Corneal blindness is a global problem to which current treatments fail to respond to the entire affected population. The lack of donors for transplant and the deficiencies of the health system in many of the countries with the highest prevalence of corneal pathology, together with the limitations of available treatments, make it necessary to find new alternatives such as those based on tissue engineering and regenerative medicine. The first time that an advanced therapy was applied at the corneal level in the clinic, it was in 1997 when Pellegrini et al implanted autologous corneal epithelial cells cultured from a limbal sample (CLET, acronym of Cultured Limbal Epithelial Transplantation) in a series of patients¹. Subsequently, other research groups have developed epithelial corneal substitutes together with artificial corneal stroma and corneal endothelial substitutes. Currently, the main objective of this discipline at the corneal level, is the generation of a complete artificial cornea that can replace the damaged one.

REGENERATION OF THE CORNEAL EPITHELIUM

Many of the diseases that affect the regeneration of the corneal epithelium are caused by the deficiency of limbal epithelial stem cells (LESC, acronym for Limbal Epithelial Stem Cells). Its definitive treatment requires a limbus transplant or CLET, from autologous or allogeneic tissue, depending on whether the LESC deficiency is complete or partial and affects one or both eyes². From the first CLET performed in 1997, more than 1000 have been carried out worldwide³.

The mechanism that underlies the efficacy of this therapy is still unknown today, given that there is no evidence that the implanted cells survive after more than 7-9 months where the cells of the receptor are replacing them⁴. The CLET studies carried out to date show promising results with a success rate that varies from 33% to 100% during follow-up periods of up to 10 years⁵. More than 75% of patients improve visual acuity, as well as transparency, integrity and stability of the corneal epithelium⁴. The culture methods for the LESC vary with respect to the preparation of the donor tissue, the culture medium used, the culture time, the substrate used and other technical aspects that include the use of feeder cells or the submission to hypoxia or air-liquid contact⁶.

LESCs can be cultured successfully by explants or enzymatic digestion, from the remaining corneo-scleral rings after a keratoplasty or limbal biopsies, this tissue also being ideal to obtain the rest of populations of adult stem cells that populate the rest of the human cornea⁷ (Figure 1). Most researchers use inactivated 3T3 fibroblasts as feeder cells or substrates such as the amniotic membrane or fibrin, on which to grow and promote the growth of the LESCs, and finally implant them in the patient using said substrate as an artificial support matrix or scaffolding⁴ (Figure 2). Recently, limbal mesenchymal stem cells have been proposed as a substitute for 3T3 cells for the in vitro expansion of LESC⁸.

The most widely used substrate in clinical practice, both at the allogeneic and autologous levels, is the amniotic membrane even in spite of the disadvantages it presents^5,9. For this reason, other substrates have been used as an alternative to this in the clinic, such as fibrin^1,10 and hydrogels with silicone¹¹. Recently, the European Drug Agency has recommended Holoclar^® (advanced therapy based on autologous human corneal epithelium grown on a sheet of fibrin) as a treatment for LESC deficiency. Some researchers have described other cellular sources as possible alternatives to LESCs, such as epithelial cells of the oral mucosa^2,12 or mesenchymal stem cells¹³.

REGENERATION OF THE CORNEAL ENDOTHELIUM

In recent years, new therapeutic strategies have been proposed to promote the proliferation of human corneal endothelial cells (HCEC), not only to treat the excessive loss of endothelial cells but to generate a complete corneal endothelium in the laboratory for clinical use. Some researchers have proposed well-defined protocols to promote proliferation of HCEC in culture, while others have proposed the use of alternative cellular sources such as mesenchymal stem cells¹⁴. Current methods for isolating and culturing HCEC vary substantially between different research groups and present different success rates¹⁵. To date, there are no published data on the application of these therapies in patients, except the use of selective inhibitors of ROCK kinase (p160-Rho-associated coiled-coil signaling pathway), which promotes the migration and proliferation of endothelial cells of the patient¹⁶.

The region of the cornea used for the isolation of HCEC depends not only on the decision of the researcher but on the availability of tissue. Many use corneo-scleral rings; however, others prefer to isolate them from complete corneas since they contain a greater number of viable HCECs¹⁷. Among the HCEC isolation methods, enzymatic digestion and explants techniques are the most used, with very similar success rates of both¹⁸. Although HCEC cultures can be established directly on uncoated plastic plates, it is recommended to cultivate them on an FNC coating (Fibronectin-Collagen Combination Coating Mix) that acts as an artificial extracellular matrix to increase adhesion and proliferation¹⁸. Other substrates have been proposed for the culture and subsequent implantation of HCEC based on natural matrices (amniotic membrane or corneal stroma) or artificial scaffolds based on diverse biomaterials^19,20.

REGENERATION OF THE CORNEAL STROMA

Although it seems that success has been achieved to provide efficient corneal cell cultures, to create a complete artificial cornea for clinical use by tissue engineering we require the generation of an artificial matrix that reproduces the three-dimensional structure and functionality of the human cornea. To this end, research has begun to develop artificial acellular matrices with biomaterials, to subsequently incorporate in them the keratocytes obtained from cultured limbal or corneal stroma samples²¹. The most used biomaterial for this purpose has been collagen type I, type III, or the mixture of both. In general, a crosslinking process is associated, or the addition of other elements such as chitosan, laminin or glycosaminoglycans, with which it is possible to increase the transparency, decrease the shrinkage of the scaffolding or promote cell adhesion both at the stromal and epithelial levels^22,23.

Most of the models described so far are partial – hemi-corneas – where the stromal component is usually associated with the epithelia^l6,24. Few researchers have been able to develop a model that contains the three layers that make up the cornea, especially the endothelium. Some complete models are based on the conjunction of non-human animal corneal cells and artificial matrices based on fibrin-agarose²⁵ (Figure 3) or collagen supplemented with other elements²⁶. Other groups have generated complete human corneal models through the use of collagen with or without glycosaminoglycans²⁷, with the use of a step-by-step technique, based on the superposition of layers of cells arranged in sheets²⁸ or using elements other than collagen such as keratin. or chitosan²⁹.

Some researchers are developing artificial matrices based on the same matrix synthesized by keratocytes or human corneal fibroblasts in culture (self-assembling technique) (Figure 4) after stimulating them with ascorbic acid²⁸ or retinoic acid³⁰. It is possible to associate molecules such as TGFβ3 in order to control the fibrotic response that can be induced³¹.

A novel approach is based on the isolation of mesenchymal stem cells from the limbal stroma, which expand faster in culture than keratocytes and can also synthesize their own extracellular matrix of ordered collagen lamellae. It is also possible to induce an optimal regeneration at the level of the damaged corneal stroma after its implantation in vivo³². Only one research group has been able, to date, to fully restore damaged corneal stroma in patients, with published results after conducting a clinical trial³³. In this study, an acellular scaffold, composed of recombinant human collagen subjected to cross-linking was used to replace the damaged corneal stroma of 10 patients. After 4 years, they presented an average corrected visual acuity of 0.37, with a gain of 5 Snellen vision lines. Recently, this group has described modifications to optimize the composition of the scaffolding and improve its biomechanical characteristics, in order to solve part of the problems found³⁴.

On the other hand, the use of biological substitutes based on decellularized tissues is increasing considerably³⁵. These tissues, once the cells they contain are removed, preserve the composition and structure of the native tissue, and therefore favor the regeneration and remodeling of the latter by the cells that will populate it³⁶. Different animal species have been used to obtain the tissue to be decellularized, being the pig the most common³⁷. Other tissues have also been used to generate a substitute for the corneal stroma such as rat intestine³⁸ or fish scales³⁹. The cornea generally requires a decellularization that combines different techniques through the use of detergents, enzymes or osmotic changes^37,40,41. In some cases, the decellularized tissues are subsequently subjected to a recellularization process, by which human corneal cells are added to the acellular matrix, in order to facilitate integration into the recipient tissue^37,41,42 (Figure 5). The efficacy of many of the corneal substitutes generated by these techniques has been evaluated in experimental animal models; however, even today, a model that can be transferred to the clinic has not been generated.

CONCLUSION

The advances achieved in more than 20 years of tissue engineering and regenerative medicine in the cornea have managed to regenerate the epithelial component through CLET. We also approach the regeneration of the endothelial and stromal components independently. However, it has not been possible to generate a complete artificial cornea that can effectively replace the damaged one. To achieve this goal, we must continue to advance in the improvement and search for biomaterials that can be correctly integrated in the recipient, not only promoting cell growth but also the optimal functionality to serve as a real corneal substitute.