Design of functional biomaterials as substrates for corneal endothelium tissue engineering

Abstract

Corneal endothelium defects are one of the leading causes of blindness worldwide. The actual treatment is transplantation, which requires the use of human cadaveric donors, but it faces several problems, such as global shortage of donors. Therefore, new alternatives are being developed and, among them, cell therapy has gained interest in the last years due to its promising results in tissue regeneration. Nevertheless, the direct administration of cells may sometimes have limited success due to the immune response, hence requiring the combination with extracellular mimicking materials. In this review, we present different methods to obtain corneal endothelial cells from diverse cell sources such as pluripotent or multipotent stem cells. Moreover, we discuss different substrates in order to allow a correct implantation as a cell sheet and to promote an enhanced cell behavior. For this reason, natural or synthetic matrixes that mimic the native environment have been developed. These matrixes have been optimized in terms of their physicochemical properties, such as stiffness, topography, composition and transparency. To further enhance the matrixes properties, these can be tuned by incorporating certain molecules that can be delivered in a sustained manner in order to enhance biological behavior. Finally, we elucidate future directions for corneal endothelial regeneration, such as 3D printing, in order to obtain patient-specific substrates.

Keywords: biomaterials; cell therapy; corneal endothelium; tissue engineering.

Graphical abstract

Figure 1.

Anatomy of the eye and cornea. The cornea is the outer layer of the eye and contains different layers: epithelium, Bowman’s membrane, stroma, Descemet’s membrane and endothelium. Current transplant are DALK replaces posterior layers; EK replaces anterior layers; and PK replaces the entire cornea. DALK, deep anterior lamellar keratoplasty; EK, endothelial keratoplasty; PK, penetrating keratoplasty.

Figure 2.

Corneal morphology and functionality in normal and abnormal conditions. Cells present an hexagonal shape in normal conditions whereas its shape and size its modified in abnormal conditions (A). Light can enter through and arrive to the inner parts of the eye in normal conditions but light is not able to arrive in abnormal conditions (B). Adapted with permission from Ref. [8].

Figure 3.

Morphology of the corneal endothelium depending on the quality of the cells and the age of the donor. Adapted with permission from Ref. [9].

Figure 4.

The anatomy of the cornea. The cornea is the outer layer of the eye and contains different layers. The inner layer is the corneal endothelium, which is in direct contact with the Descemet’s membrane and needs specific properties to have a correct functionality.

Figure 5.

Schematic representation of CEC production depending on the initial pluripotent status of cell source. On top, ESC differentiated into CEC; in the middle, somatic cells (e.g. fibroblasts), can be reprogrammed into PSC, which can be differentiated directly into CEC; at the bottom, MSC can be differentiated directly into CEC or using a two-step protocol. CEC, corneal endothelial cells; DPSC, dental pulp stem cells; ESC, embryonic stem cells; iPSC, induced pluripotent stem cells; MSC, mesenchymal stem cells; NCSC, neural crest stem cells.

Figure 6.

Thermo-responsive dishes mechanism and culture. Thermo-responsive polymers at 37°C act as hydrophobic substrate, whereas at 20°C they act as hydrophilic materials (A). Culturing corneal endothelial cells (CEC) in thermo-responsive dishes permits the formation of a monolayer of cells attached to the dish at 37°C that can be isolated through the addition of enzymes as a cell suspension or lowering the temperature and creating a cell sheet (B).

Figure 7.

Matrices for enhancing cell behavior. Cells can be cultured in decellularized matrices, in ECM mimicking materials or in advanced ECM mimicking materials.

Figure 8.

Procedure performed on live sheep corneas (a) before and (b) after insertion of the poly(ethylene glycol) hydrogel film (PHF) into anterior chamber. Images from (c–h) show evolution of transparency of control and implanted PHF, together with H&E staining of (i) control and (j) PHF after 28 d implantation. Adapted with permission from Ref. [89].

Figure 9.

Representative images of adhesion of CEC on poly-ε-lysine hydrogel uncoated or combined with the different proteins. Symbol * denotes significantly different P < 0.05 compared to poly-ε-lysine hydrogel only (A–H). The images were quantified to clearly observe the differences in cell adhesion at 24 h (I). Scale bar 100 μm. Adapted with permission from Ref. [117].

Figure 10.

Confocal microscopy images of immunofluorescence staining (A) and quantification (B) of CEC tight junction protein (ZO-1) on fibronectin and collagen mix (FNC) coated patterned substrates at 7 days (scale bar = 50 µm). Adapted with permission from Ref. [125].