3D-printed human corneas: a new era of bioengineering

Manufacturing as we know it will never be the same – three-dimensional (3D) printing has revolutionised various industries by augmenting the capabilities of existing manufacturing methods, materials and range of applications. Through creativity, customisability and sustainability, it has successfully penetrated a wide range of industries, from aerospace and automotive to healthcare and biotechnology.

Recently, the focus of the scientific community has shifted towards transformative applications of bio-nanotechnology and finding revolutionary approaches for the reconstruction and regeneration of human tissues and organs. 

The technology can use biomaterials, cells, proteins and other biological compounds as building blocks to create 3D structures of artificial organs, layer by layer.

Scientists at Newcastle University have recently developed a method to create 3D-printed corneas using a simple, low-cost 3D bio-printer that extrudes bioink in concentric circles. In combination with synthetic biology and nanotechnology, this technology has the potential to radically transform the biomedical field.

Medical background
Diseases and injuries that can damage the cornea include keratoconus, Fuchs’ dystrophy, corneal ectasia, fungal keratitis, acanthamoeba keratitis, chalazion, a corneal abrasion and previous corneal surgery (or any other eye surgery that has damaged the cornea).

According to the American Academy of Ophthalmology:

If cornea is scarred, swollen, or damaged, light is not focused properly into the eye leading to blurry vision or cause a glare. If cornea cannot be healed or repaired, ophthalmologist may recommend a corneal transplant. This is when the diseased cornea is replaced with a clear, healthy cornea from a human donor. There are different types of corneal transplants. In some cases, only the front and middle layers of the cornea are replaced. In others, only the inner layer is removed. Sometimes, the entire cornea needs to be replaced.

Reports suggest that “about 53% of the world's population have no access to corneal transplantation. There is a considerable shortage of corneal graft tissue, with only 1 cornea available for 70 needed”.

The majority of people receive a replacement cornea from a human donor. Although the surgery has a high success rate, the supply of donor tissue is limited. In the developing world, access to donor tissue is even more difficult. Further, while human donor transplants are the standard treatment for corneal blindness, their inherent complications and limitations have prompted the development of synthetic corneal substitutes. Existing synthetic corneas can be categorised into:

• fully synthetic prostheses (eg, keratoprostheses); and
• hydrogels that permit regeneration of the host tissue.

Patent filings
Until now, only three patents have been filed that explicitly disclose a method for 3D printing the human cornea, as outlined below.

University of California
In 2015 the University of California filed a patent disclosing the fabrication method of an artificial cornea by separately culturing live stromal cells, live corneal endothelial cells (CECs) and live corneal epithelial cells (CEpCs). During this method, separate stromal, CEC and CEpC layers are printed in and used to encapsulate the cells in separate hydrogel nanomeshes. The CEC layer is attached to one side of the stromal layer and the CEpC layer to the other, thereby defining the artificial cornea. The CEC layer is attached to the stromal layer by applying a thin film of hydrogel between each of the layers and curing via ultraviolet exposure. Further, the live CEpCs and CECs are cultured and differentiated from limbal stem cells (obtained from autologous tissue) and CEC progenitors (obtained from autologous tissue). The patent discloses the use of acryloyl-PEG-collagen and methacrylated hyaluronan to prepare the bioink.

Shenzhen Huaming Biological Technology
Also in 2015, Shenzhen Huaming Biological Technology filed a patent disclosing the preparation method of an artificial cornea based on 3D printing technology.

The manufacturing method involves:

• creating a human vision model;
• creating a personal eyeball model by 3D scanning;
• importing data into a 3D printer;
• preparing printing materials;
• scanning the printing materials point by point through ultraviolet laser beams with wavelength in 355 nanometres and beam quality M2 in 1.0-1.3;
• curing the liquid printing materials from point to line and line to surface;
• using a computer to move a lifting platform;
• curing the liquid printing materials layer by layer;
• stacking layer by layer to obtain a 3D-printed cured product; and
• freezing, forming and processing.

The material is a liquid prepolymer or a liquid printing HEMA PVA hydrogel with azide benzoic acid, methyl acrylate, polyethylene glycol and a volume ratio of the compound in any subunot 6:1 or 10:1 formulated with the mixture. The photo-initiator is α-hydroxy alkyl ketones.

Pohang University of Science and Technology
Pohang University of Science and Technology filed a patent disclosing the method for preparing biocompatible cornea and decellularisation composition for such biocompatible tissue. The method includes:

• providing a cornea from a tissue source through corneal incision;
• decellularising the provided cornea in purified water containing charcoal for a predetermined period; and
• post-treating the decellularised corneal extracellular matrix by stirring it in a hypotonic solution, so that the charcoal is used as a decellularising agent for the cornea. This allows the corneal tissue to imitate the original corneal extracellular matrix. Therefore, the cornea should not be rejected by the immune system and the patient should recover quickly.

After inducing gelation, the gelated bioink is injected into a 3D printer and the size of the artificial cornea is matched to that of the recipient’s.

Related filings
Several organisations such as Bacterin International have filed patents which disclose the 3D-printed bio-material-based implants and have a general focus on 3D-printed corneas.

Apart from making 3D-printed corneas, various organisations such as Qingdao Sandi Biotechnology and Johns Hopkins University have filed other applications related to 3D printing technology in cornea implantation, including:

• stroma support material for 3D-printed corneas and a method for constructing a 3D cornea stroma support;
• a cornea scaffold material and its preparation method for treating the stromal layer of the cornea;
• a 3D-printed scaffold, using a multipolymer; and
• the apparatus for isolating CECs, manufactured using 3D printing.

Comment
It is widely known that the number of corneas donated for corneal transplants is insufficient and that patients are susceptible to complications, such as infections and immune responses. The scientific community has responded to the need to develop corneal bioengineering solutions, such as 3D-printed corneas. However, research must be done into materials such as alginate and collagen, which can not only be used to prepare bioinks for printing corneas but can also support cell growth within the construct and recruit host cells for better integration.  

While 3D printing methods are unparalleled in their precision, more efforts are needed to maintain a highly homogenous cell distribution within each layer during the printing process, as well as precise control of the spatial localisation of different layers (eg, the epithelium layer, bowmans layer, stromal layer, descemet’s layer and endothelium layer).

Since the technology is relatively new and few patents have been filed, academics and organisations should be able to easily penetrate the field and file patents related to the method, material composition, 3D printing apparatus and scanning techniques for the production of human corneas.