Robotics & Bionics

Tej Kohli: Bioengineered Cornea Are About To Enter The Clinical Realm. What Does That Mean?

Tej Kohli is the founder of the not-for-profit Tej Kohli Foundation whose ‘Rebuilding You’ philosophy supports the development of scientific and technological solutions to major global health challenges whilst also making interventions to rebuild people and communities around the world. Tej Kohli is also an impact investor who backs growth-stage artificial intelligence and robotics ventures through the Kohli Ventures investment vehicle. Tej Kohli’s blog is #TejTalks and he is the author of Rebuilding You: The Philanthropy Handbook.

Twitter @MrTejKohli.
Gross appearance of the regenerated cornea of all 10 patients who received bioengineered corneal implants comprising recombinant human collagen. Left two columns: patients at 2 years post-operation. Right two columns: The same patients at 4 years post-surgery. Compiled figures adapted from Fagerholm et al. (20102014), with permission from AAAS and Elsevier.

A watershed point in corneal transplant surgery is rapidly being approached. Transplantation using human donor cornea has served us well for years and innovations in surgery have helped to improve outcomes for people with blindness or severe visual impairment.

But a chronic worldwide shortage of donor tissue, and the expense and technical challenges of creating and maintaining vital eye bank infrastructure, means that corneal transplant surgery using donor cornea is simply inaccessible to the majority of the world’s population, particularly in lower-income countries where corneal blindness is at its most pervasive.

Because of the global shortage of cornea donors, the bioengineering of corneas has become a sizeable new field in tissue engineering, regenerative medicine, and ophthalmology. Over recent years, there has been a plethora of approaches to develop biosynthetic alternatives to human donor corneas.

A cadre of ophthalmologists including scientists from the Tej Kohli Foundation Applied Research collaboration recently produced a new report about bioengineered cornea entering the clinical realm. This highly technical report summarises new innovations for bioengineering cornea.

In this post, I will share a flavour of the key elements of the report. As far as possible I will attempt to do this in inaccessible and non-technical terms. My objective is to make it easier for non-technical readers to understand both the challenges of combating corneal blindness, as well as how innovative new science and technologies are leading to novel new solutions that can improve the lives of millions of people around the world.

This is the modus operandi of the ‘Rebuilding You’ precepts of my non-for-profit Tej Kohli Foundation and something that I am immensely passionate about…

Background

Huge progress has been made in developing bioengineered corneas that can restore vision in patients who are suffering from blindness or severe visual impairment. For a long time, corneal transplants using human-donated cornea have been the only real solution, albeit a highly invasive one that requires complex surgery and sutures and carries an ongoing risk of rejection.

But worldwide there is a chronic shortage of donor cornea which, combined with the technical challenges of building eye bank infrastructure, means that an overwhelming proportion of those living with corneal blindness or visual impairment cannot access treatment. This treatment gap is particularly pervasive in lower-income nations, where corneal blindness is also most prevalent and has a disproportionately adverse social and economic impact.

But there are now many promising and different advances in the development of bioengineered substitutes for replacing the human cornea for vision restoration without relying on the human-donated cornea. 3D printing of cornea has begun, and cell-free pro-regeneration implants have already been used successfully in human trials. So what new treatments for corneal blindness can we expect to see? First, let’s take a step back to understand when the cornea and corneal blindness is really all about.

What Is The Cornea?

The cornea is the clear window at the front of the eye. Think of it a bit like a car windscreen — but for your eye. The cornea is the main refractive component of the eye which helps to focus light onto the retina. The cornea is around 11–12 mm in diameter and 550 μm thick. It is highly transparent, is an effective barrier to microorganisms, and has the ability to rapidly regenerate and repair itself in response to (minor) injury.

A recent review and meta-analysis using Bayesian hierarchical modeling estimated that 36 million people were blind worldwide, and another 217 million have a moderate-to-severe visual impairment (‘MSVI’) (Bourne et al. 2017; Flaxman et al. 2017; World Health Organization 2018a). The majority of those blind patients are found in developing countries and around 80% of overall blindness is estimated to be avoidable with early intervention.

How Many People In The World Are Blind?

Problems with corneal opacity (excluding from trachoma) are estimated to account for blindness in 1.3 million people and MSVI in 2.9 million, with corneal opacity from trachoma estimated to affect an additional 0.9 and 1.6 million worldwide. Corneal visual impairment has been named by the WHO as a priority eye disease (World Health Organization 2018b).

By definition, blindness requires loss of vision in both eyes; however, there is huge under-reporting of vision loss in only one eye. While robust data is lacking, it has been estimated that there is well over 1.5 million new cases of unilateral (both eyes) blindness each year worldwide, mainly in the developing world (Whitcher and Srinivasan 1997).

What Is Corneal Transplantation?

Cornea are the most commonly transplanted tissue globally, with 185,000 transplant surgeries estimated to have been performed in 2012 (Gain et al. 2016). Nearly 50,000 surgeries were completed at the Tej Kohli Cornea Institute in Hyderabad between 2015 and 2019 alone, mostly for free.

Corneal transplantation using human donated cornea can entail the replacement of selective layers of the cornea (known lamellar keratoplasty), or involve a full-thickness replacement (penetrating keratoplasty, PK).

Corneal transplantation was first performed at the beginning of the twentieth century but only gained popularity a few decades later with improvements in surgical technique and the use of steroid eye drops (Tan et al. 2012). Eye retrieval must be performed within 24 hours of the death of a donor and transported to an eye bank where the cornea can be stored for up to 4 weeks (Joint United Kingdom (UK) Blood Transfusion and Tissue Transplantation Services Professional Advisory Committee 2016).

The Limitations Of Human-Donated Cornea

Transplantation using human-donated cornea is fraught with inherent problems. Corneal grafts have a low short-term survival rate, with only 86 in 100 remaining successful for more than one year. This survival rate steadily declines to 73%, 62%, and 55% at 5, 10, and 15 years after transplant, which is similar to other transplanted organs (Williams et al. 2006).

Corneal graft failure in high-risk patients typically requires re-transplantation, but the rejection risk also doubles with every subsequent transplantation, and transplant survival rates reduce to below 20% after the third rejection of a corneal transplant (Claesson and Armitage 2013; Williams et al. 2008).

Perhaps one of the greater intrinsic problems of relying on the human-donated cornea to treat and cure people with blindness is the overwhelming shortage of donor cornea, particularly in the developing world. This is a significant impediment to reducing blindness from corneal disease.

A 2016 survey estimated that there are 12.7 million people worldwide who are waiting to receive a donor cornea, with only one donor cornea available for every 70 that are needed. Moreover, due to poverty, inequality, and pervasive treatment gaps, approximately 53% of the world’s population have no access to corneal transplant surgery at all (Gain et al. 2016), whilst building vital eye bank infrastructure is also highly complex, with existing eye banks face increasingly stringent regulations (Armitage 2011).

Even in well-resourced countries with an established corneal transplantation service, a lack of donor corneas presents a big challenge (Gain et al. 2016). A few countries, principally the USA, export corneas for transplant surgery, but procuring these corneas is prohibitively expensive for most healthcare systems. Corneal transplant surgery itself requires highly skilled staff and prolonged (and expensive) postoperative care that is simply inaccessible to the majority of people in the world who are suffering from corneal blindness.

Can Bioengineered Cornea Plug The Treatment Gap?

Bioengineering refers to the use of engineering principles to address issues in biology and medicine. ‘Bioengineered Cornea’ are substitutes for human donor cornea tissue that are designed to replace damaged or diseased corneas.

The bioengineered cornea can range from prosthetic devices that are designed to replace the cornea’s function to transmit light into the eye, through to tissue-engineered hydrogels and fully-reconstructed tissues that allow regeneration of tissue. There are also implantable ‘lenticules’ that can be implanted into the cornea to improve vision by altering the refractive properties of the eye as an alternative to wearing spectacles or undergoing corrective surgery.

Here we look at some of the current innovations in bioengineering cornea:

Bioengineered Corneal Epithelium

The corneal epithelium is the outermost layer of the cornea and is composed of a single layer of cells. This ‘epithelium’ part of the cornea has excellent ‘regenerative capacity, which means that as long as the stem cells are intact, surgical replacement of the epithelium is rarely necessary.

However, in situations where the stem cells are damaged or dysfunctional, such as after a severe chemical injury or due to Stevens-Johnson syndrome, a more challenging situation arises. Damage to the cells in the endothelium layers of the cornea invariably leads to vision loss.

A CLET implant was performed in Canada using a fibrin substrate (Le-Bel et al. 2019). More recently, a simple limbal epithelial transplantation (SLET) has been developed which bypasses the need for expansion of stem cells inexpensive cleanrooms (Sangwan et al. 2012). Preparation of a corneal epithelial limbal graft, showing the steps. (a) Limbal biopsy from the patient’s contralateral eye; bar: 1 mm. (b) expansion by co-culturing with irradiated human dermal fibroblasts; bar: 200 μm. © Cells on fibrin. (d) Histology (Masson’s Trichrome staining) of the tissue-engineered epithelium, showing a differentiated epithelium on a fibrin substrate; bar: 10 μm. Reproduced from Le-Bel et al. (2019) with permission from Elsevier.

But scientists have now identified a dormant population of replicating cells and have attempted to attempt to ‘activate’ these dormant cells to reinvigorate the propensity for regeneration even in extreme cases. Preliminary studies in humans have already shown promising effects, and the injection of corneal endothelial cells in combination with a ROCK inhibitor has been demonstrated in human clinical trials (Kinoshita et al. 2018).

If successful, this treatment could reduce the need for human-donor corneal transplant surgery in cases where stem cells are damaged or dysfunctional.

Bioengineered Corneal Stroma

The corneal stroma is the thickest layer of the cornea. Recent advances in stromal bioengineering include the 3D ‘bioprinting’ of stroma, with the first report by Connon and colleagues (Isaacson et al. 2018) being about a 3D bioprinted stroma that combined various ingredients into an “ink” for 3D printing corneal stromal implants.

The team printed their implants into formed molds. Subsequently, Duarte Campos et al. (2019) reported that the printing of a stroma comprising human corneal stromal keratocytes incorporated into an “ink” comprising 0.5% agarose and 0.2% type 1 rat tail collagen. A drop-on-demand technique was used to print the corneal stroma on a custom-made 3D bioprinter.

If successful, we might one day see synthetic corneal stroma being 3D-printed using a range of materials in substitution for relying on human donors.

Using support structure to facilitate the printing of a corneal structure with 3% alginate (nozzle diameter = 200 μm) and optimization of bio-inks for corneal 3D bioprinting. (a) The digital cornea is imported to the computer driving the 3D printer software slic3r and a preview of the concentric directionality of print is displayed. (b) The support structure is coated with FRESH to facilitate the 3D bioprinting of corneal structures. © View of the 3D bioprinting process. Corneal structures were printed with 3% alginate bio-ink stained with trypan blue to increase visibility. (d) Image of 3D bioprinted corneal structure captured prior to incubation. (e) FRESH is aspirated after 8 min of incubation and corneal structure is carefully removed from support, but begins to unravel 1-day post-printing once keratocytes were combined with the alginate bio-ink. (f) Images of corneal structures 3D bioprinted from composite bio-inks. (g) Relationship between nozzle diameter and printed thickness of corneal structures (left) and depiction of transparency of corneal structure 3D bioprinted from Coll-1 bio-ink. (h) Brightfield image of 3D bioprinted corneal structure containing cells at day 1 (left) and cell viability measurements over 7 days (right). (i) Representative live/dead stain images using fluorescence microscopy at days 1 and 7 after 3D bioprinting in Coll-1. Reproduced from Isaacson et al. (2018), licensed under CC BY 4.0

Bioengineered Cornea Replacements

The earliest bioengineered corneal replacements were prostheses known as keratoprostheses (KPros), designed to replace the function of the human cornea. Although a plethora of KPros has been designed and tested, only a handful are used in clinical practice. Traditional KPros was designed with a transparent core that allowed light transmission into the eye for vision and a ‘skirt’ that allowed for integration with the host’s tissues.

The Boston Keratoprosthesis is the most widely used KPro today, with over 12,000 units transplanted worldwide since 2015 (Salvador-Culla et al. 2016). However, the Boston approach still requires donor corneal tissue for attachment to the host cornea, thereby failing to overcome the problem of a chronic shortage of donor tissue. The challenge, therefore, is how to ‘connect’ these forms of the synthetic cornea without relying on donor human tissue.

New Advances In Keratoprostheses Design?

Bio-integration between the cornea and the optic stem is critical for maintaining the longevity of the KPros. Human tissue is mostly relied on as a ‘skirt’ to attach synthetic cornea, because otherwise, a lack of adhesion causes gaps between the cornea–optic stem interface, resulting in myriad problems

Various design modifications have ow been investigated to enhance bio-integration and overcome this problem. For example, Patel and colleagues (Patel et al. 2006) developed a two-prong approach to modify the surface to promote cell adhesion at the desired location. This strategy promoted bio-integration between the cornea and the KPros.

Whilst still early days, bioengineering offers the promise of redesigning the synthetic keratoprosthes to remove the need for human donated tissue.

Human Collagen Corneal Implants

Despite the successes of cell-based therapies, the various regulatory and scientific challenges — such as the need for specialized cleanrooms requiring highly trained personnel — could place limitations on their wide adoption. Therefore new innovations have also looked at bioengineering solutions t corneal blindness that is not reliant on cell-based therapies.

In 2009 a report was published of 10 patients in Sweden who had undergone corneal transplantation with cell-free, bio-responsive recombinant human collagen implants, with the 24-month and 4-year follow-up data subsequently also released (Fagerholm et al. 200920102014).

Implants were derived from the yeast Pichia pastoris, and these synthetic implants were secured in place with sutures and a bandage contact lens. Postoperatively, steroid and antibiotic drops were used three times daily for 3 weeks, at which point the bandage contact lens and sutures were removed.

Gross appearance of the regenerated cornea of all 10 patients who received bioengineered corneal implants comprising recombinant human collagen. Left two columns: patients at 2 years post-operation. Right two columns: The same patients at 4 years post-surgery. Compiled figures adapted from Fagerholm et al. (20102014), with permission from AAAS and Elsevier.

Whilst this solution did not offer a consistent improvement in vision outcomes for patients, the major achievement of the study was the excellent immune compatibility due to the process of induced tissue regeneration. The study had successfully stimulated the regeneration of the patient’s existing corneal tissues, thus removing the need for donor cornea.

This offers a promising glimpse of a future where curing blindness is not restrained by the need for human donor cornea. It is also a key element of the solutions that are being pursued by the Applied Research division of the not-for-profit Tej Kohli Foundation.

This pioneering piece of research was the first to use a cell-free biosynthetic analog of human collagen for stimulating cells from within the patients’ own eyes to regrow corneal tissue. This was a very encouraging piece of work, with the initial implant design providing a template for further improvement.

Bioengineered Corneal Implants As Medicine Delivery Systems?

Bioengineered corneal implants are also now being developed that incorporate nanoparticles or other carriers of drugs or other molecules. To help bioengineered corneal implants remain free from infection during the early healing process, anti-infective agents have been incorporated.

One potential advantage of this is to help overcome poor post-surgical compliance to multiple drops — which is a prevalent reason for corneal implant rejection in poor and developing countries.

Riau et al. (2015) have reported the inclusion of antibiotics in a hydrogel within a synthetic cornea, which when implanted into rabbits were found to have prophylactic activity against infection by S. aureus bacteria. Because of increased bacterial resistance to antibiotics, silver nanoparticles have also been proposed as alternatives to antibiotics.

(Top) From left to right: collagen-based corneal implant; green and blue implants comprising differently shaped nanoparticles within a collagen matrix. The differentially shaped nanoparticles confer different absorbance properties, and hence, different colors. (Bottom) Left: Absorption at 600 nm for bacterial suspension of Pseudomonas aeruginosa (strain PAO1) in the presence of hydrogels without and with the different types of AgNPs assessed in this work measured after 24 h. The dashed line in the plot indicates the absorbance of the samples measured at time 0. (Right) Survival colonies cultured after 24 h incubation of incubation of P. aeruginosa cultures in the presence of hydrogels containing the different types of AgNPs employed in this work. The dashed line shows the initial bacteria density. Adapted from Alarcon et al. (2016), with permission from the Royal Society of Chemistry.

Conclusion?

Cornea bioengineering is now a fast-growing and exciting field that has seen so much innovation that technologies developed for the cornea are also now being applied to other target organs in regenerative medicine.

For the patients in poorer countries who do not have eye bank infrastructure, or those high-risk patients who are not amenable to a conventional donor cornea transplantation, bioengineered corneas represent hope for eyesight restoration in corneal blind patients all over the world.