Corneal tissue engineering

Blindness is a worldwide problem. It is caused by corneal dysfunction due to various inherited or acquired diseases, or by trauma or burns. Annually, there are over 1 million new cases reported [5, 121]. Currently, corneal dysfunction is treated by transplantation of donor corneas, although this method can suffer from graft failure and immune reactions. There is also a continuous shortage of suitable donor tissue [6]. Therefore, alternative treatment options are needed, such as TE and stem cell therapy. For example, since mesenchymal stem cells (MSCs) have antiangiogenic and immunomodulatory properties, as well as the capability to inhibit corneal scarring, they have attained a great interest in corneal regeneration [122, 123]. The 3D bioengineering corneal tissues can be constructed

in vitroin many ways, for example, by using prefabricated matrices, decellularized corneal

The cornea is a transparent, avascular, collagen-rich connective tissue, and it forms

the front of the eye [6, 125]. It provides mechanical protection for the eye and protection from infections [6]. The cornea can be considered to be the window to the eye. It not only provides transparency and protects the retina from damaging UV light [6], but it is also mainly responsible for its refractive power [5]. The cornea is anisotropic, meaning that its properties are not uniform in every direction. The properties of the cornea are both non-linear elastic and viscoelastic. [126] There are three distinct cellular layers in the cornea (Fig. 2.18): endothelium, stroma, and epithelium. These layers are separated by an acellular and collagenous Bowman’s layer and Descemet’s membrane. [5, 6] Epithelium is a stratified non-keratinized squamous layer that regulates the transfer of water and other soluble components out of and into the stroma. It also provides protection for layers below from infections and chemical injuries. Together with the tear film, epithelium also allow the refraction of light (as it enters the cornea). [5, 6] The stroma makes up around 90% of the overall thickness of the cornea, and is composed of layers of highly organized collagen fibrils (lamellae, mainly heterotypic hybrids of types I and V) that together with some small leucine-rich proteoglycans (e.g., decorin, lumican, keratocan decorated with dermatan sulfate and keratan sulfate) provide mechanical support and suitable biophysical properties that transparency requires. The stroma also contains keratocytes that maintain the matrix components. [5, 6] A monolayer of specialized endothelial cells form the endothelium [5]. Endothelium serves as a metabolic pump that maintains a suitable level of stromal hydration by removing the water from the stroma. This is important for corneal transparency [6].

Cornea Iris Optic nerve Pupil Ciliary body Sclera Choroid Retina Fovea centralis Blood vessels Suspensory ligament Anterior cavity Posterior cavity Lens Epithelium (50-100 mm) Bowman’s Layer (~ 12 mm) Stroma (~ 500 mm) Descemet’s membrane (4-10 mm) Endothelium (5 mm) Keratocyte Collagen fibrils

Figure 2.18: Human eye anatomy. The specific anatomy of the cornea is magnified.

In order to mimic the functions of the cornea, there are at least three main requirements for corneal substitute material. It should provide transparency, protection, and suitable

refractive power. [6] It is difficult to rebuild corneal stroma due to its complicated structure and properties, such as transparency (light transmittance within the visible wavelength > 87% [127]) and mechanical properties (tensile strength 3.81 ± 0.40 MPa, Young’s modulus 3 MPa to 13 MPa [127]) [6]. The substitute material should be strong enough to be able to protect the cornea, and it should also withstand handling during the surgical procedure [128]. The refractive index of the cornea is n= 1.376 [111]. Although the cornea provides the strongest refractive power of the eye, it may not be necessary to replicate its exact refractive index, since refractive errors can be adjusted with spectacles. Therefore, transparency is more important. The transparency should mimic that of native tissue. A substitute material that can support the growth of an epithelial layer and serve as a vehicle for the transplantation of keratocytes would also be desired. [129] More general requirements for the hydrogel scaffold, such as suitable permeability and biodegradability have already been discussed in Section 2.4.2.

HA- and collagen-based hydrogels were studied in this thesis. Therefore, only studies related to HA- and collagen-based hydrogels used for corneal repair were chosen to be listed in Table 2.1. As previously mentioned, collagen I is the main ECM component in the corneal stroma. It provides high transparency and mechanical strength for the cornea [130]. Collagen hydrogels have many advantageous properties, such as low immunogenicity, biodegradability, and biocompatibility. Also, conventional collagen hydrogels can be chemically crosslinked or plastically compressed in order to improve their mechanical properties. [39] The feasibility of collagen-based hydrogels for use as regenerative scaffolds has already been demonstrated in many pre-clinical studies [5]. Subsequently, collagen, especially type I, is an obvious material choice for this application. Although HA is found in the eye from the vitreous, corneal epithelium, lacrimal gland, and conjunctiva, it is not normally found in the stroma [28, 131]. The benefits of HA include its anti-inflammatory properties and its ability to stimulate corneal epithelial cell migration (corneal wound healing) [28, 131]. HA also acts as a natural lubricant as it increases the corneal wettability by retaining water on the surface of the cornea. All these properties make HA suitable for ophthalmic applications. [28] At the moment, HA-based hydrogels are not as widely studied for this application as collagen hydrogels. Other hydrogels that have been used as scaffolds for corneal repair are, for example, fibrin, alginate, chitosan, gelatin, silk fibroin, and silicone hydrogels, as reviewed by Wright et al. [39] and Nguyen et al. [5].

Hydrogels

for

tissue

engineering

35

Table 2.1: Some collagen- and HA-based materials used for corneal TE, and HA- and AL-based materials used for neural TE. Abbreviations:

LESCs= limbal epithelial stem cells, CESCs= corneal epithelial cells, Ks= keratocytes, ASCs= adipose-derived stem cells, NPCs= neural progenitor cells, iPSC-NPCs= induced pluripotent stem cell-derived neural progenitor cells, NSCs= neural stem cells, PLGA=poly (lactic-co-glycolide), HA= hyaluronan, AL= alginate, PEG= polyethylene glyco, CEC-l-OSA= N-carboxyethyl chitosan-l-oxidized sodium alginate, PC= Plastic Compressed. Extracel-SS, HyStem-HP, and HyStem-CSS are commercially provided by Glycosan BioSystem Inc. (Salt Lake City, UT, USA). GMHA (thiolated hyaluronic acid) and Gelin-S (thiolated porcine gelatin) are commercially provided by BioTime Inc. (Alameda, CA, USA), GSSG (oxidized glutathione, sodium salt).

Application Polymer-basis Material Cell type Results Reference

Corneal Collagen EDC-NHS cross-linked collagen I Human CESCs Material supported confluent human epithelial and stromal-derived mesenchymal stem cell populations.

[132] Collagen I (high concentration) Human LESCs The matrices stood culture of epithelial cells coming

from a human limbal explant and remained transpar- ent during the culture.

[133]

Collagen I gel (PC) + electrospun PLGA mats

Human CESCs and Ks The cells adhered, proliferated, and maintained their phenotype well on the material.

[124] Hyaluronan Extracel-SS, HyStem-HP, and

HyStem-CSS hydrogels

Human ASCs h-ASCs were successfully grown on hydrogels in vivo and could express human cornea-specific proteins.

[134] CMHA/Gelin/GSSG hydrogels Human ASCs Supported the 3D culture of ASCs in vitro, and was

biocompatible in preliminary intracutaneous and sub- conjunctival experiments in vivo.

[135]

HyStem-C hydrogel Human CESCs The hydrogel scaffold-based xeno-free culture system supported the expansion of regenerative CESCs.

[136] Neural Hyaluronan Methacrylate-modified HA hydro-

gel, UV-crosslinked

Mouse ventral midbrain-derived NPCs

Softer hydrogels caused mostly neuronal differentia- tion, and stiffer hydrogels astrocyte differentiation.

[137]

Methacrylate-modified HA hydro- gel, UV-crosslinked

Human iPSC-derived NPCs

Layered hydrogel of different moduli influenced migra- tion and differentiation.

[138] Methacrylate-modified HA hydro-

gel, UV-crosslinked

Human iPSC-derived NPCs

Lower hydrogel stiffness promoted differentiation and robust neurite outgrowth was observed.

[139] RGD-heparin-HA hydrogel

crosslinked with PEG-diazide crosslinkers

Human PSC-derived neural progenitors

Enhanced cell survival both during and post- transplantation.

[140]

Alginate Ionically cross-linked AL hydrogel Rat NSCs The rate of cell proliferation was lower with higher hy- drogel modulus, the expression of the neuronal marker b-tubulin III was enhanced within the softest hydro- gels.

[141]

Ionically cross-linked AL hydrogel Rat embryonic corti- cal neurons

Cells encapsulated within the softest hydrogels showed excellent viability, extensive formation of ax- ons and dendrites, and long-term activity.

[142]

CEC-l-OSA hydrogels Rat NSCs Self-healing hydrogels with brain-mimicking stiffness supported the proliferation and neuronal differentia- tion of NSCs.