Corneal Stromal Regeneration: Current Status and Future Therapeutic Potential

Current Eye Research

ISSN: 0271-3683 (Print) 1460-2202 (Online) Journal homepage: https://www.tandfonline.com/loi/icey20

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

Published online: 20 Sep 2019.

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Corneal Stromal Regeneration: Current Status and Future Therapeutic Potential Neil Lagali Department of Ophthalmology, Institute for Clinical and Experimental Medicine, Faculty of Medicine, Linköping University, Linköping, Sweden; Department of Ophthalmology, Sørlandet Hospital Arendal, Arendal, Norway

ABSTRACT ARTICLE HISTORY The corneal stroma comprises 90% of the corneal thickness and is critical for the cornea’s transparency and Received 31 July 2019 refractive function necessary for vision. When the corneal stroma is altered by disease, injury, or scarring, Revised 28 August 2019 however, an irreversible loss of transparency can occur. Corneal stromal pathology is the cause of millions of Accepted 29 August 2019 cases of blindness globally, and although corneal transplantation is the standard therapy, a severe global KEYWORDS deficit of donor corneal tissue and eye banking infrastructure exists, and is unable to meet the overwhelming Corneal stroma; stromal need. An alternative approach is to harness the endogenous regenerative ability of the corneal stroma, regeneration; bioengineered which exhibits self-renewal of the collagenous extracellular matrix under appropriate conditions. To mimic cornea; acellular porcine endogenous stromal regeneration, however, is a challenge. Unlike the corneal epithelium and endothelium, cornea; stromal stem cells; the corneal stroma is an exquisitely organized extracellular matrix containing stromal cells, proteoglycans 3D bioprinting 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 recellularized, 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.

Introduction donor tissue transplantation has failed repeatedly or is contra- Therapeutic regeneration of the corneal epithelium primarily indicated – for example due to a chronically inflamed recipient using stem cells of limbal origin in various forms has received eye or high expression of enzymes that would degrade the corneal much attention and gained widespread clinical use, including the stroma – a keratoprosthesis such as the Boston KPro4 or the first European Medicines Agency approved stem cell product, osteo-odonto keratoprosthesis5 couldbeused.Theseprosthetic Holoclar.1 Likewise, endothelial regeneration therapy promoted devices are used in rare cases of severe pathology or trauma, and through the use of a donor endothelial cell suspension has entered often only after repeated failed transplantation with a donor cor- first clinical trials for bullous keratopathy.2 The bulk of the corneal nea, and are thus not suitable for the majority of cases of corneal tissue, however, consists of the corneal stroma, which comprises stromal pathology; therefore, in this review we focus only on over 90% of the corneal thickness and imparts to the cornea its primary replacements for the corneal stroma. transparency, curvature and strength, and additionally protects Stromal replacement as implemented today, however, has its the inner eye structures from the external environment. In clinical limitations. Implantation of a donor cornea into a recipient eye situations where the corneal epithelium and endothelium are induces scarring at the donor-recipient tissue interfaces, results in – healthy and functioning, the cornea can still lose its transparency incomplete nerve regeneration,6 8 incomplete cellular repopula- by various corneal stromal pathologies that include infection, tion of the graft,7 changes in refraction due to suboptimal curva- keratoconus, inflammation, neurodegeneration, neovasculariza- ture of the implanted tissue,9 and carries the potential for tion and corneal dystrophies. In these cases collectively affecting immunologic rejection due to the foreign cells10 contained within millions worldwide,3 the main therapeutic option has been to the donor tissue. Although these potential drawbacks of the use of replace the corneal stroma with human donor tissue, either by human donor stromal tissue can be significant, the main draw- full-thickness replacement of the cornea (eg., penetrating kerato- back of stromal replacement with human donor tissue is the plasty where epithelium, stroma, and endothelium are replaced) global shortage of donated corneas and the need for infrastructure or by partial thickness stromal replacement (eg., anterior lamellar to identify, harvest, transport, store and distribute donor corneas keratoplasty where epithelium and the anterior stroma are within a limited time window. The need for donated corneal tissue replaced; or posterior lamellar keratoplasty where posterior and eye banking is a global problem that leaves millions of cases of stroma and endothelium are replaced). In cases where human avoidable corneal blindness unaddressed.11,12

CONTACT Neil Lagali [email protected] 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 at www.tandfonline.com/icey. © 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. 2 N. LAGALI

Similar to epithelial and endothelial regeneration, it would approximately 300 distinct lamellae, each in turn consisting of be ideal if the stroma could be regenerated, assisted by an parallel-oriented bundles of collagen fibers,15 mainly consisting implant, scaffold, cells or other factors. Although no single of type I collagen. The major stromal cell, the keratocyte, is regenerative approach for the corneal stroma has yet emerged present throughout the stroma with the cell body often localized as an approved therapy for clinical use, several alternatives between stromal lamellae. Keratocytes have multiple functions have been proposed and are the subject of intensive research, including collagen synthesis, collagen degradation, and partici- with some having reached the clinical trial stage. The purpose pating in wound healing through transformation to a (myo) of this review is to summarize the various approaches for fibroblast phenotype.16 Natural stromal regeneration through regenerating or replacing the corneal stroma, either partially the normal keratocyte-mediated turnover of collagen is or fully, in order to treat corneal pathology. These approaches a relatively slow process, occurring over at least several years.17 aim to supplement or completely circumvent the use of This long turnover time may be a prerequisite for the regener- human donor corneas from eye banks, which are in short ated collagen to adopt a proper lamellar structure and orienta- supply and are an inadequate option for the future treatment tion to maintain corneal strength, structure and transparency of corneal stromal pathology in many parts of the world. In (stromal regeneration); by contrast, short collagen turnover this review, the concept of stromal regeneration is summar- times are often associated with aberrant collagen production by ized, while highlighting those approaches and technologies for myofibroblasts leading to stromal haze and permanent scar stromal regeneration that are closest to clinical application. tissue deposition (stromal repair).18 Besides keratocytes (and other smaller cell populations not discussed here), the corneal stroma contains proteoglycans, Evidence for endogenous stromal regeneration which are produced by keratocytes and consist of dermatan The regenerative capability of the corneal stroma (including and keratan sulfate components. These components interact the maintenance of corneal transparency during regeneration) with collagen fibrils to maintain fibril assembly and corneal is linked to the presence of various anatomic features, all transparency.19 As they are synthesized by keratocytes, the serving to maintain a strong and transparent corneal stroma proteoglycan population of the stroma can regenerate. (Figure 1). Finally, corneal nerves (axons originating from the trigeminal The most anterior part of the stroma consists of Bowman’s nerve) are present throughout the corneal stroma, with high layer, an approximately 10 µm thick layer of compacted, ran- densities present in the anterior stroma within the subepithe- domly oriented collagen fibrils13 that merges continuously with lial and subbasal nerve plexi.20 Corneal nerve regeneration is the underlying corneal stromal lamellar structure. Bowman’s an important factor in maintaining stromal integrity, restor- layer is non-regenerating, but when present is believed to facil- ing a protective blink reflex and perception of pain, tempera- itate a more rapid and complete anterior corneal nerve and ture and chemical stimuli,21 and for helping to ensure proper anterior stromal regeneration and healing after injury.14 Below tear film production and adequate wetting of the ocular sur- Bowman’s layer is the corneal stroma proper, consisting of 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. CURRENT EYE RESEARCH 3

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. healing; in corneas with a neurotrophic deficit, epithelial colleagues used SMILE lenticules with thickness over 100 µm wounds do not heal adequately, leading to stromal degrada- to seal corneal perforations by suturing the lenticules over the tion (ulceration).22 Corneal nerves within the normal, healthy perforation sites.29 Interestingly, a few weeks following the subbasal nerve plexus (subbasal nerves) are continuously operation, lenticules became incorporated into the corneal regenerating,23 and this regeneration even occurs in the con- stroma, indicating a partial regenerative effect. In another text of corneal injury or transplantation,24 although the regen- study by Ganesh and colleagues, the center of the extracted eration may take several years (Figure 2). Given their lenticules was trephined to form a donut-shaped lenticule that physiological importance, corneal nerves and their regenera- was subsequently implanted into the cornea of a small series tion should be an integral part of strategies aiming to regen- of keratoconus patients, to achieve effective central corneal erate the stroma. flattening.30 The inherent biocompatibility of human-to-human tissue implantation, with potential reduction of immunogenicity of Human donor stromal lenticules the allograft tissue by cryopreservation, is expected to be beneficial for integration of the implanted lenticule with the The refractive surgery procedure of small-incision lenticule surrounding recipient stromal tissue. A regenerative effect extraction (SMILE) has been gaining popularity in the past could be achieved by long-term quiescent stromal cell infil- decade due to the availability of accurate, high frequency tration and turnover of the implanted collagen. This effect, pulsed femtosecond lasers and excellent refractive results however, has not yet been convincingly demonstrated in the postoperatively.25 The procedure involves removal of a small case of human lenticule implantation, and thus the long-term disc (lenticule) of stromal tissue approximately 10–50 µm fate of the lenticules is unknown. Would the thin lenticules thick and 6–8 mm in diameter from the central stroma (at degrade (and therefore corneal thickness decrease) over least 100 µm from the most anterior stromal layer), in order longer time periods or would the lenticules persist? Another to flatten the cornea and correct for myopia (Figure 3). To question to be addressed with this technique is its extendibil- date over one million SMILE procedures have been ity to replace a thicker proportion of the stroma (Figure 3). performed,26 potentially making a large pool of thin stromal Could multiple lenticules be implanted simultaneously, and lenticules available for treating stromal pathology. To facilitate would such an approach be technically feasible and result in this approach, Ganesh and colleagues developed a tissue pro- an adequate transparency, strength and corneal shape? Future cessing and cryopreservation technique27 for the extracted research could address these questions. Furthermore, it has lenticules, making long term storage possible in a cryobank been proposed that a chemical decellularization method could at liquid nitrogen temperatures (−196°C). The lenticules be an alternative to cryopreservation of donor lenticules.31,32 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 Acellular porcine corneal stroma visual acuity. In a separate study, Ganesh and colleagues used similarly extracted stromal lenticules to treat a series of cases Decellularization methods for supplementing the supply of of corneal micro-perforation, corneal defect and traumatic donor corneas have gained popularity in recent years, speci- corneal tear, by using the lenticule as a bandage patch by fically in the use of non-human (typically porcine) corneal attaching it to the recipient cornea using fibrin glue.28 The tissue for human corneal transplantation. The use of non- procedure was safe and maintained corneal transparency and human corneas presents an alternative to reliance on human integrity. Extending the application of this concept, Wu and cornea donation, as porcine corneas are readily obtainable 4 N. LAGALI

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. from the food processing industry and have similar character- porcine stromal sheets resulted in an initially transparent and istics to the human cornea in terms of size, thickness and stable cornea postoperatively, but rendered the implanted sheets biomechanical properties. Decellularization is required to undetectable at the experimental endpoint. mitigate the possibility of xenogenic immunoreactivity to the Decellularized porcine corneas have also been used clini- foreign porcine cells and the possibility of porcine-to-human cally. A Chinese team in Wuhan reported a series of 47 disease transmission, and as such, applications are currently patients with prior fungal corneal infections who underwent limited to stromal replacement and anterior lamellar kerato- anterior lamellar keratoplasty, receiving acellular porcine cor- plasty in cases where recipient endothelium is functional and neal stromal grafts.39 No recurrence of infection was noted the recipient epithelium is able to regenerate to cover the and healing of ulcers was reported in most cases, although graft. with the return of neovascularization and non- Porcine corneas have been subjected to various decellulariza- epithelialization leading to graft melting in four cases. In tion protocols including sodium chloride (NaCl), sodium dode- another Chinese study, acellular porcine corneal stroma was cyl sulfate (SDS), hypoxic nitrogen, ethylene diamine tetra-acetic used in lamellar keratoplasty to treat 13 patients for herpes acid (EDTA), aprotinin, gamma irradiation and hypotonic tris simplex keratitis.40 After a mean of 15 months of postopera- – buffer.33 37 The chemical treatments are often followed by tive follow-up, seven cases had mostly transparent corneas but DNAse and RNAse treatments. Such treatments can substan- 11 of 13 cases had neovascularization, with graft melting tially remove porcine cells and DNA; however, removal of the occurring in three cases. Currently, a prospective clinical foreign immunogenic material is often not 100% complete and trial is ongoing in Guangzhou, using the acellular porcine thus the possibility of transmission of porcine pathogens cornea as a deep anterior lamellar graft in cases of infective remains. Despite this, results of decellularized porcine cornea keratitis.41 These reports, principally from China, indicate xenotransplantation in a rabbit model indicated absence of a potential future role for decellularized porcine corneas to immune rejection and good biocompatibility after one year.38 meet the global demand for suitable stromal tissue for trans- In another report, porcine corneal stroma was decellularized by plantation, which is particularly acute in China. Based on the NaCl treatment and implanted into the rabbit corneal stroma for reported results, future attention needs to be directed towards six months with or without the addition of cultured human improving epithelialization, transparency, and avoidance of corneal keratocytes.35 Without cells, a 100 µm thick stromal neovascularization of the graft. Detailed postoperative analysis implant remained stable but without reported evidence of host (or ex vivo analysis of explanted, failed grafts), however, is cellular infiltration or stromal regeneration, while the addition of currently lacking. It is unclear whether the porcine corneal human keratocytes sandwiched between five 20 µm thick stroma can stably permit stromal regeneration. Ideally for CURRENT EYE RESEARCH 5 regeneration, host keratocytes should migrate into the graft Fish scale-derived cornea and produce new human collagen as the porcine collagen is Another innovative biological source of collagen-based stro- gradually degraded, while corneal thickness and transparency mal scaffolds are the scales of the Tilapia fish. Normally are maintained. Conversely, a non-regenerative reaction considered a waste product of fish processing, the scales of would consist of inflammatory cell and fibroblast invasion, the fish can be decellularized and decalcified to produce a - stromal edema, neovasculariztion and collagen degradation, scaffold44 compatible with the culture of corneal cells.45 resulting in corneal thinning and topographic irregularities. Manufactured to an appropriate thickness, a transparent stro- A further consideration of importance is the postoperative mal scaffold derived from the fish scale was evaluated after regeneration of corneal nerves. Does the porcine stroma per- intrastromal implantation in rabbit corneas, where it was mit nerve regeneration and to what degree? Could outcomes found that the scaffold could be retained in vivo for be improved with the infiltration of host nerves into the one year, maintaining transparency without eliciting an porcine stroma? immune response.46 As the fish scales are mechanically robust, they additionally have the potential to be sutured Full-thickness corneas based on porcine corneal onto the cornea in cases of perforating trauma. This applica- stroma tion was tested using a model of full-thickness perforations in minipig corneas,47 where the fish scale-derived scaffold was Since the acellular porcine corneal stroma can only be used successfully used as an emergency patch graft (termed the therapeutically when the corneal epithelium and endothelium ‘Biocornea’), to temporarily seal the cornea and maintain the are functional, attention is being given to repopulating the integrity of the anterior chamber and eye globe for up to four stromal scaffold with human cells, to enable full-thickness pene- days (within which time a suitable donor cornea can be trating keratoplasty. A Chinese team from Shandong has sourced in an emergency trauma situation). Following posi- demonstrated the ability to seed human embryonic stem cell tive results in the minipig model, the fish scale scaffold is (hESC)-derived limbal epithelial-like cells on one side of the currently undergoing a human clinical trial as a temporary porcine scaffold and hESC-derived corneal endothelial-like patch graft for traumatic corneal perforation (J.J. cells on the other.42 After transplantation into rabbits, corneal Schuitmaker, personal communication). 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 Bioengineered acellular corneal stroma cells were observed to repopulate the implanted porcine stroma at only 8 weeks post-implantation. Whether stromal regenera- Another approach to stromal regeneration not involving the tion would eventually occur is unclear, but the given the ethical use of harvested tissue from humans or animals is the engi- debate surrounding the use of hESC, alternatives are sought. One neering of corneal stromal equivalents from raw starting alternative to the use of hESC, involves a similar porcine stromal materials. These ‘bioengineered’ stromal equivalents have the construct that was developed using human donor cornea- advantage that their properties can be tuned for a specific derived epithelial, stromal and endothelial cells that were seeded application or physiologic effect, and that they do not rely on into the construct and prepared by cell culture methods.43 One tissue harvesting or availability of corneal tissue from animals week after implantation by penetrating keratoplasty in rabbits, or from humans. Furthermore, because the stroma is engi- the construct remained almost completely transparent, before an neered from basic building block components, cells can be eventual immune response led to rejection and opacification of expressly excluded from manufacturing, to create a truly acel- thexenogenicgraftatfourweeks. lular stroma not requiring decellularization. This would The availability of appropriate animal models to test stromal greatly improve the biocompatibility of the stromal equivalent constructs containing human cells remains an issue. by avoiding the host immune response. As the implanted Additionally, initial results suggest an immune reaction of the stroma is acellular, however, stromal regeneration would host to possible cellular and/or DNA remnants within the por- require host cell infiltration and new collagen production. cine cornea that may compromise transparency and initiate One such stromal scaffold is the acellular recombinant a neovascular response. With effective strategies to remove all human collagen-based scaffold, termed the ‘biosynthetic’ cor- donor xenogenic material and sterilize the porcine corneas,37 neal stroma. The biosynthetic scaffold is composed of human along with specific postoperative regimens to suppress possible collagen (the main protein constituent of the human cornea) immune reaction, porcine stromal tissue could play a greater role produced recombinantly in yeast, and is strengthened by in the future in supplementing the human donor tissue pool for chemically crosslinking the collagen using the non-toxic and applications in therapeutic keratoplasty. It remains to be deter- water-soluble EDC/NHS system.48 We implanted this biosyn- mined, however, how the porcine corneal tissue (with or without thetic scaffold by anterior lamellar keratoplasty into ten cells) will be regulated. In particular, within the regulatory fra- Swedish patients to treat advanced keratoconus and corneal mework in the EU, the acellular stroma would be classified as scarring.49,50 After initial limited melting and haze formation a medical device while the addition of cells would render it an due to the softness of the scaffold (i.e., ‘cheese-wiring’ by the advanced therapy medicinal product (ATMP). Stringent GMP retaining sutures), the scaffolds remained stable and func- manufacturing, quality and safety controls and standards would tional for at least four years postoperatively,51 without detect- have to be met,1 encompassing the entire supply, production and able inflammation or rejection. The bulk of the stroma was distribution chains. transparent as indicated by clinical examination, however, 6 N. LAGALI some increased lamellar interface reflectivity was detected by III collagen while simultaneously degrading the scaffold optical coherence tomography (Figure 4a,b). At the cellular (Figure 5b). The new collagen produced by these cells differed level, in vivo confocal microscopy revealed that after three from the scaffold collagen but resembled the host stroma, years, host stromal cells were present at the lamellar interface based on the staining pattern. This indicates a partial regen- regions; however, a clear migration of host stromal cells into eration of host stroma by host stromal cells, facilitated by the biosynthetic scaffold could not be detected (Figure 4c,d). scaffold implantation. Interestingly, the acellular collagen Nerve regeneration, however, had occurred by three years. scaffolds retained stromal proteoglycans from the original Regenerated subbasal nerves, visualized by in vivo confocal porcine dermal source as observed by transmission electron microscopy, were present in the anterior portion of the scaf- microscopy (Figure 5c,d); however, the in vivo functionality fold (Figure 4e,f). In a patient re-transplanted after four years or integrity of these proteoglycans were not examined. This due to suboptimal vision, histologic analysis of the excised bottom-up approach of scaffold fabrication from purified tissue revealed regeneration of a stratified epithelium to cover collagen as a raw material enables stromal scaffolds to be the scaffold and increased density of stromal tissue at the manufactured with a desired diameter, thickness, or in lamellar interfaces, consistent with the presence of denser, a hybrid fashion where different parts can be tuned to have scar-type tissue (Figure 4g). Furthermore, while stromal cells differing transparency and degradation properties.53 A newer were present at the interface region, the cells did not enter the version of the BPC scaffolds have subsequently been tested in implanted scaffold. a minipig model54 and have been implanted within the stroma To overcome the limitations of the relatively soft biosyn- in patients with advanced keratoconus, with positive initial thetic scaffold and improve resistance of the scaffold to enzy- results (M. Rafat, personal communication). Besides addres- matic degradation and the robustness for surgical sing the global shortage of donor stromal tissue for transplan- implantation, we subsequently used medical-grade purified tation, the bioengineered constructs can be manufactured and porcine collagen (extracted from porcine skin) with the stored for up to two years prior to use, based on validated EDC/NHS crosslinking system to develop mechanically stron- shelf life studies (M. Rafat, personal communication). ger stromal scaffolds that were additionally more resistant to Another interesting option reported for the treatment of enzymatic degradation. These bioengineered porcine collagen corneal ulcers is the use of ‘plastic compressed collagen’ as constructs (BPC) were tested in rabbit models by intrastromal a therapeutic option where amniotic membrane grafts fail to implantation.52,53 The BPC scaffolds were biocompatible and epithelialize the ulcerated region. Schrader and colleagues maintained corneal transparency up to the three month report a commercial-grade CE-marked type I porcine dermal experimental endpoint without a host immune reaction. atelocollagen used to create a mechanically compressed col- Implantation of the scaffold was followed by stromal (myo) lagen matrix that is more dense and robust compared to fibroblasts migrating to the lamellar interface and producing typical hydrogel-based scaffolds, while retaining high tensile small amounts of type III collagen in a thin region surround- strength and elasticity.55 The compressed collagen scaffold, ing the scaffold (Figure 5a). These fibroblasts then entered the however, was not crosslinked and is therefore prone to degra- implant at the edges, where they continued to produce type 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. CURRENT EYE RESEARCH 7

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). was used to fill the ulcerated defect region (as a corneal inlay) Cell-populated engineered scaffold while a second identical matrix was used to cover the cornea 56 In cases where host cells would be unsuitable or cannot as a bandage (onlay). While the bandage onlay degraded repopulate the scaffold, an alternative approach is to provide within the first postoperative week, it facilitated coverage of appropriate cells along with the scaffold. This approach is the entire ulcerated region by recipient corneal epithelium, illustrated by the bioengineered human allogeneic nanostruc- with the inlay portion remaining intact. During the following tured anterior cornea developed by a Spanish team. The con- six month period, the inlay also degraded but with epithelium struct consists of a fibrin-agarose scaffold made from remaining intact without signs of inflammation or immune a combination of human blood plasma and 0.1% agarose rejection. Stromal regeneration was not demonstrated, and it formed into a scaffold with nanoscale features.57 Donor is not clear whether the inlay had an impact upon the host human corneal fibroblasts are implanted into the scaffold stroma. This type of stromal replacement however, although and donor corneal limbal epithelial cells are seeded on top, temporary, may provide a means to heal otherwise untreatable to create an anterior lamellar graft under full GMP conditions ulcers, enabling future vision rehabilitation (by for example, in vitro, as an authorized investigational advanced therapy keratoplasty) in these cases. It is interesting to note that even medicinal product. At the time of writing, a Phase I/IIA a temporary stromal scaffold can promote epithelial regenera- randomized multicenter clinical trial58,59 is being conducted tion in this context; stabilization of the plastic compressed in Spanish hospitals, to treat refractory neurotrophic ulcers in scaffold by crosslinking the collagen could possibly provide 20 patients by anterior lamellar keratoplasty. In the first five a means to tailor the degradability in cases where a longer- patients receiving the fibrin-agarose scaffold, complete healing lived stromal scaffold is desirable. 8 N. LAGALI of the ulcer was observed without signs of infection or allo- An alternative cell-based approach reported by Skottman geneic rejection at a two-year follow-up, and the patients and colleagues was the laser bioprinting of a 3D stromal reported relief of ocular symptoms.60 This promising equivalent using two bioinks, one containing type I human approach awaits final evaluation. It will be important to collagen and the other containing a mixture of human adipose determine the fate of the scaffold and implanted cells, includ- stem cells (hASC), collagen, human plasma and thrombin.64 ing signs of tissue regeneration, in terms of re-establishment To create the stromal equivalent, ten alternating layers of of stromal architecture, structure and thickness, and stromal acellular collagen and hASC were printed to form a 7x7 mm cell (and possibly nerve) repopulation of the lamellar graft. 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 Tissue adhesive gel the native corneal stroma. Advantages of incorporating stem For the repair of corneal defects due to traumatic injury or cells into the stroma are their repair and proliferative capacity infection that result in stromal thinning, an alternative to and the potential for non-immunogenicity, when compared to invasive corneal transplantation or use of pre-formed scaf- allogeneic differentiated stromal cells such as keratocytes or folds has been recently proposed by a US-based team. The fibroblasts. device is a stromal mimic that is a viscous gel-like substance While the above bioprinting approaches are a promising when applied to the cornea, which adapts to the form of the means to achieve stromal regeneration, they await in vivo eva- defect in situ. Once the defect is filled, the gel is ‘locked’ in luation in suitable animal models, cell fate determination post- place and stabilized into a more rigid stromal mimic through implantation, and demonstration of stromal regenerative ability photopolymerization. This technology, called GelCORE (gel such as scar-free healing and new collagen production. for corneal regeneration) has been developed as an adhesive Nevertheless, these and similar 3D printed scaffolds are attrac- biomaterial to treat corneal stromal loss.61 The biomaterial is tive as they have a highly controllable production process amen- natural and acellular in that it is derived from porcine gelatin able to mass production, or alternatively could enable that is stabilized by addition of methacrylic anhydride.62 The customized patient-specific structures to be printed, guided by result is a tough but elastic and transparent biomaterial that the condition and topography of the patient’scornea.63 can be photopolymerized using visible blue light, thereby avoiding exposure of eye structures to the potentially dama- Stromal regeneration by stem cell therapy ging UV light normally used to photocure polymers. The GelCORE was shown to be compatible with human corneal As an alternative to incorporating cells within a stromal fibroblast migration in vitro, without inducing cell death after construct, ‘stromal cell therapy’ aims to harness the direct visible light polymerization. In a rabbit model of keratectomy and/or paracrine functions of stem cell populations to with 50% of the stromal depth removed, the GelCORE was repair and regenerate the existing corneal stroma. Cell applied with four-minute photopolymerization time, and therapy can be initiated for example, by direct delivery of resulted in a transparent and smooth cornea that re- therapeutic cells into the diseased or damaged stroma epithelialized within a week. Histologic analysis at 14 days in situ. This concept has been demonstrated for treatment revealed some inflammatory cell infiltration in the implanted of corneal scars that would normally require corneal trans- zone, but the bulk of the GelCORE stromal substitute was no plantation. Instead of transplantation, Basu and colleagues longer detectable, indicating either a rapid transformation or isolated human corneal stromal stem cells (hCSSC) from degradation following epithelial coverage. Initial results are the limbal stroma of an allogenic limbal biopsy and demon- encouraging, and more detailed longer-term in vivo evalua- strated that these cells possess mesenchymal stem cell-like tion would yield further insights into the fate of the biomater- properties.65 Following isolation of these stem cells in cell ial and its potential to stimulate stromal regeneration. 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 3D bioprinting detectable four weeks post-implantation, at which time the Unlike bioengineered corneas made from raw materials that stroma had healed without scarring. At the time of writing, are dispensed or moulded into stromal tissue equivalents, this therapy is undergoing two human clinical trials in three-dimensional bioprinting offers additional flexibility for India, for the treatment of stromal injury, scars and opa- controlling the spatial organization of the fabricated stroma. cities due to pathology or surgery.66,67 A bioprinter additionally offers the possibility to combine While this approach is a promising means to support different biomaterials, cells and other factors into regenerative stromal healing, the cells are sourced from a customized matrix with high spatial resolution. As an initial the limbal stromal region of donor corneas and are there- step in this direction, Connon and colleagues reported a 3D fore still dependent on an adequate supply of donated bioprinted corneal stromal equivalent fabricated using metha- tissue. The donated tissue, however, may not necessarily crylated type I collagen and sodium alginate as bioinks, fol- need to be from the same species, as illustrated in the lowed by crosslinking using calcium chloride.63 Incorporation xenogenic human-to-mouse cell delivery above, and in of human corneal keratocytes within a collagen-alginate other studies of cell-based stromal repair68 duetotheir bioink resulted in a 3D bioprinted stromal equivalent that apparent ability to avoid a host immune response. Non- retained live keratocytes for 7 days. human derived CSSC have yet to be tested definitively for CURRENT EYE RESEARCH 9 stromal regeneration and immunomodulatory potential Conclusions and perspectives in vivo, but such cells could provide a source of CSSC Many of the therapies discussed herein are not mutually exclu- not dependent on human donors. hCSSC, however, have sive; several could be combined for optimal therapeutic effect, the ability to synthesize human stromal collagen and for example acellular stromal scaffolds and therapeutic cells. depositthiscollageninanorganizedmannertomimic Moreover, specific therapies can be personalized, for example the transparent arrangement of collagen fibrils in the by 3D bioprinting a scaffold designed to fit into a specific reci- native cornea.69 The approach of local hCSSC stromal pient bed, bioengineering a stromal equivalent to desired dimen- delivery may therefore not only provide a means to aid sions and with a tailored time to degradation, or using bio-integration and scar-free healing of implanted bioma- autologous cells to avoid immune reaction while promoting terials in the stroma, but hCSSC may also improve out- regenerative healing through paracrine processes. The future comes in xenogenic transplants (such as the acellular also holds further hope for newer advanced techniques not porcine corneal stroma described earlier) through their described here and which are still in a nascent stage, such as immunomodulatory properties. Another point worth men- the use of stem-cell derived exosomes (extracellular vescicles) for tioning is the lack of apparent effect of native hCSSC in promoting scar-free stromal healing72 and the use of iPS-derived cases of corneal scarring where the limbal niche is func- cells or organoids73,74 to promote autologous, regenerative heal- tional and intact. If CSSC exist in the stroma in close ing. The next few years will likely see the first stromal equivalents proximity to limbal epithelial stem cells (LESC) in the and cell-based technologies being approved for clinical use, Palisades of Vogt region, why do they not contribute to initially for conditions non-responsive to conventional treat- stromal wound healing, in an analogous manner as LESCs ments. As ophthalmic specialists and surgeons become more contribute to epithelial wound healing? Suboptimal wound familiar with these new therapies and as they become more healing post-transplantation may lead to stromal scarring, readily available and cost-effective, such niche applications for instance the host-to-donor interface scarring com- over time are likely to be broadened to include additional indi- monly seen after penetrating and lamellar keratoplasty. It cations affecting larger patient populations. appears that hCSSC are not able to effect optimal stromal wound healing while in their native niche to inhibit such Although many of the proposed stromal regeneration tech- interface scarring, whereas their isolation and local deliv- niques claim to be regenerative, very few demonstrate regen- ery to the wounded stroma has an observable effect. The eration or turnover of stromal collagen, restoration of current belief that CSSC provide an important support a lamellar stromal organization or regeneration of corneal function in maintaining the LESC phenotype70 may there- nerves critical for trophic support of the stroma. In addition fore mean that the primary function of the in situ CSSC is to anti-scarring treatments and repair of the extracellular to maintain the function of the limbal niche and not to matrix, neuro-regenerative factors should also be provided repair damaged corneal stroma. Nevertheless, it would be to the regenerating stroma, either in the form of specific of great interest if CSSC could be stimulated to effect axonal growth factors or as stem cell-derived paracrine factors stromal repair in situ, without requiring their extraction providing a milieu favoring nerve growth. An overview of the from a donor, purification and re-introduction into host various approaches for stromal regeneration and the evidence stroma. for stromal regeneration in vivo is given in Table 1. It is clear A promising source of cells for stromal therapy avoiding that much work is still necessary to demonstrate stromal reliance on donor corneas is being developed by Alió and regeneration in vivo in animal models and in humans. colleagues in Spain, who used autologous adipose-derived Beyond initial integration of implanted stromal scaffolds adult stem cells obtained from an elective liposuction proce- into host corneas without rejection or extensive tissue degra- dure. These stem cells were introduced mid-stromally in dation, stromal melting or scar tissue formation, the regen- advanced keratoconus patients with a thin corneal stroma.71 eration of stromal cells, matrix proteins, nerves and The cells were introduced via a femtosecond laser-created proteoglycans has been difficult to demonstrate. Part of the pocket, either alone or in combination with a 120 µm thick difficulty in demonstrating stromal regeneration may be the decellularized human donor stromal implant that had been re- time frame required, as normal stromal collagen turnover and cellularized with the autologous stem cells prior to implanta- nerve regeneration are typically slow processes that can take tion. 12 months postoperatively, all 9 corneas receiving the many years. In vivo detection of stromal regeneration in autologous adipose-derived stem cells were transparent. Five humans can also be challenging, but may be facilitated by corneas receiving only the stem cells had increased in thickness careful cellular and extracellular matrix morphologic observa- by 14.5 µm while those receiving the re-cellularized stromal tions using high-resolution imaging techniques such as implant with stem cells had maintained the stromal implant in vivo confocal microscopy and optical coherence thickness but with no further increase in thickness. While this tomography. study demonstrates the concept of autologous stromal cell Finally, stromal regeneration may represent the final piece in therapy, which has the added benefit of immune compatibility, the puzzle in the quest towards a fully engineered corneal the results suggest that cell therapy alone is insufficient to substitute. The combination of engineered epithelial and stro- replace the bulk of the corneal stroma within a reasonable mal parts into an anterior corneal equivalent, or endothelial and time frame; instead, the cells could augment healing in cases stromal parts into a posterior corneal equivalent could find where for example a non-donor stroma is used, such as application in treating a wide range of corneal pathology nor- a bioprinted or bioengineered stromal equivalent. mally requiring donor tissue transplantation. An engineered full 10 N. LAGALI

Table 1. Summary of various approaches for achieving stromal regeneration and evidence for regeneration in animal models and in human studies. Stromal Regenerative Stromal regeneration in animal Approach Key advantages models Stromal regeneration in humans Decellularized scaffolds Availability, native stromal architecture, strength Human donor stromal Availability, human stromal tissue Integration with rabbit31 and Integration with host stroma28–30 lenticules (SMILE) macaque32 stroma Acellular porcine corneal Abundant, low cost, thick stromal Integration with rabbit stroma, host No evidence provided39,40 stroma tissue cells in scaffold35,38 Recellularized scaffolds Cells can actively effect stromal repair Human embryonic stem Pluripotent, high proliferative No evidence provided in rabbit cells potential model42 Adipose-derived MSC Abundant, autologous, non- Integration with rabbit stroma, Integration with host stroma, cell survival71 immunogenic delivered cells remain in scaffold75 Human donor corneal Corneal cell type, isolation from donor Scaffold not detected in rabbits35, No evidence available57,58 cells corneas no evidence in rabbits43, scar suppression in mice76 Cell-free bioengineered Bottom-up design, low scaffolds immunogenicity, no reliance on donor eyes Fish-scale collagen Abundant, low cost, natural Poor integration in rats44, tolerated biomaterial in rabbits with macrophage infiltration46,no integration in mini-pigs47 Bioengineered collagen Natural biomaterial, biocompatibility Integration with rabbit stroma, host Integration with host stroma,50,51 nerve regeneration51 cells in scaffold, scaffold breakdown and new collagen production52 Tissue adhesives Strength, biocompatibility, ease of application GelCORE Natural biomaterial, biocompatibility, Rapid scaffold degradation in photopolymerization rabbits, rapid ECM deposition hypothesized61 Cell-based without Cell-mediated, no biomaterial scaffold needed, direct delivery Bone-marrow-derived Abundant, autologous source, stromal Cells survive and differentiate into MSC lineage, keratocytes in immunomodulatory mouse stroma77 Adipose-derived MSC Autologous MSC source, abundant Cells survive in rabbit stroma, Cells survive, slight stromal thickness increase, possible differentiate to new collagen production71 keratocytes, new collagens produced78 Umbilical cord-derived Availability, cryopreservation for later Cells survive in mouse stroma, MSC use, differentiate to young cells highly potent keratocytes, synthesize ECM components79,80 Induced pluripotent stem Multipotent, high potency, autologous Recovery of stromal transparency cells following wounding in mice, suppresses inflammation81 Human corneal stromal Isolation from donor cornea rims, New ECM components produced in No evidence available66,67 stem cells stromal origin/fate healthy68 and injured65 stroma in mice Abbreviations: SMILE: small-incision lenticule extraction; MSC: mesenchymal stem cells; GelCORE: gelatin-based adhesive for corneal regeneration; ECM: extracellular matrix corneal equivalent including epithelium, stroma, and endothe- 50–52) are acknowledged. For Figure 5, the prior scientific contributions of lium would be beneficial in treating a further subset of cases Mehrdad Rafat and co-authors (references 53–55) are acknowledged. where only penetrating keratoplasty can restore corneal transparency. Although ambitious, these goals are within reach given the current pace in the development of corneal regenerative Funding technologies and the parallel developments in clinical evaluation This work was supported by the European Commission funded and regulatory approvals of advanced therapeutic modalities. Horizon2020 framework project ARREST BLINDNESS, Grant No. [667400] (www.arrestblindness.eu).

Acknowledgments Declaration of interest The author would like to acknowledge the kind assistance of Anthony Mukwaya in the preparation of Figures 1 and 3.ForFigure 4, the prior The author reports no conflicts of interest. The author alone is respon- scientific contributions of Prof. Per Fagerholm and co-authors (references sible for the content and writing of the paper. CURRENT EYE RESEARCH 11

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