Full Terms & Conditions of access and use can be found at
https://www.tandfonline.com/action/journalInformation?journalCode=icey20
ISSN: 0271-3683 (Print) 1460-2202 (Online) Journal homepage: https://www.tandfonline.com/loi/icey20
Corneal Stromal Regeneration: Current Status and
Future Therapeutic Potential
Neil Lagali
To cite this article: Neil Lagali (2019): Corneal Stromal Regeneration: Current Status and Future Therapeutic Potential, Current Eye Research, DOI: 10.1080/02713683.2019.1663874
To link to this article: https://doi.org/10.1080/02713683.2019.1663874
© 2019 The Author(s). Published with license by Taylor & Francis Group, LLC. Published online: 20 Sep 2019.
Submit your article to this journal
Article views: 738
View related articles
Corneal Stromal Regeneration: Current Status and Future Therapeutic Potential
Neil LagaliDepartment of Ophthalmology, Institute for Clinical and Experimental Medicine, Faculty of Medicine, Linköping University, Linköping, Sweden; Department of Ophthalmology, Sørlandet Hospital Arendal, Arendal, Norway
ABSTRACT
The corneal stroma comprises 90% of the corneal thickness and is critical for the cornea’s transparency and refractive function necessary for vision. When the corneal stroma is altered by disease, injury, or scarring, however, an irreversible loss of transparency can occur. Corneal stromal pathology is the cause of millions of cases of blindness globally, and although corneal transplantation is the standard therapy, a severe global deficit of donor corneal tissue and eye banking infrastructure exists, and is unable to meet the overwhelming need. An alternative approach is to harness the endogenous regenerative ability of the corneal stroma, which exhibits self-renewal of the collagenous extracellular matrix under appropriate conditions. To mimic endogenous stromal regeneration, however, is a challenge. Unlike the corneal epithelium and endothelium, the corneal stroma is an exquisitely organized extracellular matrix containing stromal cells, proteoglycans and corneal nerves that is difficult to recapitulate in vitro. Nevertheless, much progress has recently been made in developing stromal equivalents, and in this review the most recent approaches to stromal regeneration therapy are described and discussed. Novel approaches for stromal regeneration include human or animal corneal and/or non-corneal tissue that is acellular or is decellularized and/or re-cellularized, acellular bioengineered stromal scaffolds, tissue adhesives, 3D bioprinting and stromal stem cell therapy. This review highlights the techniques and advances that have achieved first clinical use or are close to translation for eventual therapeutic application in repairing and regenerating the corneal stroma, while the potential of these novel therapies for achieving effective stromal regeneration is discussed.
ARTICLE HISTORY
Received 31 July 2019 Revised 28 August 2019 Accepted 29 August 2019
KEYWORDS
Corneal stroma; stromal regeneration; bioengineered cornea; acellular porcine cornea; stromal stem cells; 3D bioprinting
Introduction
Therapeutic regeneration of the corneal epithelium primarily using stem cells of limbal origin in various forms has received much attention and gained widespread clinical use, including the first European Medicines Agency approved stem cell product, Holoclar.1 Likewise, endothelial regeneration therapy promoted through the use of a donor endothelial cell suspension has entered first clinical trials for bullous keratopathy.2The bulk of the corneal tissue, however, consists of the corneal stroma, which comprises over 90% of the corneal thickness and imparts to the cornea its transparency, curvature and strength, and additionally protects the inner eye structures from the external environment. In clinical situations where the corneal epithelium and endothelium are healthy and functioning, the cornea can still lose its transparency by various corneal stromal pathologies that include infection, keratoconus, inflammation, neurodegeneration, neovasculariza-tion and corneal dystrophies. In these cases collectively affecting millions worldwide,3 the main therapeutic option has been to replace the corneal stroma with human donor tissue, either by full-thickness replacement of the cornea (eg., penetrating kerato-plasty where epithelium, stroma, and endothelium are replaced) or by partial thickness stromal replacement (eg., anterior lamellar keratoplasty where epithelium and the anterior stroma are replaced; or posterior lamellar keratoplasty where posterior stroma and endothelium are replaced). In cases where human
donor tissue transplantation has failed repeatedly or is contra-indicated– for example due to a chronically inflamed recipient eye or high expression of enzymes that would degrade the corneal stroma – a keratoprosthesis such as the Boston KPro4 or the osteo-odonto keratoprosthesis5 could be used. These prosthetic devices are used in rare cases of severe pathology or trauma, and often only after repeated failed transplantation with a donor cor-nea, and are thus not suitable for the majority of cases of corneal stromal pathology; therefore, in this review we focus only on primary replacements for the corneal stroma.
Stromal replacement as implemented today, however, has its limitations. Implantation of a donor cornea into a recipient eye induces scarring at the donor-recipient tissue interfaces, results in incomplete nerve regeneration,6–8 incomplete cellular repopula-tion of the graft,7changes in refraction due to suboptimal curva-ture of the implanted tissue,9 and carries the potential for immunologic rejection due to the foreign cells10contained within the donor tissue. Although these potential drawbacks of the use of human donor stromal tissue can be significant, the main draw-back of stromal replacement with human donor tissue is the global shortage of donated corneas and the need for infrastructure to identify, harvest, transport, store and distribute donor corneas within a limited time window. The need for donated corneal tissue and eye banking is a global problem that leaves millions of cases of avoidable corneal blindness unaddressed.11,12
CONTACTNeil Lagali neil.lagali@liu.se Department of Ophthalmology, Institute for Clinical and Experimental Medicine, Faculty of Medicine, Linköping University, Linköping 581 83, Sweden
Color versions of one or more of the figures in the article can be found online atwww.tandfonline.com/icey.
https://doi.org/10.1080/02713683.2019.1663874
© 2019 The Author(s). Published with license by Taylor & Francis Group, LLC.
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License (http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way.
Similar to epithelial and endothelial regeneration, it would be ideal if the stroma could be regenerated, assisted by an implant, scaffold, cells or other factors. Although no single regenerative approach for the corneal stroma has yet emerged as an approved therapy for clinical use, several alternatives have been proposed and are the subject of intensive research, with some having reached the clinical trial stage. The purpose of this review is to summarize the various approaches for regenerating or replacing the corneal stroma, either partially or fully, in order to treat corneal pathology. These approaches aim to supplement or completely circumvent the use of human donor corneas from eye banks, which are in short supply and are an inadequate option for the future treatment of corneal stromal pathology in many parts of the world. In this review, the concept of stromal regeneration is summar-ized, while highlighting those approaches and technologies for stromal regeneration that are closest to clinical application.
Evidence for endogenous stromal regeneration
The regenerative capability of the corneal stroma (including the maintenance of corneal transparency during regeneration) is linked to the presence of various anatomic features, all serving to maintain a strong and transparent corneal stroma (Figure 1).
The most anterior part of the stroma consists of Bowman’s layer, an approximately 10 µm thick layer of compacted, ran-domly oriented collagen fibrils13that merges continuously with the underlying corneal stromal lamellar structure. Bowman’s layer is non-regenerating, but when present is believed to facil-itate a more rapid and complete anterior corneal nerve and anterior stromal regeneration and healing after injury.14Below
Bowman’s layer is the corneal stroma proper, consisting of
approximately 300 distinct lamellae, each in turn consisting of parallel-oriented bundles of collagen fibers,15mainly consisting of type I collagen. The major stromal cell, the keratocyte, is present throughout the stroma with the cell body often localized between stromal lamellae. Keratocytes have multiple functions including collagen synthesis, collagen degradation, and partici-pating in wound healing through transformation to a (myo) fibroblast phenotype.16 Natural stromal regeneration through the normal keratocyte-mediated turnover of collagen is a relatively slow process, occurring over at least several years.17 This long turnover time may be a prerequisite for the regener-ated collagen to adopt a proper lamellar structure and orienta-tion to maintain corneal strength, structure and transparency (stromal regeneration); by contrast, short collagen turnover times are often associated with aberrant collagen production by myofibroblasts leading to stromal haze and permanent scar tissue deposition (stromal repair).18
Besides keratocytes (and other smaller cell populations not discussed here), the corneal stroma contains proteoglycans, which are produced by keratocytes and consist of dermatan and keratan sulfate components. These components interact with collagen fibrils to maintain fibril assembly and corneal transparency.19 As they are synthesized by keratocytes, the proteoglycan population of the stroma can regenerate. Finally, corneal nerves (axons originating from the trigeminal nerve) are present throughout the corneal stroma, with high densities present in the anterior stroma within the subepithe-lial and subbasal nerve plexi.20Corneal nerve regeneration is an important factor in maintaining stromal integrity, restor-ing a protective blink reflex and perception of pain, tempera-ture and chemical stimuli,21and for helping to ensure proper tear film production and adequate wetting of the ocular sur-face. Corneal nerves additionally facilitate epithelial wound
Figure 1.The human corneal stroma. A 10 µm-thick Bowman’s layer consisting of randomly oriented compacted collagen fibrils comprises the anterior portion of the stroma. Posterior to Bowman’s layer lies the corneal stroma proper, consisting of less compact but regularly arranged collagen fibrils organized in lamellae. Stromal cells called keratocytes are regularly interspersed throughout the stroma, preferentially between adjacent lamellae. Distributed within the lamellae in the extracellular matrix are proteoglycans produced by the keratocytes. Finally, corneal nerves traverse through the stroma and anteriorly form a dense subbasal nerve plexus.
healing; in corneas with a neurotrophic deficit, epithelial wounds do not heal adequately, leading to stromal degrada-tion (ulceradegrada-tion).22Corneal nerves within the normal, healthy subbasal nerve plexus (subbasal nerves) are continuously regenerating,23and this regeneration even occurs in the con-text of corneal injury or transplantation,24although the regen-eration may take several years (Figure 2). Given their physiological importance, corneal nerves and their regenera-tion should be an integral part of strategies aiming to regen-erate the stroma.
Human donor stromal lenticules
The refractive surgery procedure of small-incision lenticule extraction (SMILE) has been gaining popularity in the past decade due to the availability of accurate, high frequency pulsed femtosecond lasers and excellent refractive results postoperatively.25The procedure involves removal of a small
disc (lenticule) of stromal tissue approximately 10–50 µm
thick and 6–8 mm in diameter from the central stroma (at
least 100 µm from the most anterior stromal layer), in order to flatten the cornea and correct for myopia (Figure 3). To date over one million SMILE procedures have been performed,26potentially making a large pool of thin stromal lenticules available for treating stromal pathology. To facilitate this approach, Ganesh and colleagues developed a tissue
pro-cessing and cryopreservation technique27 for the extracted
lenticules, making long term storage possible in a cryobank
at liquid nitrogen temperatures (−196°C). The lenticules
extracted from myopic patients after SMILE were cryopre-served and subsequently implanted into hyperopic corneas, with no reported allogeneic rejection or loss of best corrected visual acuity. In a separate study, Ganesh and colleagues used similarly extracted stromal lenticules to treat a series of cases of corneal micro-perforation, corneal defect and traumatic corneal tear, by using the lenticule as a bandage patch by attaching it to the recipient cornea using fibrin glue.28 The procedure was safe and maintained corneal transparency and integrity. Extending the application of this concept, Wu and
colleagues used SMILE lenticules with thickness over 100 µm to seal corneal perforations by suturing the lenticules over the perforation sites.29 Interestingly, a few weeks following the operation, lenticules became incorporated into the corneal stroma, indicating a partial regenerative effect. In another study by Ganesh and colleagues, the center of the extracted lenticules was trephined to form a donut-shaped lenticule that was subsequently implanted into the cornea of a small series of keratoconus patients, to achieve effective central corneal flattening.30
The inherent biocompatibility of human-to-human tissue implantation, with potential reduction of immunogenicity of the allograft tissue by cryopreservation, is expected to be beneficial for integration of the implanted lenticule with the surrounding recipient stromal tissue. A regenerative effect could be achieved by long-term quiescent stromal cell infil-tration and turnover of the implanted collagen. This effect, however, has not yet been convincingly demonstrated in the case of human lenticule implantation, and thus the long-term fate of the lenticules is unknown. Would the thin lenticules degrade (and therefore corneal thickness decrease) over longer time periods or would the lenticules persist? Another question to be addressed with this technique is its extendibil-ity to replace a thicker proportion of the stroma (Figure 3). Could multiple lenticules be implanted simultaneously, and would such an approach be technically feasible and result in an adequate transparency, strength and corneal shape? Future research could address these questions. Furthermore, it has been proposed that a chemical decellularization method could be an alternative to cryopreservation of donor lenticules.31,32
Acellular porcine corneal stroma
Decellularization methods for supplementing the supply of donor corneas have gained popularity in recent years, speci-fically in the use of non-human (typically porcine) corneal tissue for human corneal transplantation. The use of non-human corneas presents an alternative to reliance on non-human cornea donation, as porcine corneas are readily obtainable
Figure 2.Regeneration of subbasal nerves following penetrating keratoplasty (PK) as documented by in vivo confocal microscopy in the same corneal region 3 and 4 years postoperatively. (a) After 3 years, only a single regenerated nerve is visible in this region, following a path around the peripheral postoperative scar tissue (hyper-reflective white region). (b) One year later upon examination of the same scar feature, increased nerve regeneration was apparent with nerve paths differing over time, indicative of continuous axonal growth and regeneration. Image size is 400 x 400 µm.
from the food processing industry and have similar character-istics to the human cornea in terms of size, thickness and biomechanical properties. Decellularization is required to mitigate the possibility of xenogenic immunoreactivity to the foreign porcine cells and the possibility of porcine-to-human disease transmission, and as such, applications are currently limited to stromal replacement and anterior lamellar kerato-plasty in cases where recipient endothelium is functional and the recipient epithelium is able to regenerate to cover the graft.
Porcine corneas have been subjected to various decellulariza-tion protocols including sodium chloride (NaCl), sodium dode-cyl sulfate (SDS), hypoxic nitrogen, ethylene diamine tetra-acetic acid (EDTA), aprotinin, gamma irradiation and hypotonic tris
buffer.33–37 The chemical treatments are often followed by
DNAse and RNAse treatments. Such treatments can substan-tially remove porcine cells and DNA; however, removal of the foreign immunogenic material is often not 100% complete and thus the possibility of transmission of porcine pathogens remains. Despite this, results of decellularized porcine cornea xenotransplantation in a rabbit model indicated absence of immune rejection and good biocompatibility after one year.38 In another report, porcine corneal stroma was decellularized by NaCl treatment and implanted into the rabbit corneal stroma for six months with or without the addition of cultured human corneal keratocytes.35 Without cells, a 100 µm thick stromal implant remained stable but without reported evidence of host cellular infiltration or stromal regeneration, while the addition of human keratocytes sandwiched between five 20 µm thick
porcine stromal sheets resulted in an initially transparent and stable cornea postoperatively, but rendered the implanted sheets undetectable at the experimental endpoint.
Decellularized porcine corneas have also been used clini-cally. A Chinese team in Wuhan reported a series of 47 patients with prior fungal corneal infections who underwent anterior lamellar keratoplasty, receiving acellular porcine cor-neal stromal grafts.39 No recurrence of infection was noted and healing of ulcers was reported in most cases, although
with the return of neovascularization and
non-epithelialization leading to graft melting in four cases. In another Chinese study, acellular porcine corneal stroma was used in lamellar keratoplasty to treat 13 patients for herpes simplex keratitis.40After a mean of 15 months of postopera-tive follow-up, seven cases had mostly transparent corneas but 11 of 13 cases had neovascularization, with graft melting occurring in three cases. Currently, a prospective clinical trial is ongoing in Guangzhou, using the acellular porcine cornea as a deep anterior lamellar graft in cases of infective keratitis.41 These reports, principally from China, indicate a potential future role for decellularized porcine corneas to meet the global demand for suitable stromal tissue for trans-plantation, which is particularly acute in China. Based on the reported results, future attention needs to be directed towards improving epithelialization, transparency, and avoidance of neovascularization of the graft. Detailed postoperative analysis (or ex vivo analysis of explanted, failed grafts), however, is currently lacking. It is unclear whether the porcine corneal stroma can stably permit stromal regeneration. Ideally for
Figure 3.Concept of donor tissue lenticule-based stromal regeneration. Lenticules obtained after small-incision lenticule extraction (SMILE) refractive procedures can be made acellular (and thereby less immunogenic) using procedures such as cryopreservation and chemical decellularization. Cryopreservation additionally enables the long-term storage of lenticules for future use. The acellular lenticules can subsequently be inserted into corneas as an additive therapy to correct for hyperopia (as an alternative to laser ablation) or to flatten and/or thicken the cornea in keratoconus. The long-term stromal regeneration ability of the lenticules, however, as well as their long-term fate after implantation is unknown. In the future, multiple lenticules (typically less than 50 µm thick) could be inserted for example as a stack, into a thin cornea or to replace a portion of scarred or diseased stroma. The regenerative potential of this therapy, and its ability to restore and maintain transparency, however, are unknown.
regeneration, host keratocytes should migrate into the graft and produce new human collagen as the porcine collagen is gradually degraded, while corneal thickness and transparency are maintained. Conversely, a non-regenerative reaction would consist of inflammatory cell and fibroblast invasion, stromal edema, neovasculariztion and collagen degradation, resulting in corneal thinning and topographic irregularities. A further consideration of importance is the postoperative regeneration of corneal nerves. Does the porcine stroma per-mit nerve regeneration and to what degree? Could outcomes be improved with the infiltration of host nerves into the porcine stroma?
Full-thickness corneas based on porcine corneal stroma
Since the acellular porcine corneal stroma can only be used therapeutically when the corneal epithelium and endothelium are functional, attention is being given to repopulating the stromal scaffold with human cells, to enable full-thickness pene-trating keratoplasty. A Chinese team from Shandong has demonstrated the ability to seed human embryonic stem cell (hESC)-derived limbal epithelial-like cells on one side of the porcine scaffold and hESC-derived corneal endothelial-like cells on the other.42After transplantation into rabbits, corneal transparency was gradually improved through 8 weeks of follow-up, although residual haze and neovascularization were present. Nevertheless, along with vessels and inflammatory cells, stromal cells were observed to repopulate the implanted porcine stroma at only 8 weeks post-implantation. Whether stromal regenera-tion would eventually occur is unclear, but the given the ethical debate surrounding the use of hESC, alternatives are sought. One alternative to the use of hESC, involves a similar porcine stromal construct that was developed using human donor cornea-derived epithelial, stromal and endothelial cells that were seeded into the construct and prepared by cell culture methods.43One week after implantation by penetrating keratoplasty in rabbits, the construct remained almost completely transparent, before an eventual immune response led to rejection and opacification of the xenogenic graft at four weeks.
The availability of appropriate animal models to test stromal
constructs containing human cells remains an issue.
Additionally, initial results suggest an immune reaction of the host to possible cellular and/or DNA remnants within the por-cine cornea that may compromise transparency and initiate a neovascular response. With effective strategies to remove all donor xenogenic material and sterilize the porcine corneas,37 along with specific postoperative regimens to suppress possible immune reaction, porcine stromal tissue could play a greater role in the future in supplementing the human donor tissue pool for applications in therapeutic keratoplasty. It remains to be deter-mined, however, how the porcine corneal tissue (with or without cells) will be regulated. In particular, within the regulatory fra-mework in the EU, the acellular stroma would be classified as a medical device while the addition of cells would render it an advanced therapy medicinal product (ATMP). Stringent GMP manufacturing, quality and safety controls and standards would have to be met,1encompassing the entire supply, production and distribution chains.
Fish scale-derived cornea
Another innovative biological source of collagen-based stro-mal scaffolds are the scales of the Tilapia fish. Norstro-mally considered a waste product of fish processing, the scales of the fish can be decellularized and decalcified to produce a -scaffold44 compatible with the culture of corneal cells.45 Manufactured to an appropriate thickness, a transparent stro-mal scaffold derived from the fish scale was evaluated after intrastromal implantation in rabbit corneas, where it was
found that the scaffold could be retained in vivo for
one year, maintaining transparency without eliciting an
immune response.46 As the fish scales are mechanically
robust, they additionally have the potential to be sutured onto the cornea in cases of perforating trauma. This applica-tion was tested using a model of full-thickness perforaapplica-tions in minipig corneas,47 where the fish scale-derived scaffold was successfully used as an emergency patch graft (termed the ‘Biocornea’), to temporarily seal the cornea and maintain the integrity of the anterior chamber and eye globe for up to four days (within which time a suitable donor cornea can be sourced in an emergency trauma situation). Following posi-tive results in the minipig model, the fish scale scaffold is currently undergoing a human clinical trial as a temporary
patch graft for traumatic corneal perforation (J.J.
Schuitmaker, personal communication).
Bioengineered acellular corneal stroma
Another approach to stromal regeneration not involving the use of harvested tissue from humans or animals is the engi-neering of corneal stromal equivalents from raw starting materials. These‘bioengineered’ stromal equivalents have the advantage that their properties can be tuned for a specific application or physiologic effect, and that they do not rely on tissue harvesting or availability of corneal tissue from animals or from humans. Furthermore, because the stroma is engi-neered from basic building block components, cells can be expressly excluded from manufacturing, to create a truly acel-lular stroma not requiring decelacel-lularization. This would greatly improve the biocompatibility of the stromal equivalent by avoiding the host immune response. As the implanted stroma is acellular, however, stromal regeneration would require host cell infiltration and new collagen production.
One such stromal scaffold is the acellular recombinant human collagen-based scaffold, termed the‘biosynthetic’ cor-neal stroma. The biosynthetic scaffold is composed of human collagen (the main protein constituent of the human cornea) produced recombinantly in yeast, and is strengthened by chemically crosslinking the collagen using the non-toxic and
water-soluble EDC/NHS system.48We implanted this
biosyn-thetic scaffold by anterior lamellar keratoplasty into ten Swedish patients to treat advanced keratoconus and corneal scarring.49,50After initial limited melting and haze formation due to the softness of the scaffold (i.e.,‘cheese-wiring’ by the retaining sutures), the scaffolds remained stable and func-tional for at least four years postoperatively,51without detect-able inflammation or rejection. The bulk of the stroma was transparent as indicated by clinical examination, however,
some increased lamellar interface reflectivity was detected by optical coherence tomography (Figure 4a,b). At the cellular level, in vivo confocal microscopy revealed that after three years, host stromal cells were present at the lamellar interface regions; however, a clear migration of host stromal cells into the biosynthetic scaffold could not be detected (Figure 4c,d). Nerve regeneration, however, had occurred by three years. Regenerated subbasal nerves, visualized by in vivo confocal microscopy, were present in the anterior portion of the scaf-fold (Figure 4e,f). In a patient re-transplanted after four years due to suboptimal vision, histologic analysis of the excised tissue revealed regeneration of a stratified epithelium to cover the scaffold and increased density of stromal tissue at the lamellar interfaces, consistent with the presence of denser, scar-type tissue (Figure 4g). Furthermore, while stromal cells were present at the interface region, the cells did not enter the implanted scaffold.
To overcome the limitations of the relatively soft biosyn-thetic scaffold and improve resistance of the scaffold to
enzy-matic degradation and the robustness for surgical
implantation, we subsequently used medical-grade purified porcine collagen (extracted from porcine skin) with the EDC/NHS crosslinking system to develop mechanically stron-ger stromal scaffolds that were additionally more resistant to enzymatic degradation. These bioengineered porcine collagen constructs (BPC) were tested in rabbit models by intrastromal implantation.52,53The BPC scaffolds were biocompatible and maintained corneal transparency up to the three month experimental endpoint without a host immune reaction. Implantation of the scaffold was followed by stromal (myo) fibroblasts migrating to the lamellar interface and producing small amounts of type III collagen in a thin region surround-ing the scaffold (Figure 5a). These fibroblasts then entered the implant at the edges, where they continued to produce type
III collagen while simultaneously degrading the scaffold (Figure 5b). The new collagen produced by these cells differed from the scaffold collagen but resembled the host stroma, based on the staining pattern. This indicates a partial regen-eration of host stroma by host stromal cells, facilitated by scaffold implantation. Interestingly, the acellular collagen scaffolds retained stromal proteoglycans from the original porcine dermal source as observed by transmission electron microscopy (Figure 5c,d); however, the in vivo functionality or integrity of these proteoglycans were not examined. This bottom-up approach of scaffold fabrication from purified collagen as a raw material enables stromal scaffolds to be manufactured with a desired diameter, thickness, or in a hybrid fashion where different parts can be tuned to have differing transparency and degradation properties.53A newer version of the BPC scaffolds have subsequently been tested in a minipig model54and have been implanted within the stroma in patients with advanced keratoconus, with positive initial results (M. Rafat, personal communication). Besides addres-sing the global shortage of donor stromal tissue for transplan-tation, the bioengineered constructs can be manufactured and stored for up to two years prior to use, based on validated shelf life studies (M. Rafat, personal communication).
Another interesting option reported for the treatment of corneal ulcers is the use of ‘plastic compressed collagen’ as a therapeutic option where amniotic membrane grafts fail to epithelialize the ulcerated region. Schrader and colleagues report a commercial-grade CE-marked type I porcine dermal atelocollagen used to create a mechanically compressed col-lagen matrix that is more dense and robust compared to typical hydrogel-based scaffolds, while retaining high tensile strength and elasticity.55 The compressed collagen scaffold, however, was not crosslinked and is therefore prone to degra-dation. In a first clinical case, the compressed collagen matrix
Figure 4.Postoperative assessment of stromal regeneration following biosynthetic corneal implantation into keratoconus patients by anterior lamellar keratoplasty, three years postoperatively (A– F) and four years postoperatively in one patient (G). (a) The anterior lamellar scaffold remained transparent, with border region visible in the slit lamp (border indicated by arrows). (b) Optical coherence tomography reveals the intact scaffold with increased stromal density and reflectivity at implant-to-host interfaces (arrow). (c, d) By in vivo confocal microscopy, stromal cells from the host have not migrated to occupy the implanted scaffold (dark, transparent region). (e, f) Subbasal nerves anterior to the scaffold have partially regenerated (arrows). (g) Hematoxylin and eosin stained section from an excised corneal button indicates an intact scaffold (white asterisk) that remains acellular. Host stromal cells (black arrows) are present at the scaffold-to-host lamellar interface, within a region of denser, more compacted collagen (black asterisk) indicated by stronger eosinophilic staining. Stromal fibroblasts also migrated anteriorly to the implant under the epithelium and deposited a layer of denser collagen, consistent with the light-scattering scar tissue (white arrows) also observed anteriorly in the subbasal plexus (F). Images (C– F) are 400 x 400 µm.
was used to fill the ulcerated defect region (as a corneal inlay) while a second identical matrix was used to cover the cornea
as a bandage (onlay).56 While the bandage onlay degraded
within the first postoperative week, it facilitated coverage of the entire ulcerated region by recipient corneal epithelium, with the inlay portion remaining intact. During the following six month period, the inlay also degraded but with epithelium remaining intact without signs of inflammation or immune rejection. Stromal regeneration was not demonstrated, and it is not clear whether the inlay had an impact upon the host stroma. This type of stromal replacement however, although temporary, may provide a means to heal otherwise untreatable ulcers, enabling future vision rehabilitation (by for example, keratoplasty) in these cases. It is interesting to note that even a temporary stromal scaffold can promote epithelial regenera-tion in this context; stabilizaregenera-tion of the plastic compressed scaffold by crosslinking the collagen could possibly provide a means to tailor the degradability in cases where a longer-lived stromal scaffold is desirable.
Cell-populated engineered scaffold
In cases where host cells would be unsuitable or cannot repopulate the scaffold, an alternative approach is to provide appropriate cells along with the scaffold. This approach is illustrated by the bioengineered human allogeneic nanostruc-tured anterior cornea developed by a Spanish team. The con-struct consists of a fibrin-agarose scaffold made from a combination of human blood plasma and 0.1% agarose
formed into a scaffold with nanoscale features.57 Donor
human corneal fibroblasts are implanted into the scaffold and donor corneal limbal epithelial cells are seeded on top, to create an anterior lamellar graft under full GMP conditions in vitro, as an authorized investigational advanced therapy medicinal product. At the time of writing, a Phase I/IIA randomized multicenter clinical trial58,59 is being conducted in Spanish hospitals, to treat refractory neurotrophic ulcers in 20 patients by anterior lamellar keratoplasty. In the first five patients receiving the fibrin-agarose scaffold, complete healing
Figure 5.Evidence for stromal regeneration following implantation of a bioengineered porcine construct (BPC) scaffold into rabbit corneas for 8 weeks. (a) Immunohistochemistry of the cornea with staining for scar-type collagen III indicates limited new collagen production by host stromal fibroblasts at the lamellar interface (arrows). Note that the fibroblasts migrate along the lamellar surface until the edge of the scaffold is reached, before entering the scaffold at the edge (asterisk). (b) At the scaffold edge, fibroblasts enter the scaffold (arrows), breaking down the scaffold collagen (hematoxylin stained, gray color) and producing new host-derived collagen, as indicated by the eosinophilic pink color. (c) Transmission electron microscopy image of host rabbit stromal collagen stained for proteoglycans, visible as dark electron-dense spots (arrows). Note the distribution of proteoglycans in parallel lines, following the parallel orientation of collagen fibrils in the native rabbit stroma. (bd) In the scaffold, proteoglycans are also present but appear randomly distributed (arrows).
of the ulcer was observed without signs of infection or allo-geneic rejection at a two-year follow-up, and the patients
reported relief of ocular symptoms.60 This promising
approach awaits final evaluation. It will be important to determine the fate of the scaffold and implanted cells, includ-ing signs of tissue regeneration, in terms of re-establishment of stromal architecture, structure and thickness, and stromal cell (and possibly nerve) repopulation of the lamellar graft.
Tissue adhesive gel
For the repair of corneal defects due to traumatic injury or infection that result in stromal thinning, an alternative to invasive corneal transplantation or use of pre-formed scaf-folds has been recently proposed by a US-based team. The device is a stromal mimic that is a viscous gel-like substance when applied to the cornea, which adapts to the form of the defectin situ. Once the defect is filled, the gel is ‘locked’ in place and stabilized into a more rigid stromal mimic through photopolymerization. This technology, called GelCORE (gel for corneal regeneration) has been developed as an adhesive biomaterial to treat corneal stromal loss.61The biomaterial is natural and acellular in that it is derived from porcine gelatin that is stabilized by addition of methacrylic anhydride.62The result is a tough but elastic and transparent biomaterial that can be photopolymerized using visible blue light, thereby avoiding exposure of eye structures to the potentially dama-ging UV light normally used to photocure polymers. The GelCORE was shown to be compatible with human corneal fibroblast migration in vitro, without inducing cell death after visible light polymerization. In a rabbit model of keratectomy with 50% of the stromal depth removed, the GelCORE was applied with four-minute photopolymerization time, and resulted in a transparent and smooth cornea that re-epithelialized within a week. Histologic analysis at 14 days revealed some inflammatory cell infiltration in the implanted zone, but the bulk of the GelCORE stromal substitute was no longer detectable, indicating either a rapid transformation or degradation following epithelial coverage. Initial results are
encouraging, and more detailed longer-term in vivo
evalua-tion would yield further insights into the fate of the biomater-ial and its potentbiomater-ial to stimulate stromal regeneration.
3D bioprinting
Unlike bioengineered corneas made from raw materials that are dispensed or moulded into stromal tissue equivalents, three-dimensional bioprinting offers additional flexibility for controlling the spatial organization of the fabricated stroma. A bioprinter additionally offers the possibility to combine
different biomaterials, cells and other factors into
a customized matrix with high spatial resolution. As an initial step in this direction, Connon and colleagues reported a 3D bioprinted corneal stromal equivalent fabricated using metha-crylated type I collagen and sodium alginate as bioinks, fol-lowed by crosslinking using calcium chloride.63Incorporation of human corneal keratocytes within a collagen-alginate bioink resulted in a 3D bioprinted stromal equivalent that retained live keratocytes for 7 days.
An alternative cell-based approach reported by Skottman and colleagues was the laser bioprinting of a 3D stromal equivalent using two bioinks, one containing type I human collagen and the other containing a mixture of human adipose
stem cells (hASC), collagen, human plasma and thrombin.64
To create the stromal equivalent, ten alternating layers of acellular collagen and hASC were printed to form a 7x7 mm and 500 µm thick stromal equivalent. After 14 days, thickness had reduced to 300 µm but cells were viable, proliferating, and had aligned themselves in a lamellar organization mimicking the native corneal stroma. Advantages of incorporating stem cells into the stroma are their repair and proliferative capacity and the potential for non-immunogenicity, when compared to allogeneic differentiated stromal cells such as keratocytes or fibroblasts.
While the above bioprinting approaches are a promising means to achieve stromal regeneration, they awaitin vivo eva-luation in suitable animal models, cell fate determination post-implantation, and demonstration of stromal regenerative ability such as scar-free healing and new collagen production. Nevertheless, these and similar 3D printed scaffolds are attrac-tive as they have a highly controllable production process amen-able to mass production, or alternatively could enamen-able customized patient-specific structures to be printed, guided by the condition and topography of the patient’s cornea.63
Stromal regeneration by stem cell therapy
As an alternative to incorporating cells within a stromal construct, ‘stromal cell therapy’ aims to harness the direct and/or paracrine functions of stem cell populations to repair and regenerate the existing corneal stroma. Cell therapy can be initiated for example, by direct delivery of therapeutic cells into the diseased or damaged stroma in situ. This concept has been demonstrated for treatment of corneal scars that would normally require corneal trans-plantation. Instead of transplantation, Basu and colleagues isolated human corneal stromal stem cells (hCSSC) from the limbal stroma of an allogenic limbal biopsy and demon-strated that these cells possess mesenchymal stem cell-like properties.65 Following isolation of these stem cells in cell culture, they were then introduced into the anterior stroma of the mouse cornea after stromal debridement, and kept in place using standard fibrin glue. The stem cells were still detectable four weeks post-implantation, at which time the stroma had healed without scarring. At the time of writing, this therapy is undergoing two human clinical trials in India, for the treatment of stromal injury, scars and opa-cities due to pathology or surgery.66,67
While this approach is a promising means to support regenerative stromal healing, the cells are sourced from the limbal stromal region of donor corneas and are there-fore still dependent on an adequate supply of donated tissue. The donated tissue, however, may not necessarily need to be from the same species, as illustrated in the xenogenic human-to-mouse cell delivery above, and in
other studies of cell-based stromal repair68 due to their
apparent ability to avoid a host immune response. Non-human derived CSSC have yet to be tested definitively for
stromal regeneration and immunomodulatory potential in vivo, but such cells could provide a source of CSSC not dependent on human donors. hCSSC, however, have the ability to synthesize human stromal collagen and deposit this collagen in an organized manner to mimic the transparent arrangement of collagen fibrils in the
native cornea.69 The approach of local hCSSC stromal
delivery may therefore not only provide a means to aid bio-integration and scar-free healing of implanted bioma-terials in the stroma, but hCSSC may also improve out-comes in xenogenic transplants (such as the acellular porcine corneal stroma described earlier) through their immunomodulatory properties. Another point worth men-tioning is the lack of apparent effect of native hCSSC in cases of corneal scarring where the limbal niche is func-tional and intact. If CSSC exist in the stroma in close proximity to limbal epithelial stem cells (LESC) in the Palisades of Vogt region, why do they not contribute to stromal wound healing, in an analogous manner as LESCs contribute to epithelial wound healing? Suboptimal wound healing post-transplantation may lead to stromal scarring, for instance the host-to-donor interface scarring com-monly seen after penetrating and lamellar keratoplasty. It appears that hCSSC are not able to effect optimal stromal wound healing while in their native niche to inhibit such interface scarring, whereas their isolation and local deliv-ery to the wounded stroma has an observable effect. The current belief that CSSC provide an important support
function in maintaining the LESC phenotype70 may
there-fore mean that the primary function of the in situ CSSC is to maintain the function of the limbal niche and not to repair damaged corneal stroma. Nevertheless, it would be of great interest if CSSC could be stimulated to effect stromal repair in situ, without requiring their extraction from a donor, purification and re-introduction into host stroma.
A promising source of cells for stromal therapy avoiding reliance on donor corneas is being developed by Alió and colleagues in Spain, who used autologous adipose-derived adult stem cells obtained from an elective liposuction proce-dure. These stem cells were introduced mid-stromally in advanced keratoconus patients with a thin corneal stroma.71 The cells were introduced via a femtosecond laser-created pocket, either alone or in combination with a 120 µm thick decellularized human donor stromal implant that had been re-cellularized with the autologous stem cells prior to implanta-tion. 12 months postoperatively, all 9 corneas receiving the autologous adipose-derived stem cells were transparent. Five corneas receiving only the stem cells had increased in thickness by 14.5 µm while those receiving the re-cellularized stromal implant with stem cells had maintained the stromal implant thickness but with no further increase in thickness. While this study demonstrates the concept of autologous stromal cell therapy, which has the added benefit of immune compatibility, the results suggest that cell therapy alone is insufficient to replace the bulk of the corneal stroma within a reasonable time frame; instead, the cells could augment healing in cases where for example a non-donor stroma is used, such as a bioprinted or bioengineered stromal equivalent.
Conclusions and perspectives
Many of the therapies discussed herein are not mutually exclu-sive; several could be combined for optimal therapeutic effect, for example acellular stromal scaffolds and therapeutic cells. Moreover, specific therapies can be personalized, for example by 3D bioprinting a scaffold designed to fit into a specific reci-pient bed, bioengineering a stromal equivalent to desired dimen-sions and with a tailored time to degradation, or using autologous cells to avoid immune reaction while promoting regenerative healing through paracrine processes. The future also holds further hope for newer advanced techniques not described here and which are still in a nascent stage, such as the use of stem-cell derived exosomes (extracellular vescicles) for promoting scar-free stromal healing72and the use of iPS-derived cells or organoids73,74to promote autologous, regenerative heal-ing. The next few years will likely see the first stromal equivalents and cell-based technologies being approved for clinical use, initially for conditions non-responsive to conventional treat-ments. As ophthalmic specialists and surgeons become more familiar with these new therapies and as they become more readily available and cost-effective, such niche applications over time are likely to be broadened to include additional indi-cations affecting larger patient populations.
Although many of the proposed stromal regeneration tech-niques claim to be regenerative, very few demonstrate regen-eration or turnover of stromal collagen, restoration of a lamellar stromal organization or regeneration of corneal nerves critical for trophic support of the stroma. In addition to anti-scarring treatments and repair of the extracellular matrix, neuro-regenerative factors should also be provided to the regenerating stroma, either in the form of specific axonal growth factors or as stem cell-derived paracrine factors providing a milieu favoring nerve growth. An overview of the various approaches for stromal regeneration and the evidence for stromal regeneration in vivo is given inTable 1. It is clear that much work is still necessary to demonstrate stromal regeneration in vivo in animal models and in humans. Beyond initial integration of implanted stromal scaffolds into host corneas without rejection or extensive tissue degra-dation, stromal melting or scar tissue formation, the regen-eration of stromal cells, matrix proteins, nerves and proteoglycans has been difficult to demonstrate. Part of the difficulty in demonstrating stromal regeneration may be the time frame required, as normal stromal collagen turnover and nerve regeneration are typically slow processes that can take many years. In vivo detection of stromal regeneration in humans can also be challenging, but may be facilitated by careful cellular and extracellular matrix morphologic observa-tions using high-resolution imaging techniques such as
in vivo confocal microscopy and optical coherence
tomography.
Finally, stromal regeneration may represent the final piece in the puzzle in the quest towards a fully engineered corneal substitute. The combination of engineered epithelial and stro-mal parts into an anterior corneal equivalent, or endothelial and stromal parts into a posterior corneal equivalent could find application in treating a wide range of corneal pathology nor-mally requiring donor tissue transplantation. An engineered full
corneal equivalent including epithelium, stroma, and endothe-lium would be beneficial in treating a further subset of cases where only penetrating keratoplasty can restore corneal trans-parency. Although ambitious, these goals are within reach given the current pace in the development of corneal regenerative technologies and the parallel developments in clinical evaluation and regulatory approvals of advanced therapeutic modalities.
Acknowledgments
The author would like to acknowledge the kind assistance of Anthony Mukwaya in the preparation ofFigures 1and3. ForFigure 4, the prior scientific contributions of Prof. Per Fagerholm and co-authors (references
50–52) are acknowledged. ForFigure 5, the prior scientific contributions of Mehrdad Rafat and co-authors (references 53–55) are acknowledged.
Funding
This work was supported by the European Commission funded Horizon2020 framework project ARREST BLINDNESS, Grant No. [667400] (www.arrestblindness.eu).
Declaration of interest
The author reports no conflicts of interest. The author alone is respon-sible for the content and writing of the paper.
Table 1.Summary of various approaches for achieving stromal regeneration and evidence for regeneration in animal models and in human studies. Stromal Regenerative
Approach Key advantages
Stromal regeneration in animal
models Stromal regeneration in humans Decellularized scaffolds Availability, native stromal
architecture, strength Human donor stromal
lenticules (SMILE)
Availability, human stromal tissue Integration with rabbit31and
macaque32stroma Integration with host stroma 28–30
Acellular porcine corneal stroma
Abundant, low cost, thick stromal tissue
Integration with rabbit stroma, host cells in
scaffold35,38
No evidence provided39,40
Recellularized scaffolds Cells can actively effect stromal repair
Human embryonic stem cells
Pluripotent, high proliferative potential
No evidence provided in rabbit model42
Adipose-derived MSC Abundant, autologous, non-immunogenic
Integration with rabbit stroma, delivered cells
remain in scaffold75
Integration with host stroma, cell survival71
Human donor corneal cells
Corneal cell type, isolation from donor corneas
Scaffold not detected in rabbits35,
no evidence in
rabbits43, scar suppression in mice76
No evidence available57,58
Cell-free bioengineered scaffolds
Bottom-up design, low immunogenicity, no reliance on donor eyes Fish-scale collagen Abundant, low cost, natural
biomaterial
Poor integration in rats44, tolerated in rabbits with
macrophage infiltration46, no integration in mini-pigs47
Bioengineered collagen Natural biomaterial, biocompatibility Integration with rabbit stroma, host cells in scaffold,
scaffold breakdown and new collagen production52
Integration with host stroma,50,51nerve regeneration51
Tissue adhesives Strength, biocompatibility, ease of application
GelCORE Natural biomaterial, biocompatibility, photopolymerization
Rapid scaffold degradation in rabbits, rapid ECM
deposition hypothesized61
Cell-based without scaffold
Cell-mediated, no biomaterial needed, direct delivery Bone-marrow-derived
MSC
Abundant, autologous source, stromal lineage,
immunomodulatory
Cells survive and differentiate into keratocytes in
mouse stroma77
Adipose-derived MSC Autologous MSC source, abundant Cells survive in rabbit stroma, differentiate to
keratocytes, new collagens produced78
Cells survive, slight stromal thickness increase, possible new collagen production71
Umbilical cord-derived MSC
Availability, cryopreservation for later use,
young cells highly potent
Cells survive in mouse stroma, differentiate to
keratocytes, synthesize ECM components79,80
Induced pluripotent stem cells
Multipotent, high potency, autologous Recovery of stromal transparency following
wounding in mice, suppresses inflammation81
Human corneal stromal stem cells
Isolation from donor cornea rims, stromal origin/fate
New ECM components produced in healthy68and
injured65stroma in mice
No evidence available66,67
Abbreviations: SMILE: small-incision lenticule extraction; MSC: mesenchymal stem cells; GelCORE: gelatin-based adhesive for corneal regeneration; ECM: extracellular matrix
ORCID
Neil Lagali http://orcid.org/0000-0003-1079-4361
References
1. Pellegrini G, Ardigò D, Milazzo G, Iotti G, Guatelli P, Pelosi D, De Luca M. Navigating market authorization: the path holoclar took to become the first stem cell product approved in the European Union. Stem Cells Transl Med. 2018;7(1):146–54. doi:10.1002/sctm.17-0003.
2. Kinoshita S, Koizumi N, Ueno M, Okumura N, Imai K, Tanaka H, Yamamoto Y, Nakamura T, Inatomi T, Bush J, et al. Injection of cultured cells with a ROCK inhibitor for bullous keratopathy. N Engl J Med. 2018 Mar 15;378(11):995–1003. doi:10.1056/ NEJMoa1712770.
3. Whitcher JP, Srinivasan M, Upadhyay MP. Corneal blindness: a global perspective. Bull World Health Organ.2001;79:214–21. 4. Zerbe BL, Belin MW, Ciolino JB. Boston type 1 keratoprosthesis
study group. Results from the multicenter Boston type 1 kerato-prosthesis study. Ophthalmology. 2006 Oct 1;113(10):1779–84. doi:10.1016/j.ophtha.2006.05.015.
5. Liu C, Paul B, Tandon R, Lee E, Fong K, Mavrikakis I, Herold J, Thorp S, Brittain P, Francis I, et al. The osteo-odonto-keratoprosthesis (OOKP). Seminars in ophthalmology. 2005;20(2):113–28.
6. Tervo T, Vannas A, Tervo K, Holden BA. Histochemical evidence of limited reinnervation of human corneal grafts. Acta Ophthalmol (Copenh). 1985;63(2):207–14. doi: 10.1111/j.1755-3768.1985.tb01535.x.
7. Niederer RL, Perumal D, Sherwin T, McGhee CN. Corneal inner-vation and cellular changes after corneal transplantation: an in vivo confocal microscopy study. Invest Ophthalmol Vis Sci.
2007;48(2):621–26. doi:10.1167/iovs.06-0538.
8. Al-Aqaba MA, Otri AM, Fares U, Miri A, Dua HS. Organization of the regenerated nerves in human corneal grafts. Am J Ophthalmol.2012;153(1):29–37. doi:10.1016/j.ajo.2011.06.006. 9. Nubile M, Carpineto P, Lanzini M, Calienno R, Agnifili L,
Ciancaglini M, Mastropasqua L. Femtosecond laser arcuate kera-totomy for the correction of high astigmatism after keratoplasty. Ophthalmology. 2009;116(6):1083–92. doi:10.1016/j.ophtha.2009. 01.013.
10. Khodadoust AA, Silverstein AM. Transplantation and rejection of individual cell layers of the cornea. Invest Ophthalmol Vis Sci.
1969;8:180–95.
11. Oliva MS, Schottman T, Gulati M. Turning the tide of corneal blindness. Indian J Ophthalmol. 2012;60(5):423. doi:10.4103/ 0301-4738.100540.
12. Gain P, Jullienne R, He Z, Aldossary M, Acquart S, Cognasse F, Thuret G. Global survey of corneal transplantation and eye banking. JAMA Ophthalmol. 2016;134(2):167–73. doi:10.1001/ jamaophthalmol.2015.4776.
13. Komai Y, Ushiki T. The three-dimensional organization of col-lagen fibrils in the human cornea and sclera. Invest Ophthalmol Vis Sci.1991;32:2244–58.
14. Lagali N, Germundsson J, Fagerholm P. The role of Bowman’s layer in corneal regeneration after phototherapeutic keratectomy: a prospective study using in vivo confocal microscopy. Invest Ophthalmol Vis Sci.2009;50(9):4192–98. doi:10.1167/iovs.09-3781. 15. Bron AJ. The architecture of the corneal stroma. Br J Ophthalmol.
2001;85:379–81. doi:10.1136/bjo.85.4.379.
16. West-Mays JA, Dwivedi DJ. The keratocyte: corneal stromal cell with variable repair phenotypes. Int J Biochem Cell Biol.2006;38 (10):1625–31. doi:10.1016/j.biocel.2006.03.010.
17. Paik DC, Trokel SL, Suh LH. Just what do we know about corneal collagen turnover? Cornea. 2018;37(11):e49–50. doi:10.1097/ ICO.0000000000001685.
18. Wilson SL, El Haj AJ, Yang Y. Control of scar tissue formation in the cornea: strategies in clinical and corneal tissue engineering. J Funct Biomater.2012;3(3):642–87. doi:10.3390/jfb3030642. 19. Michelacci YM. Collagens and proteoglycans of the corneal
extra-cellular matrix. Braz J Med Biol Res. 2003;36(8):1037–46. doi:10.1590/s0100-879x2003000800009.
20. Marfurt CF, Cox J, Deek S, Dvorscak L. Anatomy of the human corneal innervation. Exp Eye Res.2010;90(4):478–92. doi:10.1016/ j.exer.2009.12.010.
21. Belmonte C, Acosta MC, Gallar J. Neural basis of sensation in intact and injured corneas. Exp Eye Res. 2004;78(3):513–25. doi:10.1016/j.exer.2003.09.023.
22. Müller LJ, Marfurt CF, Kruse F, Tervo TM. Corneal nerves: structure, contents and function. Exp Eye Res. 2003;76 (5):521–42. doi:10.1016/s0014-4835(03)00050-2.
23. Patel DV, McGhee CN. In vivo laser scanning confocal micro-scopy confirms that the human corneal sub-basal nerve plexus is a highly dynamic structure. Invest Ophthalmol Vis Sci. 2008;49 (8):3409–12. doi:10.1167/iovs.08-1951.
24. Lagali N, Peebo BB, Germundsson J, Edén U, Danyali R, Rinaldo M, Fagerholm P. Laser-scanning in vivo confocal micro-scopy of the cornea: imaging and analysis methods for preclinical and clinical applications. In: Lagali N, editor. Confocal laser microscopy principles and applications in medicine, biology, and the food sciences. Rijeka (Croatia): InTech;2013. p. 51–80. 25. Ivarsen A, Asp S, Hjortdal J. Safety and complications of more than
1500 small-incision lenticule extraction procedures. Ophthalmology.
2014;121(4):822–28. doi:10.1016/j.ophtha.2013.11.006.
26. Larkin H. Hyperopia innovations: adding donor stroma is new frontier for hyperopia, presbyopia, keratoconus treatment. Posted: Monday; 2019 Apr 1. Eurotimes. https://www.eurotimes.org/ hyperopia-innovations/.
27. Ganesh S, Brar S, Rao PA. Cryopreservation of extracted corneal lenticules after small incision lenticule extraction for potential use in human subjects. Cornea. 2014;33(12):1355–62. doi:10.1097/ ICO.0000000000000276.
28. Bhandari V, Ganesh S, Brar S, Pandey R. Application of the SMILE-derived glued lenticule patch graft in microperforations and partial-thickness corneal defects. Cornea.2016;35(3):408–12. doi:10.1097/ICO.0000000000000741.
29. Wu F, Jin X, Xu Y, Yang Y. Treatment of corneal perforation with lenticules from small incision lenticule extraction surgery: a preliminary study of 6 patients. Cornea. 2015;34(6):658–63. doi:10.1097/ICO.0000000000000397.
30. Ganesh S, Brar S. Femtosecond intrastromal lenticular implanta-tion combined with accelerated collagen cross-linking for the treatment of keratoconus–initial clinical result in 6 eyes. Cornea.
2015;34(10):1331–39. doi:10.1097/ICO.0000000000000539. 31. Yam GH, Yusoff NZ, Goh TW, Setiawan M, Lee XW, Liu YC,
Mehta JS. Decellularization of human stromal refractive lenticules for corneal tissue engineering. Sci Rep. 2016 May 23;6:26339. doi:10.1038/srep26339.
32. Liu YC, Teo EPW, Ang HP, Seah XY, Lwin NC, Yam GHF, Mehta JS. Biological corneal inlay for presbyopia derived from small incision lenticule extraction (SMILE). Sci Rep. 2018;8 (1):1831. doi:10.1038/s41598-018-20267-7.
33. Lee W, Miyagawa Y, Long C, Cooper DK, Hara H. A comparison of three methods of decellularization of pig corneas to reduce immunogenicity. Int J Ophthalmol.2014;7:587–93.
34. Yoeruek E, Bayyoud T, Maurus C, Hofmann J, Spitzer MS, Bartz-Schmidt K-U, Szurman P. Decellularization of porcine corneas and repopulation with human corneal cells for tissue-engineered xenografts. Acta Ophthalmol. 2012;90:e125–e131. doi:10.1111/ j.1755-3768.2011.02261.x.
35. Ma XY, Zhang Y, Zhu D, Lu Y, Zhou G, Liu W, Cao Y, Zhang WJ. Corneal stroma regeneration with acellular corneal stroma sheets and keratocytes in a rabbit model. PLoS One.
36. Gonzalez-Andrades M, de la Cruz Cardona J, Ionescu AM, Campos A, Del Mar Perez M, Alaminos M. Generation of bioen-gineered corneas with decellularized xenografts and human keratocytes. Invest Ophthalmol Vis Sci. 2011;52:215–22. doi:10.1167/iovs.09-4773.
37. Mirazul Islam M, Sharifi R, Mamodaly S, Islam R, Nahra D, Abusamra DB, Chuen Hui P, Adibnia Y, Goulamaly M, Paschalis EI, et al. Effects of gamma radiation sterilization on the structural and biological properties of decellularized corneal xenografts. Acta Biomater. 2019;96:330–344. doi: 10.1016/j. actbio.2019.07.002.
38. Du L, Wu X. Development and characterization of a full-thickness acellular porcine cornea matrix for tissue engineering. Artif Organs.2011;35:691–705. doi:10.1111/j.1525-1594.2010.01174.x. 39. Zhang MC, Liu X, Jin Y, Jiang DL, Wei XS, Xie HT. Lamellar
keratoplasty treatment of fungal corneal ulcers with acellular porcine corneal stroma. Am J Transplant. 2015;15(4):1068–75. doi:10.1111/ajt.13096.
40. Zheng J, Huang X, Zhang Y, Wang Y, Qin Q, Lin L, Jin X, Lam C, Zhang J. Short-term results of acellular porcine corneal stroma keratoplasty for herpes simplex keratitis. Xenotransplantation.
2019;26(4):e12509. doi:10.1111/xen.12509.
41. ClinicalTrials.gov identifier: NCT03105466. https://clinicaltrials. gov/ct2/show/NCT03105466
42. Zhang C, Du L, Sun P, Shen L, Zhu J, Pang K, Wu X. Construction of tissue-engineered full-thickness cornea substitute using limbal epithelial cell-like and corneal endothelial cell-like cells derived from human embryonic stem cells. Biomaterials.
2017;124:180–94. doi:10.1016/j.biomaterials.2017.02.003. 43. Zhang K, Ren XX, Li P, Pang KP, Wang H. Construction of a
full-thickness human corneal substitute from anterior acellular porcine corneal matrix and human corneal cells. Int J Ophthalmol.2019;12(3):351–62. doi:10.18240/ijo.2019.03.01. 44. van Essen TH, Lin CC, Hussain AK, Maas S, Lai JL, Linnartz H, van Den Berg TJTP, Salvatori DCF, Luyten GPM, Jager MJ. A fish scale-derived collagen matrix as artificial cornea in rats: properties and potential. Invest Ophthalmol Vis Sci. 2013;54:3224–33. doi:10.1167/iovs.13-11799.
45. Lin CC, Ritch R, Lin SM, Ni MH, Chang YC, Lu YL, Lai HJ, Lin FH. A new fish scale-derived scaffold for corneal regeneration. Eur Cell Mater.2010;19:50–57.
46. Van Essen TH, Van Zijl L, Possemiers T, Mulder AA, Zwart SJ, Chou CH, Lin CC, Lai HJ, Luyten GP, Tassignon MJ, et al. Biocompatibility of a fish scale-derived artificial cornea: cytotoxi-city, cellular adhesion and phenotype, and in vivo immunogenicity. Biomaterials. 2016;81:36–45. doi:10.1016/j. biomaterials.2015.11.015.
47. Chen SC, Telinius N, Lin HT, Huang MC, Lin CC, Chou CH, Hjortdal J. Use of fish scale-derived BioCornea to seal full-thickness corneal perforations in pig models. PLoS One.
2015;10(11):e0143511. doi:10.1371/journal.pone.0143511. 48. Liu Y, Gan L, Carlsson DJ, Fagerholm P, Lagali N, Watsky MA,
Munger R, Hodge WG, Priest D, Griffith M. A simple, cross-linked collagen tissue substitute for corneal implantation. Invest Ophthalmol Vis Sci. 2006;47(5):1869–75. doi:10.1167/ iovs.05-1339.
49. Fagerholm P, Lagali NS, Carlsson DJ, Merrett K, Griffith M. Corneal regeneration following implantation of a biomimetic tis-sue-engineered substitute. Clin Transl Sci. 2009;2(2):162–64. doi:10.1111/j.1752-8062.2008.00083.x.
50. Fagerholm P, Lagali NS, Merrett K, Jackson WB, Munger R, Liu Y, Polarek JW, Söderqvist M, Griffith M. A biosynthetic alternative to human donor tissue for inducing corneal regenera-tion: 24-month follow-up of a phase 1 clinical study. Sci Transl Med.2010;2(46):46ra61. doi:10.1126/scitranslmed.3001022. 51. Fagerholm P, Lagali NS, Ong JA, Merrett K, Jackson WB,
Polarek JW, Suuronen EJ, Liu Y, Brunette I, Griffith M. Stable corneal regeneration four years after implantation of a cell-free recombinant human collagen scaffold. Biomaterials. 2014;35 (8):2420–27. doi:10.1016/j.biomaterials.2013.11.079.
52. Koulikovska M, Rafat M, Petrovski G, Veréb Z, Akhtar S, Fagerholm P, Lagali N. Enhanced regeneration of corneal tissue via a bioengineered collagen construct implanted by a nondisruptive surgical technique. Tissue Eng Part A.2015;21(5–6):1116–30. doi:10.1089/ten.TEA.2014. 0562.
53. Rafat M, Xeroudaki M, Koulikovska M, Sherrell P, Groth F, Fagerholm P, Lagali N. Composite core-and-skirt collagen hydrogels with differential degradation for corneal therapeutic applications. Biomaterials. 2016;83:142–55. doi:10.1016/j.biomaterials.2016.01. 004.
54. Lagali NS, Xeroudaki M, Thangavelu M, Fagerholm P, Mukwaya A, Lennikov A, Rafat M. Double-crosslinked bioengineered collagen implants for corneal stromal transplantation: evaluation in a porcine model. ARVO annual meeting abstract 2018. Invest Ophthalmol Vis Sci.2018;59(9):2251. doi:10.1167/iovs.17-23678.
55. Drechsler CC, Kunze A, Kureshi A, Grobe G, Reichl S, Geerling G, Daniels JT, Schrader S. Development of a conjunctival tissue substitute on the basis of plastic compressed collagen. J Tissue Eng Regen Med.2017;11:896–904. doi:10.1002/ term.1991.
56. Schrader S, Witt J, Geerling G. Plastic compressed collagen trans-plantation–a new option for corneal surface reconstruction? Acta Ophthalmol (Copenh).2018Sep 1;96:6. doi:10.1111/aos.13801. 57. González-Andrades M, Mata R, González-Gallardo MDC,
Medialdea S, Arias-Santiago S, Martínez-Atienza J, Ruiz-García A, Pérez-Fajardo L, Lizana-Moreno A, Garzón I, et al. A study protocol for a multicentre randomised clinical trial evaluating the safety and feasibility of a bioengineered human allogeneic nanos-tructured anterior cornea in patients with advanced corneal trophic ulcers refractory to conventional treatment. BMJ Open.
2017;7(9):e016487. doi:10.1136/bmjopen-2017-016487.
58. Andrades MG, Martinez-Atienza J, Campos A, Arias-Santiago S, Gallardo CG, Mataix B, Medialdea S, Ruiz-Garcia A, Mata R, Cuende N, et al. Preliminary results of a multicenter randomized clinical trial evaluating the safety and feasibility of an allogeneic nanostructured artificial anterior human cornea. Cytotherapy.
2017;19(5):S26. doi:10.1016/j.jcyt.2017.02.052.
59. ClinicalTrials.gov identifier: NCT01765244. https://clinicaltrials. gov/ct2/show/NCT01765244
60. Gonzalez Andrades M, González Gallardo MC, Mataix B, Medialdea S, Martinez-Atienza J, Ruiz-Garcia A, Arias S, Campos A, Mata R, Cuende N, et al. Results of a Phase I-IIA multicentre clinical trial evaluating an allogeneic nanostructured artificial anterior human cornea. Oral session presented at: asso-ciation for Research in Vision and Ophthalmology Annual Meeting;2019; Vancouver (Canada); Abstract Number: 2231. 61. Sani ES, Kheirkhah A, Rana D, Sun Z, Foulsham W, Sheikhi A,
Khademhosseini A, Dana R, Annabi N. Sutureless repair of cor-neal injuries using naturally derived bioadhesive hydrogels. Sci Adv.2019;5(3):eaav1281. doi:10.1126/sciadv.aav1281.
62. Assmann A, Vegh A, Ghasemi-Rad M, Bagherifard S, Cheng G, Sani ES, Ruiz-Esparza GU, Noshadi I, Lassaletta AD, Gangadharan S, et al. A highly adhesive and naturally derived sealant. Biomaterials.2017;140:115–27. doi:10.1016/j.biomaterials. 2017.06.004.
63. Isaacson A, Swioklo S, Connon CJ. 3D bioprinting of a corneal stroma equivalent. Exp Eye Res.2018;173:188–93. doi:10.1016/j. exer.2018.05.010.
64. Sorkio A, Koch L, Koivusalo L, Deiwick A, Miettinen S, Chichkov B, Skottman H. Human stem cell based corneal tissue mimicking structures using laser-assisted 3D bioprinting and functional bioinks. Biomaterials. 2018;171:57–71. doi:10.1016/j. biomaterials.2018.04.034.
65. Basu S, Hertsenberg AJ, Funderburgh ML, Burrow MK, Mann MM, Du Y, Du Y, Lathrop KL, Syed-Picard FN, Adams SM, et al. Human limbal biopsy-derived stromal stem cells prevent corneal scarring. Sci Transl Med. 2014;6 (266):266ra172. doi:10.1126/scitranslmed.3009644.
66. ClinicalTrials.gov identifier: NCT02948023. https://clinicaltrials. gov/ct2/show/NCT02948023
67. ClinicalTrials.gov identifier: NCT03295292, https://clinicaltrials. gov/ct2/show/NCT03295292
68. Du Y, Carlson EC, Funderburgh ML, Birk DE, Pearlman E, Guo N, Kao WW-Y, Funderburgh JL. Stem cell therapy restores transparency to defective murine corneas. Stem Cells.
2009;27:1635–42. doi:10.1002/stem.v27:7.
69. Wu J, Du Y, Watkins SC, Funderburgh JL, Wagner WR. The engineering of organized human corneal tissue through the spatial guidance of corneal stromal stem cells. Biomaterials.
2012;33:1343–52. doi:10.1016/j.biomaterials.2011.10.068. 70. Polisetty N, Fatima A, Madhira SL, Sangwan VS, Vemuganti GK.
Mesenchymal cells from limbal stroma of human eye. Mol Vis.
2008;14:431–42.
71. Alió JL, Del Barrio JL, El Zarif M, Azaar A, Makdissy N, Khalil C, Harb W, El Achkar I, Jawad ZA, De Miguel MP. Regenerative surgery of the corneal stroma for advanced keratoconus: 1-year outcomes. Am J Ophthalmol.2019;203:53–68. doi:10.1016/j.ajo.2019.02.009. 72. Shojaati G, Khandaker I, Funderburgh ML, Mann MM, Basu R,
Stolz DB, Geary ML, Dos Santos A, Deng SX, Funderburgh JL. Mesenchymal stem cells reduce corneal fibrosis and inflammation via extracellular vesicle-mediated delivery of miRNA. Stem Cells Transl Med. Jul 102019. doi:10.1002/sctm.18-0297
73. Hayashi R, Ishikawa Y, Sasamoto Y, Katori R, Nomura N, Ichikawa T, Araki S, Soma T, Kawasaki S, Sekiguchi K, et al. Co-ordinated ocular development from human iPS cells and recovery of corneal function. Nature.2016;531(7594):376. doi:10.1038/nature17000.
74. Foster JW, Wahlin K, Adams SM, Birk DE, Zack DJ, Chakravarti S. Cornea organoids from human induced pluripo-tent stem cells. Sci Rep.2017;7:41286. doi:10.1038/srep41286. 75. Del Barrio JL, Chiesa M, Garagorri N, Garcia-Urquia N,
Fernandez-Delgado J, Bataille L, Rodriguez A, Arnalich-Montiel F, Zarnowski T, de Toledo JP, et al. Acellular
human corneal matrix sheets seeded with human adipose-derived mesenchymal stem cells integrate functionally in an experimental animal model. Exp Rye Res.
2015;132:91–100. doi:10.1016/j.exer.2015.01.020.
76. Shojaati G, Khandaker I, Sylakowski K, Funderburgh ML, Du Y, Funderburgh JL. Compressed collagen enhances stem cell therapy for corneal scarring. Stem Cells Transl Med. 2018;7(6):487–94. doi:10.1002/sctm.17-0258.
77. Liu H, Zhang J, Liu CY, Hayashi Y, Kao WW. Bone marrow mesenchymal stem cells can differentiate and assume corneal keratocyte phenotype. J Cell Mol Med. 2012;16:1114–24. doi:10.1111/j.1582-4934.2011.01306.x.
78. Arnalich-Montiel F, Pastor S, Blazquez-Martinez A, Fernandez-Delgado J, Nistal M, Alio JL, De Miguel MP. Adipose-derived stem cells are a source for cell therapy of the corneal stroma. Stem Cells. 2008 Feb;26(2):570–79. doi:10.1634/stemcells.2007-0653.
79. Liu H, Zhang J, Liu CY, Wang IJ, Sieber M, Chang J, Jester JV, Kao WW. Cell therapy of congenital corneal diseases with umbilical mesenchymal stem cells: lumican null mice. PLoS One. 2010;5(5):e10707. doi:10.1371/journal.pone.0010 707.
80. Coulson-Thomas VJ, Caterson B, Kao WW. Transplantation of human umbilical mesenchymal stem cells cures the corneal defects of mucopolysaccharidosis VII mice. Stem Cells.2013;31 (10):2116–26. doi:10.1002/stem.1481.
81. Yun YI, Park SY, Lee HJ, Ko JH, Kim MK, Wee WR, Reger RL, Gregory CA, Choi H, Fulcher SF, et al. Comparison of the anti-inflammatory effects of induced pluripotent stem cell-derived and bone marrow-derived mesenchymal stromal cells in a murine model of corneal injury. Cytotherapy.2017;19 (1):28–35. doi:10.1016/j.jcyt.2016.10.007.
