dysfunction. The concept of a temporary biodegradable implant is particularly attractive in the context of endothelial dysfunction. While awaiting the availability of a definitive cell-based transplant, the fibroin membrane can provide the barrier function necessary to maintain corneal transparency and vision, "buying time" and preserving corneal structure until a definitive intervention becomes possible.
Clinical and translational perspectives
Clinical studies on ocular tissue engineering utilizing biomaterials have shown promising results, with collagen-based materials showing the greatest potential since they closely resemble the natural composition of corneal stroma, although each material presents distinct advantages and limitations. Fibroin positions itself within this panorama as an alternative that can overcome some of collagen's limitations, particularly regarding mechanical resistance and controllable degradation speed. While animal collagen requires cross-linking processes to improve its mechanical properties and slow enzymatic degradation, fibroin can obtain appropriate mechanical characteristics through simple control of processing conditions.
