**4. Cell-free regeneration-inducing biomaterials**

Recently biomaterials have shown enormous potential in tissue engineering due to their desirable physical, chemical, and mechanical properties, which can guide cells to grow and promote functionality. Biomaterials developed as corneal substitutes should replicate the structure and functionality of the native cornea. They should mimic the in vivo microenvironment, e.g., extracellular matrix, to support resident cell growth, proliferation, and integration within the host tissue for bifunctional regeneration of damaged cornea. Biomaterials should also possess desirable characteristics such as biodegradability, biocompatibility, non-immunogenicity, and seamless integration with host tissues. In addition, biomaterials should possess sufficient mechanical (tensile) strength to support suturing or gluing and to withstand the intraocular pressure to support the fluctuations. Besides this, it is important to have optical transparency and a refractive index like that of a healthy cornea, plus allow the diffusion of nutrients into the scaffold and waste out of it [23]. Thus, it is important to have these considerations in mind for designing corneal construct mimicking native cornea. Griffith and coworkers have provided an extensive guide to consider beforehand while designing biosynthetic alternatives for clinical applications [24].

Natural, synthetic, and composite polymers are the main biomaterials used for corneal tissue engineering. Natural polymers tend to have inherent biocompatibility and bio-integration compatibilities, whereas synthetic polymers allow for customization of desired chemical and mechanical properties. Composite polymers have the advantage of both natural and synthetic polymers, i.e., they are both biocompatible and tunable to allow for desirable mechanical and chemical properties for corneal tissue engineering.

## **4.1 Naturally derived and synthetic hydrogels**

The main advantage of using naturally derived polymers for corneal tissue engineering is their biocompatibility. Commonly used natural polymers for corneal regeneration include collagen, silk fibroin, gelatin, chitosan, cellulose, hyaluronic acid (HA), and decellularized cornea. They are formed by chemical cross-linking and formation of physical bonds and networks.

## *4.1.1 Collagen and collagen-based hydrogels*

One of the most attractive natural polymers is collagen, the most abundant extracellular matrix component in several tissues including the human cornea. Collagen is the main component of corneal tissue. It possesses unique properties such as biocompatibility, bio-adhesiveness, suitable biodegradability, and low immunogenicity, which is to support corneal regeneration. In addition, collagen supports the corneal epithelial growth and cell adhesion without eliciting toxicity. The source of collagen plays important role in its physiochemical properties. Type I and type II collagen possess adequate tensile strength, whereas type III collagen is superior in mechanical strength and optical clarity [25]. Collagen from different sources such as porcine, bovine, rat, recombinant human collagen type I and III are commercially available. On their own, collagen hydrogels are soft and unstable. Attempts to improve the physicochemical properties of collagen-based hydrogels include chemical cross-linking and development of composite hydrogels with synthetic polymers to add mechanical strength. Examples include cross-linking by glutaraldehyde, 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC), and multifunctional dendrimers to improve the elastic and mechanical strength of collagen scaffolds [26]. One limitation of using chemical linkers is that they can be cytotoxic and not cell-friendly, limiting their use to fabricating cell-free scaffolds. A naturally derived cross-linker such as genipin was tested to reduce the cytotoxic effect of cross-linker, but the concentration

## *Advances in Biomaterials for Corneal Regeneration DOI: http://dx.doi.org/10.5772/intechopen.106966*

of genipin required to enhance the mechanical strength resulted in dark-blue-colored collagen scaffold [27].

Further, to reduce the heterogenicity of collagen derived from animal source, Fagerholm and team [28] performed the first successful clinical trials where they used the cell-free implant made from recombinant human collagen type III (RHCIII). Trial includes the treatment of 10 patients with keratoconus or central scarring with RHCIII implant, which showed stable regeneration of epithelium, stroma, and nerves after 2 years of implantation. The long-term observation also revealed the immune compatibility of implants. Cell-free implant containing carbodiimide cross-linked recombinant human collagen (RHC) was grafted into patients and revealed stable integration of regenerated neo-corneas without rejection. In addition, nerve and stromal cell regeneration observed over 4 years mimic microarchitecture of healthy corneas without recruiting inflammatory dendritic cells into the implant area [29]. Next, Griffith and coworkers conducted a clinical study in cornea patients with a high risk of implant rejection [30]. 2-methacryloyloxyethyl phosphorylcholine (MPC), a synthetic phosphorylcholine with reported inflammation suppressing properties, was incorporated into RHCIII to form interpenetrating networks. The resulting RHCIII-MPC hydrogels were implanted into the corneas of unilaterally blind seven patients (one dropout) by anterior lamellar keratoplasty after excision of the pathologic tissue. The patients were followed up for an average of 24 months (**Figure 1**). The implants supported the stable regeneration of the epithelium, stroma, and nerves over the observation period [30]. Short collagen-like peptides (CLPs) conjugated to polyethylene glycol (PEG) showed promising functionality equivalent to recombinant human collagen. CLP-PEG hydrogel supported stable regeneration of corneal tissue and nerve in preclinical animal testing [31].

Recently, a cross-linker-free supramolecular gel strategy has been explored to make collagen gel where collagen molecules were intertwined inside a pyrene conjugated dipeptide amphiphile (PyKC) without any functional group modification. The newly developed collagen implant was optically transparent, mechanically stable, and supported the growth of all corneal cells. It also triggered anti-inflammatory differentiation while suppressing the pro-inflammatory differentiation of human monocytes [32]. Plant-derived recombinant human collagen I was used to make hydrogel-based corneal graft to support mechanical stability, biocompatibility, and transparency in mini-pigs. Thus, showed the potential of plant-based RHC1 as an alternative to animal-derived collagen or allografts [33].

## **Figure 1.**

*Clinical trial of RHCIII MPC corneal implant in high-risk patient at the last follow-up showing three groups of patients as per the preoperative diagnosis: Infection (herpes and fungal keratitis), burn (alkali or thermal), and other (failed graft and post-stroke neurotrophic keratitis). Patient with infection showed best recovery with mostly clear regenerative cornea followed by cornea with burns. Reproduced from [30].*

## *4.1.2 Gelatin-based hydrogel*

Recently, methacrylated gelatin (GelMA)-based hydrogels have gained attention for corneal tissue engineering because of their appropriate mechanical strength and optical properties. GelMA hydrogels showed good biocompatibility with 98% survival rate of keratocyte after 21 days of culture. In vivo studies of Gel MA in rabbit model showed no signs of ulcer, edema, or infection when hydrogel was implanted in mid-stromal pocket and observed for 8 weeks. Hematoxylin and Eosin staining showed hydrogel integration within the host tissue with negligible foreign body reaction [34]. The efficacy of GelMA in repairing rat corneas compared with lamellar keratoplasty (LKP) showed significant differences between the two groups in terms of inflammatory cell infiltrates, corneal cell thickness, and expression of α-SMA and TGF-β after 3 months of observation. Thus, supporting the ability of Gel MA in alleviating the corneal stroma fibrosis and reducing the loss of corneal refractive power due to fibrosis [35]. Electrospinning was used to make Gel MA fibers that were then complexed with poly (2-hydroxymethyl methacrylate) (p(HEMA)) through UV photo-polymerization to form GelMA-p(HEMA) composite hydrogels. The composite hydrogel showed optical transmittance equivalent to that of the human cornea, highly promising mechanical strength, improved structural integrity, and supported proliferation of BCE C/D¬1b corneal endothelial cells in 3D culture [36].

Gelatin-based hydrogels have also been explored for the drug delivery applications to overcome the poor bioavailability of traditional eye drop method. Dual cross-linking reactions prepared gelatin hydrogel/contact lens composite via in situ free radical polymerization and carboxymethyl cellulose/N-hydroxysulfosuccinimide to encapsulate rutin for corneal wound healing. The composite hydrogel showed sustained release of rutin for 14 days and without eliciting toxicity. In vivo studies in rabbit cornea injury model showed that the rutin-encapsulating composite hydrogel supported faster healing (98.3% ± 0.7%) at 48 h post-operation compared with the composite alone (healing rate, 87.0% ± 4.5%). Results also revealed the role of ERK/ MAPK and PI3K/AKT signal pathways in corneal wound healing [37]. Injectable photo-cross-linked gelatin hydrogels were formed using acrylated gelatin and thiolated gelatin with tunable mechanical properties, biocompatibility, and biological properties that support the repair of corneal wound. The hydrogel showed promising cell viability in cell seeding and cell encapsulation studies and supported the regeneration of new tissues under focal corneal wounds in rabbit corneas [38]. Collagen/ gelatin/alginate (CGA) hydrogels were used for the sustained delivery of moxifloxacin (MFX) and dexamethasone (DEX) loaded into nanoparticles, for the treatment of infectious ocular keratitis. Lipid nanocarrier loaded with MFX/DEX (Lipo-MFX/ DEX) and encapsulated within the hydrogel showed promising cell proliferation ability when co-cultured with ocular epithelial cells. CGA-Lipo-MFX/DEX composite hydrogel showed the sustained release of drug for up to 12 h and exhibit the ability to inhibit bacterial growth to improve corneal wound healing [39]. Thus, gelatin-based hydrogels are not only promising alternatives for stimulating corneal regeneration but could also be a viable option for ocular drug delivery applications.

## *4.1.3 Silk-fibrion-based hydrogels*

Silk fibroin is a structural protein extracted from the silkworm's cocoon, Bombyx mori. Silk represents a unique choice to use as a biomaterial for tissue engineering due to its tunable and robust mechanical properties, controlled degradation, and
