Exciting Australian research into corneal tissue bioengineering could provide sight saving options for patients who would previously have lost their vision. Mimicking the complex corneal anatomy while remaining biologically functional is the key to success.
CORNEAL DISEASE: A SIGNIFICANT PROBLEM
Corneal disease and trauma contribute a significant portion of the global burden of vision loss, with an estimated 10 million cases of blindness or severe visual impairment.1 The relative incidence of corneal-related visual impairment remains variable across geographic regions and age groups, with literature indicating between 2.2% to 18.8% of cases are attributable to cornea disease.2,3 Within Oceania, corneal indications represent the fourth leading cause of moderate to severe visual impairment.4
Although Australian Tissue Banks continue to meet current tissue demand, the availability of allogeneic corneal tissue remains limited globally
Corneal transplantation remains the gold standard for visual rehabilitation with nearly three-quarters of patients showing visual improvement following surgery.5 Although lamellar transplant techniques appear to further reduce the risk of immunologic rejection, it remains a significant concern with around a quarter of patients experiencing at least a single case of rejection within the first five years of follow-up.6 This number increases in patients with pre-existing conditions, such as infectious keratitis and trauma, which remain at high-risk for transplant failure.7 Early rejection episodes are treatable with topical steroids, however in severe cases transplant failure will occur, requiring additional graft procedures that further impact the availability of donor corneal tissue. Although Australian Tissue Banks continue to meet current tissue demand, the availability of allogeneic corneal tissue remains limited globally.8 A 2012 review found that 53% of countries had limited or no access to corneal tissue, effectively suggesting that a single cornea was available for every 70 patients requiring transplantation.8 The anatomical and practical limitations of corneal transplant surgery, therefore, have driven research into bioengineered alternative approaches.9
CORNEAL ANATOMY: KEY ROLES IN DETERMINING BIOENGINEERED APPROACHES
Consisting of three primary layers divided by two extracellular matrix interfaces, the cornea is a transparent, avascular tissue with several fundamental functions; to maintain transparency and allow light to enter the eye, to provide adequate refractive power for optimal visual acuity, and to protect the intraocular contents from trauma and infection.10 The composition and architecture of each layer remains relatively unique, providing different challenges for researchers in developing separate bioengineered corneal layers. The complex interplay between layers and cumulative properties represents a further challenge in providing a functional, full thickness corneal tissue alternative.
The corneal epithelium is the outermost surface and represents the major refractive element of the cornea. Approximately 40-50 microns thick, it consists of four to six layers of stratified and nonkeratinised squamous epithelial cells. The superficial cell layers form tight junctions that prevent toxins and microbes from entering the eye. The presence of the surface glycocalyx is essential to tear film stability and therefore optical clarity. The innermost basal layer contributes almost half the total epithelial thickness and consists of wing and superficial cells. These cells are capable of mitosis and contribute, alongside limbal stem cells and amplifying cells, to constant epithelial regeneration, a process which occurs every seven to 10 days. The epithelium basement membrane is attached to the basal layer and remains an essential component for both adhesion and modulating cellular signaling between the epithelium and stromal layers11 (Figure 1).
The stromal layer accounts for between 80–90% of corneal thickness. The cell composition and architecture significantly contribute to the tensile strength, stability and transparency of the cornea.12
The stroma is predominantly collagen Type I which, combined with laminin and additional collagen types (III and V), form fibrils that in turn develop as triple-helix bundles. These bundles align as flat lamellar sheets (between 250–300 sheets in total). The structural complexity of the stroma supports the key properties with anterior lamellar interwoven in the anterior stroma for greater stability, and then in parallel within the mid and posterior stroma to optimise transparency. Keratocyte cells fill the intervals between layers and are responsible for the continued lamellar organisation and both renewal and regulation of the extracellular matrix components.
The endothelial layer is composed of a single layer of tightly bound, hexagonalshaped cells with a total thickness of five microns. Although progenitors are suspected to reside in the posterior limbal area, once formed, endothelial cells are considered to become mitotically inactive.13 The endothelial layer is attached to Descemet’s Membrane by hemidesmosomes.
The role of the endothelial layer is to maintain a stable water content and prevent excessive hydration through the passive pump-leak process between the stroma and aqueous humour.
Extracellular Matrix Interfaces (Bowman’s and Descemet’s Membranes)
Bowman’s membrane is an acellular basement layer connecting epithelial cells and the stromal layer. The function of Bowman’s membrane remains unclear, although a protective role for the sub-epithelial nerve layer has been hypothesised.14 Descemet’s membrane is a basement layer continuously secreted by corneal endothelial cells.10 Descemet’s membrane maintains endothelium structure and may play a further role in maintaining corneal dehydration as evidenced by stromal oedema following damage to the structure through surgery or disease process (e.g. corneal hydrops and keratoconus).
Both membranes are predominantly composed of collagen (Types I, III – VIII), although fibronectin and laminins may also be found within these layers.
ARTIFICIAL CORNEAS AND TISSUE BIOENGINEERING
Although primary corneal transplant surgery is broadly successful, patients with recurrent or chronic inflammation and corneal vascularisation remain at high risk for graft failure. Similarly, the odds of success for additional transplant procedures recede with each repeated graft.15 As suggested previously, the lack of access globally to donor corneal tissue represents a further roadblock for patients and surgeons alike. These issues have largely driven the development of alternatives including the concept of the artificial cornea and, in general, the development of bioengineered alternatives.
The initial concept of an artificial cornea for the rehabilitation of corneal disease was described in 1789 by Pellier de Quengsy, a French ophthalmologist who suggested replacing the opaque cornea with a silver-rimmed glass window.16 Although impractical from a materials perspective, he correctly identified the need for a skirt to anchor the optic which remains an ongoing consideration in most recent models. Modern artificial corneas favour two distinct models; a central optic with hard skirt plates, which sandwich existing donor corneal tissue, or a soft optic and skirt which is combined in an integrated design.17
The difficulties faced when incorporating artificial corneas include the need for biocompatibility with the skirt material used, the long-term concerns with integration of polymer material within living tissue, and the permanent exposure to air of the outer component of the keratoprosthesis.10 Although these keratoprostheses have provided good short-term outcomes in high-risk cases, few studies are available that indicate either visual outcomes or retention rates at extended follow-up, thereby demanding further investigation as an ongoing option. Priddy et al, in a recent meta-analysis of medium to longterm outcomes of the popular Boston Type 1 Keratoprosthesis, found that only 51% of eyes had vision > 6/60 at five years (Figure 2). The combined retention rate was estimated at 74% for a similar time period.18 Retroprosthetic membrane and glaucoma represented the main postoperative complications occurring in between 36.6% and 39.3% of eyes respectively. Of interest, patients with autoimmune diseases and cicatrising conditions continued to show a higher incidence of postoperative complications requiring further management.19 In a small series of nine eyes, a modified osteo-odonto-keratoprosthesis indicated encouraging results in these difficult patients, with all eyes retaining the devices at final visit, however cost and extended follow-up remain a concern, thus highlighting the need for a further option.20
Locally, the AlphaCor artificial cornea was developed with reasonable outcomes, however similar concerns suggest this remains a niche option also21 (Figure 2).
Bioengineered Corneal Tissue
Bioengineering aims to overcome existing limitations by fabricating tissue in the lab environment with patient-specific cells.22 Traditional tissue engineering is based on the interplay between cells, the fabrication of biocompatible scaffolds for additional stability, the transfer of cells and biological material, and the possible additional application of external stimuli including mechanical, chemical or biological stimuli e.g. collagen cross-linking.22 A number of key requisites are required for a fully-functioning bioengineered cornea, including comparative transparency, immuno-compatibility, strength and grade manufacturing processes (Table 1). Cost is an additional consideration, particularly as this option will primarily benefit patients in developing nations with minimal current access to donor corneal tissue.
In the field of corneal tissue engineering, a range of natural and synthetic biomaterials have been investigated with variable impact and properties (Tables 2 and 3). Due to its abundance within the cornea, collagenbased materials remain the most common substitute and have been developed as hydrogels, compressed matrices and membranes.11,23 Other natural polymers, such as chitosan, silk fibroin and fibrin have been investigated. The relative advantages and disadvantages of these materials are listed in Table 2.
The term ‘3D bioprinting’ refers to an additive manufacturing process based on a layer-by-layer approach by the deposition of bioinks in a precise arrangement.24
Bioinks represent a more recent alternative, although the main challenge remains in replicating the complex corneal fibril arrangements and extracellular matrix architecture of the corneal stroma. A range of bioprinting methods are currently in use with the main types listed in Table 4. The use of the techniques will predominantly be driven by the structures under investigation e.g. epithelium, stroma, and endothelium, however practical considerations such as cost and availability continue to play a role (Table 4). Additional techniques, such as electrospinning, are being developed and may provide additional advantages. Electrospinning, for example appears to be able to more closely replicate the shape and structure of the human cornea. However, until this technique can be used to develop tissue with a comparable composition, in vivo use may not be possible, at least in the long term.
The development of fully integrated corneal tissue remains a significant goal of researchers. Breaking down the current developments by corneal layers provides an understanding of the challenges facing scientists and clinicians.
Each year in Australia, approximately 55,000 hospital emergency presentations are due to corneal injuries including foreign bodies and abrasions. These cases lead to direct and indirect costs of more than AU$155 million annually, representing a significant economic and practical burden.25 Although minor defects will regenerate quickly, more significant surface injuries may result in non-healing defects, neovascularisation, corneal melting and or scarring.26 In addition to routine antibiotics and anti-inflammatory topical treatment, blood-derived treatments including platelet-rich plasma and platelet lysate have become increasingly common. The proposed benefits over standard treatment include the addition of a diverse range of growth factors and cytokines that have been shown to facilitate wound healing in these cases.27 In more significant cases, amniotic membrane or autologous fibrin membranes may be used in conjunction with the blood-derived products.28 Potential benefits however, remain offset by the required preparation, lack of transparency and variable mechanical strength allowing the treatment to remain in place. Subsequently, the use of bioinks incorporating cell lines have been identified as playing a potential role to support faster and improved healing in surface injuries and further, in cases of corneal perforation.27
Two main epithelial approaches are used; 3D in-situ bio-printing direct to the corneal surface, or as part of a multi-layer approach applied to a printed structure or scaffold. The use of in-situ printing may be most beneficial in providing early treatment for acute surface injuries in the absence of specialist care. To be successful, a bioink must be capable of shear thinning to be distributed adequately by the delivery system and further, must be able to undergo phase transition to the desired form upon printing, most routinely as a gel. The benefit of a multi-layer approach is increased support and mechanical strength, however reduced light transmittance may represent a trade-off leading to reduced visual outcomes. Decellularised corneas as the base of the structure have previously been used with some success, although transparency was reduced to approximately 75%, well below the 90% of the standard cornea.29 Several bioinks have successfully used materials of non-human origin (bovine and porcine materials) to generate a working product although this provides additional potential concerns30,31 (Table 5). The use of additional cross-linking at the point of extrusion may be required to maximise the mechanical strength of the bioink material and help retain positioning over the defect. Although successful, this represents an additional step and long-term safety of the use of cross-linking in corneal injuries is yet to be elucidated.
Our team recently developed a human platelet-lysate based bioink containing fibrinogen and thrombin, both products widely used within tissue glues in ocular surgery.27 The combination appeared to represent a positive balance between shear thinning and gelation, allowing for in-situ extrusion printing onto the wounded cornea with transparency equivalent to healthy corneal tissue (Figure 3). Initial animal tests have suggested faster recovery and increased pain relief compared to standard treatment, representing a further positive step in the use of bioengineered alternatives (Figure 4).
The stroma is a crucial structure for corneal bioengineering due to both the volume and complexity of the tissue.13 Mimicking the intricate structure of the stroma is difficult and several approaches have been taken previously. Sorkio et al introduced a laserbased bioprinting technique using a donor slide coated with collagen type IV and type I layers. This is treated with laser pulses to create expanding vapour bubbles which are then collected on a collector slide with arbitrary geometric designs and maintained. Although these cultured substrates have shown increased cell proliferation, they currently lack the ability to integrate corneal stromal keratocytes (CSK) which remain essential to develop a feasible alternate, functional stromal layer.32 More recent structures printed onto various 3D moulds have provided geometrics closer to the native cornea, however incorporation of CSKs remain a challenge that impact the potential microstructure and mechanical strength of the derived tissue. Relative to this laserbased printing, a drop-on-demand approach appears to be more successful in propagating the introduction to CSKs.33 Although this technique appears successful at reproducing CSKs with similar characteristics of native cells, the constructs have lacked both mechanical stability and light transmittance, highlighting the potential trade-off across various solutions. The use of decellularised corneal extracellular matrix tissue in stromal engineering may provide improved cellular morphological features and mechanical properties, however they still do not appear to approach the elastic modulus of normal stromal tissue (bioengineered tissue ~15- 100 KPa vs 150-700kPa of normal stromal tissue). It is likely that future options may represent a hybrid approach.
Recently, researchers at the ARC Centre of Excellence for Electromaterials Science at the University of Wollongong have provided an electro-compaction model which has shown increased mechanical stability while retaining the ability to incorporate CSK within the structure. This further suggests that collaboration outside of ophthalmology may provide additional, improved options.
In-vivo human corneal cells have long been considered as non-proliferative, and approaches to treat endothelial disease have traditionally represented full thickness or lamellar procedures. More recently, in-vitro modified endothelial cells have been reinjected into patients with a requirement of face-down positioning during the immediate recovery period.34 To aid this approach, endothelial cells have been seeded on biological matrices prior to implantation. Biological and synthetic carriers have been used as substrates with variable success, with poor or suboptimal degradation of the substrate and inflammatory responses representing key issues requiring further research.13 Incorporation into an expanded corneal structure requires consideration of an adequate mechanism to promote adherence to the bioengineered corneal stroma.
FULL STRUCTURE CORNEA
The goal of scientists is to convert traditional 2D cell constructs conceived in-vitro to a more intricate, 3D structure capable of mimicking natural corneal properties while maintaining optimal biocompatibility. Corneal innervation and cell immunity are also required.10 Early results with lamellar transplantation of biosynthetic corneal stromal implants indicated that tissue remained fully integrated, however increasing corneal astigmatism and surface steepness changes over the initial healing suggest that optimisation of the full corneal approach is required.35
PRACTICAL AND ETHICAL CONSIDERATIONS OF BIOENGINEERED CORNEAS
Current techniques may employ primary cell cultures to support the existing bioengineered tissue which provides a range of potential concerns. Practically, large scale manufacture of cell lines requires significant base product including human corneal endothelial cells for example. Inter-donor variation and the risk of tissue contamination represents a key issue demanding the development of good manufacturing practice to help standardise quality and safety applications.36 Providing adequate storage and transportation mechanisms is a further consideration for companies and end-user surgeons. The use of biological inks or compositions raises ethical questions also. Although this may be ameliorated by using the patient’s autologous cells in preparation, little is known about the future biological behaviour of cells, either taken from non-ocular tissue or from cells that have undergone significant mechanical stress as may occur through preparation.24 As this may impact future use with unwanted side effects, it requires further investigation and consideration. The risk of non-autologous material represents additional considerations for the possible development of zoonosis diseases.13 From both a practical and ethical perspective, the use of randomised controlled trials within medicine supports the introduction of new products and technology through phased studies. The use of biological tissue alternatives, particularly customised approaches, represents a niche potential role initially, which may impact the ability to complete these essential large-scale projects. Close collaboration with national regulatory bodies, such as the Therapeutic Goods Administration, remains essential to develop these options further.
Corneal tissue bioengineering can potentially bypass a number of existing limitations of conventional corneal transplantation and treatment. This technology will continue to provide a range of exciting opportunities for both surgeons and particularly, patients who previously may not have had access to sight-saving options. Ensuring that products adequately mimic the complex corneal anatomy while remaining biologically functional represents a key goal for researchers and will likely determine the potential use and applicability of these options for the broader market. Maintaining mechanical, physiological and biochemical properties through long-term use continues to require additional consideration, as does the development of adequate good manufacturing practice procedures.
Locally, the Australian government has recognised the potential applications of bioengineering to both eye and general disease by supporting a range of initiatives. Combined with our increased knowledge, this bodes well for positive outcomes sooner, rather than later.
Professor Gerard Sutton MBBS MMed FRANZCO is internationally recognised as an expert in cataract surgery, laser vision correction and corneal transplantation. He has performed over 20,000 surgical procedures, and is the Sydney Medical School Foundation Professor of Clinical Ophthalmology and Eye Health. Prof Sutton consults at Vision Eye Institute Chatswood and is the Medical Director of the Lions NSW Eye Bank. He remains actively involved in research collaborations with University of Sydney and University of Wollongong.
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- Gunnlaugsdottir E, Arnarsson A, Jonasson F. Five-year incidence of visual impairment and blindness in older Icelanders: the Reykjavik Eye Study. Acta Ophthalmol. 2010 May;88(3):358-66.
- Keeffe JE, Casson RJ, Pesudovs K, et al. Prevalence and causes of vision loss in South-east Asia and Oceania in 2015: magnitude, temporal trends and projections. Br J Ophthalmol. 2019 Jul;103(7):878-884.
- Williams KA, Lowe M, Bartlett C, et al. Risk factors for human corneal graft failure within the Australian corneal graft registry. Transplantation. 2008 Dec 27;86(12):1720-4
- Stulting RD, Sugar A, Beck R, et al. Effect of donor and recipient factors on corneal graft rejection. Cornea. 2012 Oct;31(10):1141-7
- Tran TM, Duong H, Bonnet C, et al. Corneal Blindness in Asia: A Systematic Review and Meta-Analysis to Identify Challenges and Opportunities. Cornea. 2020 Sep;39(9):1196-1205.
- Gain P, Jullienne R, He Z, et al. Global Survey of Corneal Transplantation and Eye Banking. JAMA Ophthalmol. 2016 Feb;134(2):167-73.
- Barbosa AP, Alves M, Furtado JMF, et al. Corneal blindness in Plato’s cave: the acting forces to prevent and revert corneal opacity. Part I: epidemiology and new physiopathological concepts. Arq Bras Oftalmol. 2020 Sep-Oct;83(5):437-446.
- Guérin LP, Le-Bel G, Desjardins P, et al. The Human Tissue-Engineered Cornea (hTEC): Recent Progress. Int J Mol Sci. 2021 Jan 28;22(3):1291.
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- Fuest M, Yam GH, Mehta JS, Duarte Campos DF. Prospects and Challenges of Translational Corneal Bioprinting. Bioengineering (Basel). 2020 Jul 6;7(3):71.
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- Williams KA, Esterman AJ, Bartlett C, et al. How effective is penetrating corneal transplantation? Factors influencing long-term outcome in multivariate analysis. Transplantation 2006; 81: 896-901.
- Chirila TV, Hicks CR. The origins of the artificial cornea: Pellier de Quengsy and his contribution to the modern concept of keratoprosthesis. Gesnerus. 1999;56(1-2):96-106
- Fu L, Hollick EJ. Artificial Cornea Transplantation. 2021 Mar 16. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2021 Jan–. PMID: 33760451.
- Priddy J, Bardan AS, Tawfik HS, Liu C. Systematic Review and Meta-Analysis of the Medium- and Long-Term Outcomes of the Boston Type 1 Keratoprosthesis. Cornea. 2019 Nov;38(11):1465-1473.
- Nonpassopon M, Niparugs M, Cortina MS. Boston Type 1 Keratoprosthesis: Updated Perspectives. Clin Ophthalmol. 2020 Apr 29;14:1189-1200.
- Bakshi SK, Graney J, Paschalis EI, Agarwal S, Basu S, Iyer G, Liu C, Srinivasan B, Chodosh J. Design and Outcomes of a Novel Keratoprosthesis: Addressing Unmet Needs in End-Stage Cicatricial Corneal Blindness. Cornea. 2020 Apr;39(4):484-490
- Hicks CR, Crawford GJ, Tan OT, Snibson GR, Sutton GL, Gondhowiardjo TD, Lam OS, Downie N. Outcome of implantation of an artificial cornea, AlphaCor: effects of prior ocular herpes simplex infection. Cornea; 2002 Oct, 21(7):685-90
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- Fagerholm P, Lagali NS, Merrett K, et al. A biosynthetic alternative to human donor tissue for inducing corneal regeneration: 24-month follow-up of a phase 1 clinical study. Sci Transl Med. 2010 Aug 25;2(46):46ra61.
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- Frazer H, You J, Chen Z, Sayyar S, Liu X, Taylor A, Hodge C, Wallace G, Sutton G. Development of a Platelet Lysate-Based Printable, Transparent Biomaterial With Regenerative Potential for Epithelial Corneal Injuries. Transl Vis Sci Technol. 2020 Dec 23;9(13):40.
- Alio JL, Rodriguez AE, Martinez LM, Rio AL. Autologous fibrin membrane combined with solid platelet-rich plasma in the management of perforated corneal ulcers: a pilot study. JAMA Ophthalmol. 2013 Jun;131(6):745-51.
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- Ong JA, Auvinet E, Forget KJ, et al. 3D Corneal Shape After Implantation of a Biosynthetic Corneal Stromal Substitute. Invest Ophthalmol Vis Sci. 2016 May 1;57(6):2355-65.
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