An Essential But Challenging Journey

Figure 5. Bioengineered cornea example (BIENCO).

WRITER Professor Gerard Sutton

Corneal disease accounts for one in eight cases of total blindness globally. It is a significant burden for patients, their support network, and the function of their community and country.1

And while corneal transplantation offers excellent outcomes, a global shortage of skills and donor corneal tissue, along with limited transplant survival, makes this approach unsustainable for all countries. Professor Gerard Sutton and his team at BIENCO (Bio-Engineered Cornea) are preparing to commence clinical trials that may offer a solution.

Corneal transplantation continues to offer an excellent opportunity for visual rehabilitation in many patients, with reported median postoperative best-corrected visual acuity of 6/12 across the significant Australian Corneal Registry database.2

Many transplant indications exceed this median value, representing a clear improvement over preoperative acuity and promoting impactful vision-related quality of life benefits.2,3 Transplant survival over time is a critical measure of success, and survival probability at five years remains positive between 54% to 78%.

Long-term survival is more variable, however, with reported rates at 20 years between 14% and 64% depending on the indication and the procedure.2 These outcomes suggest that while transplantation remains central to corneal surgical practice, there is room for improvement, particularly as many patients undergo surgery in early adulthood and are thereby likely to require further procedures

over time.4 The paradox is somewhat difficult to ignore. Corneal transplantation is still one of the most successful tissue transplants in medicine, but it remains biologically imperfect, requiring lifelong vigilance. Accordingly, failed previous grafts now represent the most common indication for corneal transplantation in Australia, accounting for 26% of procedures performed.2

Worldwide, the problem is magnified exponentially by the lack of available corneal tissue. In Australia, as in many developed countries, we have outstanding Eye Bank infrastructure that supports the ready availability of corneal tissue for patients with minimal waiting times.5 This is not universal, with only one in 70 patients globally able to access corneal tissue. So, patients in developing countries remain blind because there are not enough corneas to go around.6 A final challenge is ensuring that even when there is corneal tissue available, ophthalmic surgeons are trained to the high skill level required to perform the surgery, and to provide the lifelong follow up required. (Figure 1)

THE SOLUTIONS

This problem, I believe, is solvable (hopefully in my lifetime).

Eyebanks and Surgical Training Programs

In the past 20 years, eye banks around the world have become more efficient in the education of potential donors, recovery of tissue, technological adaptation, and innovation. In New South Wales, for example, when I was Medical Director of the NSW Eye Bank, with a concentrated effort from scientists such as Raj Devasahayam, we were able to reduce the waiting time for corneal transplant from two years to three months. This level of success has been replicated in many countries with political will and economic capacity.

Attempts to establish and develop eye banks in the developing world have been less straightforward. Supported by Vision Eye Institute and together with a team of dedicated eye bankers and surgeons, including Associate Professor Con Petsoglou (University of Sydney), I established and supervised an eye bank and surgical training programs in Myanmar over 15 years, between 2005 and 2020, but with moderate success.

Ultimately, we were not able to adequately address the decades’ long backlog of corneal blindness before our program ground to a halt in 2020 when the Burmese military executed a coup, ousting the democratically elected government.

Prevention of Blindness Programs and New Technology

These are critical, and I would argue the most effective long-term solution to eradicating treatable corneal blindness, but they can only be run in stable geopolitical environments. In the past decade, innovation in technology and surgery has provided hope.

This is a basic review outlining the many challenges, the progress thus far, and the future as I see it.

The ‘Artificial’ Cornea This problem isn’t new. The concept of an artificial cornea was first proposed as far back as 1789. French ophthalmologist, Guillaume Pellier de Quengsy had the insight to not only conceive a device, but also to understand the need for a porous prosthetic skirt for biointegration. This remains a fundamental concept of more modern versions.7 Fast forward to the 1960s, when we saw the introduction of the Boston keratoprosthesis type 1 (KPro). This is still the most widely implanted artificial corneal device, with more than 19,000 devices implanted worldwide to date.8

Reflecting Pellier de Quengsy’s initial observations, the KPro includes a front plate and central optics. This allows light to be transmitted, which is enabled by donor corneal tissue providing the skirt to the native cornea. Although short-term visual and retention outcomes remain reasonable, with 70% of eyes achieving 6/60 or better at two years and 90% survival or retention rates, artificial corneas largely remain a niche procedure for very complex cases and will not, on their own, solve the need for more corneas worldwide.

The complications and risks are not insubstantial; progressive glaucomatous disease, retinal detachments, and retroprosthetic membranes occur in approximately one third of cases.8,9

Figure 1. Bilateral corneal blindness in Myanmar.

Alternatives for extreme cases, such as the osteo-odonto-keratoprosthesis (OOKP), which uses autologous tooth-root-alveolar bone complex as the skirt material, provide a superior long-term option for device retention. However, the surgery is complex, extensive, and uncosmetic.10 Survival rates for OOKP devices or derivatives vary considerably, with published data suggesting 30-year survival rates for 49% to 94% of cases, indicating significant potential.11,12

In a cohort of 90 patients undergoing OOKP for significant anterior disease, including Stevens-Johnson syndrome, chemical injury, and ocular cicatricial pemphigoid, best-corrected visual acuity of 6/18 or better was achieved in a third of the eyes.13 However, vitreoretinal complications occurred in 45% of patients at follow up, an issue consistent across numerous OOKP cohorts.

The surgical complexity of inserting artificial corneal implants remains a significant barrier, particularly in developing countries where cost and availability remains a paramount consideration. Australians have been at the forefront of innovation in this area, with Professor Geoff Crawford and the

Lions Eye Institute in Perth developing the Alphacor keratoprosthesis,14 which received approval from the United States Food and Drug Administration (FDA) back in 2002. More recently, Dr Greg Moloney and Dr Tanya Trinh have established an Artificial Cornea Program at the Sydney Eye Hospital. These are challenging surgeries for end-stage disease, and while I predict continued improvement in the technology and surgical innovation, artificial corneas will not be the major surgical solution.

Corneal Bioengineering

Restoring corneal physiology, transparency, and biomechanics will, I believe, occur in the near future through regenerative medicine and bioengineering. In fact, it is already happening. In 2023, the Australian

Figure 2. BIENCO Collaboration.

Figure 3. Stromal layer organisation.

Corneal Bioengineering Collaboration was granted AU$35m by the Australian federal government through its Medical Research Future Fund scheme to develop bioengineered products to address the shortage of corneal tissue globally and impact corneal blindness in Australia and worldwide. The consortium brings together the University of Sydney, University of Wollongong, University of Queensland, University of Melbourne, the Centre for Eye Research Australia and NSW Health under the umbrella of BIENCO (Figure 2).

Characteristics of a Human Cornea

The human cornea has unique characteristics that support continued clarity and optical properties. The ability to adequately reproduce these will underline the success of any bioengineered replacement tissue and subsequent surgical procedure. They are worth highlighting:

Transparency. Corneal transparency is defined as the ‘cornea’s capability for light transmission’ and is critical for optimal visual function.15 The main corneal properties that support transparency include stromal thickness, collagen fibril organisation, and the continued function of the endothelial layer. Corneal transparency is measured as a percentage, with the normal cornea exhibiting light transmission between 80–98%

depending on the wavelength spectrum.16 Incident light interacting with collagen fibrils can cause light scattering, thereby reducing transparency, even in normal corneas. This is ameliorated by the highly organised, lattice-like arrangement of the collagen fibrils within the cornea. The presence of scarring or other corneal irregularities will impact transparency, and therefore, potential vision.

Bio-mechanical strength. The primary factors driving corneal biomechanics are thickness, curvature, and composition. The human cornea has a Young’s modulus (strength) that varies between 115 and 520 kPa, depending on age and conditions. Materials must approach the native corneal strength properties to successfully maintain optimal shape and properties when placed in the eye. The mechanical properties of the cornea reflect a complex interplay between many factors. Substrates, such as gelatin, silk, and of course collagen, have been used in different corneal models. Our understanding of the most appropriate stromal material remains incomplete, but we believe human collagen is the logical option. Already, we have successfully produced transparent collagen I and collagen IV (the main components of the human cornea) from donor human skin and placenta. Similar to crosslinking in keratoconus, collagen crosslinking on bioengineered tissue can aid biomechanical strength. We have currently produced a corneal substrate that mimics the transparency and strength of the human cornea.

Curvature. Any biomimetic corneal material must be able to be manufactured in the same curvature as the cornea. This is a critical structural factor that guides both stability and optical properties.17 In addition to corneal curvature providing the main refractive power of the eye, it also plays a significant role with respect to cellular behaviour through variations in surface tension and substrate strain. The presence of an irregular surface or suboptimal curvature can impact optical transparency. More prosaically, replicating the curved surface of the natural cornea presents procedural challenges, particularly for extrusion gel techniques. Our current strategies attempt to mimic the corneal lamellae’s thickness and orientation.

Metabolic function. The cornea is a relatively dehydrated structure that relies on the pump function of a viable endothelial layer to maintain transparency. Excess corneal hydration, for example in Fuchs’ dystrophy, can adversely impact the tensile and compressive properties of the cornea.16 Corneal permeability is also critical, allowing the passive diffusion of oxygen from the tear film and aqueous humour, and aiding the transport of glucose across the cornea. As we know from contact lens investigations, low permeability can impact function, leading to hypoxia and again, loss of corneal transparency.18 This is relevant to alternative tissue sources, which must maintain an adequate balance between low and high permeability states.

As you can see, even with this relatively superficial overview, the challenges facing scientists and surgeons in adequately replicating natural tissue are significant – but not impossible.

THE BIOENGINEERED JOURNEY SO FAR

Relative to the complexity of other transplanted organs, such as the kidney, liver or heart, the cornea – at first blush – appears to perhaps represent a simpler challenge for biofabrication. Indeed, several corneal properties readily lend themselves to the concept of fabrication, namely minimal thickness, small size, and lack of vascularisation.19 (Whenever I suggest this to BIENCO’s Lead Scientist Professor Gordon Wallace, he is quick to remind me that perhaps surgeons should stick to surgery.)

A major breakthough in corneal bioengineering and cell therapy occurred in Japan in 1993, when researchers isolated bovine corneal cells from the three main corneal layers (epithelium, stroma, and endothelium) before inserting them into a basic in vitro three-dimensional collagen gel culture.20 The authors noted further cell differentiation and proliferation. By the turn of the century, Swedish researchers had developed human cell lines from cells

isolated from individual corneal layers before placement in a collagen-based substrate.21,22 The corneal equivalent grossly resembled natural corneal morphology, transparency, and histology. These early model systems provided encouragement and a new exciting research direction.

Moving from the lab to the eye with even the most rudimentary modes proved challenging in replicating the form, strength, and function of the native cornea. The concurrent transition to lamellar corneal transplant procedures and the novel work in tissue repair provided further direction, both for understanding the optimal approach and providing additional opportunities. Lamellar techniques focus on replacing diseased corneal tissue, and similar to this procedure, clinicians found benefit in applying specialised bioinks directly onto injured sites. Our group developed a human-platelet lysate-based biomaterial (bioink) that showed greater adhesiveness than commercially available fibrin glue, along with faster recovery of corneal ulcers and lower pain scores in early animal experiments. Known as the iFIX system, it was generously supported by the NSW Government Medical Device Fund, and is now capable of treating epithelial defects in an animal model. Collagen and other bioinks have been developed by the BIENCO team and will ultimately form the basis for the development of a broader bioengineered cornea.23

Bioprinting corneal layers can overcome some of the aforementioned challenges, including form, strength, and transparency.17 With respect to the bioprinted cornea, the selection of materials and printing methods play a key role in the fabrication of corneal layers, and the combined corneal replacement tissue.

Most current research groups utilise a form of material extrusion. This is an additive technique that involves extruding a bioink through a nozzle in a predetermined path. This approach allows for a layer-by-layer deposition of the bioinks and facilitates increasingly complex tissue constructs. As we saw with our prior bioink experience, optimising the speed, pressure, and temperature of the extrusion process remains critical to establishing the corneal tissue and its properties, such as the maintenance of cell integrity and the shape construct itself. Different 3D printing modalities may offer respective advantages, for example light-based printing can help facilitate the polymerisation of bioinks at the time of printing (i.e. crosslinking), which potentially provides additional strength and may allow for more streamlined curvature of the construct.14

Orientation of the cells and fibril layers is critical for transparency and to further optimise overall strength (Figure 3). This may be enhanced using a support structure to maintain shape during printing, however the use of alternative technologies may also support an improved process. The BIENCO approach involves strengthened corneal lamellae interspersed with printed keratocytes. Although some properties, such as water content, were lower, general permeability was maintained. The most significant benefit, however, was increased strength, which now approaches the native cornea. (Figure 4)

Our team recently published novel findings of a dual-layered corneal structure using collagen-based bioinks, which supported high cell viability and appropriate morphological features. Having demonstrated retention of curvature, transparency, and integrity through the early response, we have established a physiological example capable of the next step: A true bioengineered corneal tissue capable of implantation and continued visual and structural benefit.24 (Figure 5, page 28).

The BIENCO team has now developed prototypes for an endokeratoplasty (BIENCO-EK) and full-thickness penetrating keratoplasty (BIENCo-PK). In addition to a further product that facilitates descemet membrane endothelial keratoplasty (DMEK) surgery, we expect to have the BIENCO-EK in preclinical trials this year.

FRIENDS OVERSEAS

Corneal bioengineering is an exciting area of research and, as well as our work in Australia, there are promising results from our colleagues internationally. It would be remiss of me not to mention the success of two of the other groups who have also had success in corneal bioengineering and cell therapy, which you will hear about very soon. Professor Shigeru Kinoshita in Japan has developed an endothelial cell-based therapy that can be used to treat Fuchs’ dystrophy. It is already available in Japan. In Israel, Dr Mimouni has successfully inserted a bioengineered endokeratoplasty in rabbits.

Our progress at BIENCO, and these successes internationally, make me confident that we will have solutions to address the shortage of corneal tissue and impact corneal blindness around the world.

Figure 4. Bioengineered cornea with and without cells highlighting continued transparency and potential for cell incorporation.

The role of eye banks will remain critical but will evolve. Manufacture of these products in Australia will, I’m sure, lead to further innovation and seed application to challenges in other areas of medicine.

It has been an exciting and challenging journey thus far, and I suspect we are about halfway there. Whenever my BIENCO team gets sidetracked by setbacks (or bureaucratic red tape), I always bring them back to the ‘WHY’. It is the patient.

When I first went to Myanmar in 2004, at the request of Dr Geoff Cohn, to assess the causes of corneal blindness, I took with me four corneas from the NSW EyeBank. I used them to begin teaching and transferring skills to a local ophthalmologist who would go on to become the second surgeon to perform a corneal transplant in Myanmar. When I arrived at the eye centre in one Buddhist monastery, there were 500 patients, all needing a corneal transplant. Yet I only had four.

It remained one of the most difficult times of my life but reaffirmed for me of the importance of translational research.

Professor Gerard Sutton MBBS MD FRANZCO is regarded internationally as an expert in cataract, refractive, and corneal transplant surgery and is in private practice at Vision Eye Institute, Chatswood, NSW.

A Professor of Ophthalmology at the University of Sydney, he established the world’s first university course in laser eye surgery, has published more than 150 scientific papers, and been invited to present internationally on over 100 occasions.

His surgical expertise has been recognised with numerous awards, including the XOVA Award for Excellence in Ophthalmology, The Lions International Award for Service in Ophthalmology, and both the Doug Coster and John Parr Medals. He is involved in clinical and translational research in modern cataract and refractive surgery and has introduced many new surgical techniques to Australia. He also currently directs a research team within the Faculty of Medicine and Health at The University of Sydney.

The author acknowledges assistance of Christopher Hodge PhD, Clinical Research Coordinator at Vision Eye Institute, in writing this article.

References available at mivision.com.au.