The Limbal Epithelium of the Eye – a Review of Limbal Stem Cell Biology Disease and Treatment
Severe ocular surface disease tin upshot in limbal stem cell deficiency (LSCD), a status leading to decreased visual vigil, photophobia, and ocular pain. To restore the ocular surface in avant-garde stem jail cell scarce corneas, an autologous or allogenic limbal stem cell transplantation is performed. In recent years, the adventure of secondary LSCD due to removal of large limbal grafts has been significantly reduced past the optimization of cultivated limbal epithelial transplantation (CLET). Despite the groovy successes of CLET, there yet is room for improvement as overall success charge per unit is 70% and visual acuity often remains suboptimal after successful transplantation. Simple limbal epithelial transplantation reports higher success rates but has non been performed in as many patients yet. This review focuses on limbal epithelial stem cells and the pathophysiology of LSCD. State-of-the-art therapeutic management of LSCD is described, and new and evolving techniques in ocular surface regeneration are being discussed, in particular, advantages and disadvantages of alternative cell scaffolds and cell sources for jail cell based ocular surface reconstruction.
1. Introduction
Located at the anterior segment of the eye, the cornea is highly organised transparent tissue consisting of multiple cellular and noncellular layers [i]. The corneal epithelium covers the corneal surface and plays a major part in protection and transparency [2, three]. Epithelial cells are shed regularly and replaced by stem cell sources located at the limbus, a rim of tissue located at the junction of the cornea and sclera (Figures one(A) and 1(B)). The limbal epithelial stem cells (LESCs) reside in specific regions at the limbus known equally the limbal stem cell niches [4]. Harm to the stem cells or disruption of the niches may lead to Limbal Stem Prison cell Deficiency (LSCD). In the absence of a healthy corneal epithelium, the conjunctiva proliferates over the cornea resulting in opacification and vascularization, which in turn may lead to reduced vision, pain, and photophobia [5, 6]. LSCD can be caused past a wide variety of chief and secondary causes (Tabular array i) but is most frequently seen associated with severe chemical or thermal burns.
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| 5-FU: 5-fluorouracil. | ||||||||||||||||||||||||||||||||||||||||||||||||
Diagnosis of LSCD is ofttimes on the bases of history and clinical findings, which include loss of limbal anatomy, corneal conjunctivalization, persistent epithelial defects, and scar formation [vii, 8]. In partial LSCD clinical signs are present but express to specific regions, which may be quantified by the number of limbal clock hours involved. The diagnosis is confirmed by impression cytology [ix], illustrating the presence of goblet cells, increased cytokeratin 19 (CK19) expression, and reduced CK3/12 expression [10]. More recently CK7, mucin1, and mucin5AC have been reported as more specific than CK19 for diagnostic purposes [11–xiv].
In vivo confocal microscopy (IVCM) and anterior optical coherence tomography (OCT) are promising techniques that may aid in diagnosing and quantifying LSCD and guiding therapeutic management. IVCM provides high-resolution images of anatomical structures at the cellular level [xv, sixteen]. A number of applied factors limit its use; firstly at that place is no consensus on the definitive morphological advent of LESCs, surrounding niche cells or goblet cells on IVCM [17, eighteen]. Secondly, in the presence of a hazy cornea, the technique is less constructive in defining structures due to high degree of backscatter, and finally it requires the prolonged cooperation of the patient [19]. Anterior OCT, and in particular Fourier Domain Oct (FD-OCT), is a more rapid and user-friendly method of imaging limbal, scleral, and conjunctival structures, though, with significantly lower resolution than IVCM [20]. 3D guided reconstructions of the limbus can be fabricated and may assist guided limbal biopsy [20]. Furthermore, FD-OCT can be applied in imaging hazy corneas and facilitates intraoperative dissection of fibrovascular pannus.
2. Treatment of LSCD
Therapeutic options for LSCD range from bourgeois to invasive and depend on the severity of the pathology (Table 2). Bourgeois therapeutic options include supportive management, corneal scraping, and amniotic membrane patching. In these cases, recovery depends on the presence of some remaining LESCs that tin can be rehabilitated to restore the epithelium. If there are no remaining stem cell reserves, the cornea must exist reseeded with new LESCs [7, 21]. Over the past 18 years, optimizing reseeding techniques has been a major focus of corneal tissue engineering. The earliest techniques required large sections of donor tissue either from the patient's fellow centre (autograft) or from a salubrious donor or cadaver (allograft). Taking such large biopsies places the donor centre at take a chance of developing LSCD. In 1997, Pellegrini et al. reported the start awarding of ex vivo expansion of a very small-scale stem jail cell biopsy in the treatment of LSCD [22]. The ex vivo technique significantly reduced the gamble to the donor eye. Since the original report, numerous clinical trials have reported outcomes of tissue engineered corneal surface reconstruction [22–60]. This review will focus on the nature of LESCs and the evolution and optimization of cultivated limbal epithelial stem jail cell transplantation (CLET) as well as possible hereafter directions.
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| Procedure | Machinery of action and remarks | References |
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| Bourgeois nonsurgical options | ||
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| Autologous serum drops | Serum drops promote migration and proliferation of healthy epithelium while lubricating the ocular surface, preventing epithelial adhesion to the tarsal conjunctiva, and reducing shear stress. | [86–88] |
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| Therapeutic soft contact lens | Therapeutic lenses promote healing of persistent epithelial defects (PED) and prevent the germination of new defects. | [89] |
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| Therapeutic scleral lens | Scleral lenses promote healing of PED while improving vision (optical effect) and reducing pain and photophobia (therapeutic result). They also prevent germination of new epithelial defects. | [90] |
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| Eye lubrication | Ocular surface lubrication prevents epithelial adhesion to the tarsal conjunctiva and reduces shear stress. Unlike autologous serum drops, residual stalk prison cell migration and proliferation is not enhanced. | [89] |
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| Bourgeois surgical options | ||
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| Corneal scraping | During scraping the overgrown conjunctiva is removed, enabling reepithelialisation past islands of functioning corneal epithelial stalk cells. Even so, because the conjunctival epithelium migrates more rapidly than the corneal epithelium, it may be necessary to echo the procedure two to three times. | [91] |
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| Amniotic membrane transplantation (AMT) | AMT promotes proliferation and migration of residue LESCs, contributing to the recovery of the corneal surface, improved visual acuity, and alleviation of pain and photophobia. Low immunogenicity, and anti-inflammatory, antiangiogenic, antifibrotic, antimicrobial, and antiapoptotic properties of the amniotic membrane assist in its therapeutic result. An AMT is performed immediately after corneal scraping equally the overgrown conjunctiva is removed and the amnion membrane is patched over the epithelial defect. Variable clinical outcome may exist attributed to inter- and intradonor variation of the biologically sourced membrane. | [88, 92] |
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| Limbal epithelial stem cell transplantation | ||
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| Conjunctival limbal autograft (CLAU) | Autologous graft derived from the patient'due south salubrious eye, using the conjunctiva equally carrier tissue. Equally this procedure involves dissecting 2 clock hours each of limbal tissue superiorly and inferiorly, CLAU holds the risk of inducing LSCD in the healthy donor middle. | [6, 21, fifty, 89] |
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| Conjunctival limbal allograft (CLAL) | Allogenic graft derived from a living related (lr-CLAL) or deceased donor (c-CLAL), using the conjunctiva as carrier tissue. CLAL comes with an increased risk of transmitting infectious disease and promoting neoplasia due to the long-term use of immunosuppressants. The surgical procedure and number of clock hours to be dissected are like to that for CLAU. Lr-CLAL may induce LSCD in the good for you donor middle. | [6, 21, 50, 89] |
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| Keratolimbal allograft (KLAL) | Allogenic graft derived from a deceased donor, using the cornea as carrier tissue. Equally in CLAL, in that location is an increased hazard of disease transmission and formation of neoplasia. KLAL requires approximately 6 clock hours of tissue to exist removed from the donor limbus and transplanted onto the stem cell deficient eye. | [6, 21, l, 89] |
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| Ex vivo cultivated limbal epithelial stalk cells (CLET) | Autologous or allogenic transplantation of cultivated stem cells, most commonly using the human amniotic membrane or fibrin every bit a carrier for the blended graft. The major advantage of this technique is the reduced risk of inducing LSCD in the healthy donor middle, and the decreased incidence of immunological rejection as Langerhans cells are not cultured in the composite graft. All the same, the utilize of HAM or the transplantation of allogenic LESCs bears the take chances of affliction manual. Furthermore, the utilize of immunosuppressants may be necessary in allogenic transplantation with limited HLA-compatibility. Finally, some civilization protocols use animate being-derived products, which pose the theoretical risk of zoonosis and/or elicit an immune response in the acceptor. | [21, 22, 59, 93] |
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| Uncomplicated limbal epithelial transplantation (SLET) | Autologous transplantation of tiny limbal grafts that are distributed and glued evenly over a HAM. Circumventing difficulties of ex vivo culture techniques, epithelialisation is achieved in vivo. As seen in CLET, at that place is limited risk of immunological rejection or induction of LSCD in the healthy donor eye. Furthermore difficulties of due eastten vivo culturing are avoided, promoting toll-effectiveness. Nonetheless, the rate of LESC expansion in vivo must be greater than that of the speedily proliferative conjunctiva to reach successful engraftment. | [94] |
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3. Limbal Epithelial Stalk Jail cell Niches and Markers
A stalk prison cell niche is the unique microenvironment that surrounds stem cells and modulates their function and fate through internal and external factors. LESCs reside in a such well-protected microenvironment, the limbal stalk cell niche. The niches are protected from UV-radiation by (i) melanocytes that reside in the basal layers of the limbal epithelium and (ii) the upper and lower eyelid that offer cover to the superior and inferior limbus [8, 61, 62]. The niche's undulated basement membrane protects LESCs from shear strength, whereas limbal stromal claret vessels and mesenchymal cells supply it with oxygen, cytokines, growth factors (e.g., the keratinocyte growth cistron), and other nutrients [16, 63–65]. The niche also regulates the LESC cell cycle to go on them in an undifferentiated resting state [sixteen, 66]. Proliferation of a LESC gives rise to two daughter cells, where one remains an oligopotent LESC and the other differentiates into a transient amplifying cell (TAC). Afterward a high but limited number of mitoses, TACs differentiate into "postmitotic cells" and afterwards "terminally differentiated cells" [67–69] (Figure i(C)). During this differentiation procedure, cells migrate centripetally from the niche to the corneal surface [4] according to the -hypothesis [70], that is, proliferation of basal epithelial cells (), differentiation and centripetal migration (), and isolation/desquamation ().
Recently, Molvaer et al. localized and described the three different limbal stem cell niches, (i) the limbal epithelial crypts (LECs), (ii) the limbal crypts (LCs), and (iii) the focal stromal projections (FSPs) (Effigy 2) [98]. LECs were start described in 2005 as projections extending from the undersurface of the limbal epithelium into the stroma. These projections extend radially into the conjunctival stroma parallel to the palisade or circumferentially along the limbus at right angles to the palisade (Effigy 2(a)) [99]. In 2007, LCs and FSPs were described as additional stem jail cell niches. LCs are projections of the limbal epithelium into the stroma, which are laterally enclosed by the palisades of Vogt [16]. The defined area corresponds in function to the previously described interpalisades (Figure two(b)). FSPs are finger-shaped projections of stroma containing a primal claret vessel, which extend upward into the limbal epithelium [16]. More recently, a farther subdivision was fabricated betwixt basal and superficial LCs, the former containing LESCs with melanocytes, the latter containing TACs [100]. It has been proven that all three limbal stem cell niches are mainly present at the superior, and to bottom extent, the inferior limbus. There is no consensus, nevertheless, about the exact number and location of niches in the limbus [98].
(a)
(b)
Stemness and differentiation of LESCs have been investigated through the assay of various jail cell markers. Though no specific marking for LESCs has been identified [101, 102], ABCG2 (also known as BRCP1) [103], p63 [104], and Np63 [105] isoforms are the leading markers used in putative LESC identification. Additional stem jail cell markers take been described, with integrins v3/5 and the ABCB5 cistron virtually recently [106, 107]. Ordonez et al. identified integrin vthree/5 in less than iv% of cells nowadays in the limbal epithelial niche. However, these cells had phenotypic and functional LESC properties [106].
4. Cultured Limbal Epithelial Stem Cell Transplantation
As a technique, cultured limbal epithelial stem cells transplantation (CLET) is in its infancy. The overall success rate is estimated to be 76% [21], though direct comparison of clinical trials is hard due to the wide multifariousness of pathologies treated, culture protocols, surgical approach, and subjective and objective event parameters. When recently published clinical reports are taken into business relationship, success rate decreases slightly to seventy%. Details on culture methods and clinical results of published reports are described (Table iii). No meaning differences were found in the clinical outcomes based on initial crusade of LSCD, source of donor tissue (autologous or allogenic), or culture technique (explants or suspension) [21, 93]. Some culture protocols crave the use of lethally irradiated or Mitomycin C-treated 3T3 feeder cells, either in direct contact or in coculture with the LESCs [25, 29, 31, 35, 37, 41–45, 47, fifty–52, 55, 58–60]. The feeder layers are involved in promoting niche regulation and stemness of cultivated cells. Though no adverse reactions have been reported in the use of 3T3 feeder layers in large case series [108, 109], fugitive xenogenic textile may help reduce the take a chance of animal-derived infection and graft rejection. The search for alternatives to bovine and other animal products in cultivation protocols, for case, fetal bovine serum and animate being-derived growth factors, has led to recent clinical studies cultivating LESCs under nonxenogenic weather condition [52, 54, 55, 57–59]. Other advances in the field that may likewise interpret to a college success rate in time to come trials include feeder layers of human fibroblasts or Mesenchymal Stalk Cells (MSCs) [110–114], standardized GMP (Good Manufacturing Do) protocols [115] for HAM preparation and ex vivo culture, use of autologous serum drops postoperatively, and minimal manipulation of the graft during transplantation [116–118].
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| GMP: good manufacturing practice; HAM: homo amniotic membrane; CL: contact lens; KP: keratoplasty; AMT: amnion membrane transplantation; PTK: phototherapeutic keratectomy; CRVO: central retinal vein occlusion; PED: persistent epithelial defect. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
In 2012, uncomplicated limbal epithelial transplantation (SLET) was described as a novel surgical technique for the handling of unilateral LSCD [94]. During SLET surgery, a small strip of donor limbal tissue (e.g., mm) is divided into several smaller pieces, which are then distributed evenly over a HAM placed on the cornea [94]. The surgery obviates the need for a culture protocol entirely. Although each clinical study reported a success rate of 100% in a small case serial (Table iv) [94–97, 119, 120], the long-term effectiveness of the technique is yet to exist proven.
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| HAM: human amniotic membrane. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
five. Alternative Cell Carriers
In clinical trials, HAM is the virtually commonly used cell carrier for ocular surface reconstruction [23–27, 29–33, 35–42, 44, 45, 47, 48, fifty–threescore]. Withal, in that location are risks associated with the utilize of HAM including possible transfer of infectious agents, variable tissue quality, and express transparency, which is why alternative seeding scaffolds accept been proposed [42, 121].
5.1. Modified HAM
Chemical crosslinking of HAM may raise mechanical and thermal stability, optical transparency, and resistance to collagenase digestion [122–126]. The crosslinking agents that take been investigated are Glutaraldehyde, (L-Lysine-modulated) Carbodiimide, and Altwo(So4)3 [122–126]. In vitro experiments showed that Glutaraldehyde conferred a college degree of cytotoxicity than Carbodiimide [123], whereas the improver of L-lysine to the Carbodiimide crosslinking enhanced mechanical and thermal strength, the ability to back up LESCs, and resistance to enzymatic digestion, though higher concentrations could compromise transparency and biocompatibility [126].
5.2. Collagen
Collagen is the chief extracellular matrix protein of the cornea and has been widely investigated in the evolution of biomimetic carrier materials. It is naturally biocompatible and relatively inexpensive to isolate [127, 128]. LESCs tin can exist successfully cultivated on collagen carriers, while maintaining normal phenotype and achieving multilayered stratification when transplanted in vivo [127, 129, 130]. Jail cell attachment and proliferation tin can be further improved, by coating scaffolds with extracellular matrix proteins (due east.g., laminin, type IV collagen, and fibronectin) or derivative adhesion peptides (eastward.thousand., YIGSR, IKVAV, and RGC) [131–137]. Nigh experimental studies accept been performed using animal-derived collagen (due east.yard., porcine collagen blazon I, rat tail collagen type I, bovine dermal collagen, and fish scale) [127, 138–144]. This collagen may transmit diseases or induce allowed reactions, and therefore the more expensive recombinant homo collagen (RHC) type I and type III are being investigated further for clinical translation [145–151]. Despite the advantages associated with their apply, collagen hydrogels are inherently weak due to the loftier h2o content [152]. Several methods accept been proposed to improve the mechanical properties of collagen hydrogels.
5.2.1. Chemically Crosslinked Collagen
Griffith et al. have reported the structure of biosynthetic collagen scaffolds consisting of full-bodied type I and type III RHC solutions, crosslinked with 1-ethyl-3-(3-dimethyl aminopropyl) Carbodiimide (EDC) and N-hydroxysuccinimide (NHS) [153–155]. When LESCs were cultivated in vitro on the optically transparent constructs, a stratified epithelium formed and covered the surface within 3 weeks. The constructs were sufficiently robust to provide acceptable mechanical stability and elasticity for surgical manipulation. Type III collagen hydrogels tended to be mechanically superior. In vivo verification and validation showed that the acellular scaffolds stayed optically clear and promoted regeneration of corneal cells, nerves, and tear flick, without the need for long-term immunosuppression [149]. However, the mechanical properties of the constructs were significantly lower than man corneas and the long-term stability withal needs to be ascertained.
To improve the mechanical properties of the constructs, Griffith et al. have investigated reinforced membranes fabricated from EDC/NHS crosslinked type Three RHC and PEG-diacrylate crosslinked 2-methacryloyloxyethyl phosphorylcholine (MPC) [151, 156–158]. These hydrogels showed increased mechanical strength and stability against enzymatic digestion and UV degradation and promoted corneal cell and nerve regeneration while optical properties were comparable to a normal cornea [156]. Cell-free RHC-MPC implants take been grafted in 7 optics, in which patients showed stable epithelia 12 months postoperatively and the best corrected vision improved past ane-ii lines [151, 158]. Another class of collagen hydrogel, genipin-crosslinked chitosan-collagen and PEG-Carbodiimide chitosan-collagen hydrogel, has likewise been examined for ocular surface reconstruction [139, 159]. In vitro experiments with these constructs bear witness maintenance of regular stratified multilayered epithelium [159], while initial creature testing shows expert biocompatibility [139]. Use in human corneal regeneration has not yet been reported.
five.ii.2. Plastic Compression Collagen
In 2010, Mi et al. improved the mechanical forcefulness of collagen hydrogels past compressing and blotting the constructs between paper sheets and a nylon mesh thereby reducing the water content of the gels [160]. LESCs cultivated on this construct displayed a smooth and homogenous morphology, whereas cells cultured on conventional hydrogels were distributed more heterogeneously. Subsequent studies confirmed that plastically compressed collagen gels are optically transparent and like shooting fish in a barrel to handle, had improved mechanical strength, and support LESC adhesion, proliferation, and stratification [160–163]. Mechanical force could further be improved by photochemical crosslinking [164]. Kits that enable the production of 3D plastic compressed cultures have recently become commercially available (RAFT, TAP Biosystems, Hertfordshire, UK).
five.iii. Fibrin
Fibrin is the biodegradable product formed during coagulation. Fibrin membranes tin exist fabricated by combining fibrinogen and thrombin, both harvested from human plasma. Fibrin derivates take been used extensively in ophthalmology, typically as a glues or membranes [165–168].
Four clinical studies have reported the use of fibrin every bit a substrate in CLET surgery [28, 46, 48, 49]. In fauna studies, fibrin gels were found to degrade completely later on 3 days [169]. After gel deposition, the transplanted cells adhered directly to the host corneal stroma. In early 2015, Holoclar (Chiesi, Italian republic) has been conditionally approved to exist released in Italia as the first commercially available stalk prison cell therapy for LSCD treatment. Existing data on Holoclar have been obtained past retrospective patient follow-up, and almanac renewal of approval will be guided by results of a current multicenter, prospective phase Four clinical trial. All the same, applied use of this fibrin-based Advanced Therapeutic Medicinal Product (ATMP) is limited to autologous stem cell transplantation in unilateral cases after chemical or thermal burn. Notably, the technique still utilizes lethally irradiated murine 3T3-J2 fibroblast feeder cells and bovine serum during graft generation, which brings into question the safety of the xeno-based cell production [49].
5.4. Siloxane Hydrogel Contact Lenses
In the initial CLET clinical trial past Lu et al., a 3T3 cocultured human epithelial sheet was mounted on a soft contact lens, prior to transplantation as a carrier [170]. In a subsequent study by Di Girolamo et al., the LESCs were cultivated straight on the contact lens [171]. Gore et al. investigated cultivation of LESCs on contact lenses that were coated with a 3T3 feeder layer [172]. In this study, in vitro cultivated LESCs formed a multilayered corneal epithelium, while some basal cells maintained their stemness. Plasma polymer-coated contact lenses also promoted in vitro LESC adhesion and proliferation [173]. Transplantation of these LESCs in a LSCD rabbit model gave rising to patches of stratified epithelium; however, recipient corneas showed only fractional reconstruction, possibly due to brusk-term follow-up (26 days).
five.five. Poly(-caprolactone)
Poly(-caprolactone) is a highly flexible and stiff material that has already been used as a scaffold for skin, bone, and MSC applications. The biocompatibility and optical transparency of poly(-caprolactone) may exist improved by electrospinning and surface modification, and such modified sheets can back up LESC cultivation [174]. The in vivo use of the fabric has not yet been reported.
5.6. Chitosan-Gelatin
Chitosan is a stiff crystalline polysaccharide that is extracted from chitin from arthropod exoskeletons. Membranes of pure chitosan are too potent for ocular purposes but the improver of gelatine and crosslinkers can ameliorate the material handling [175]. Chitosan-gelatine membranes have extensively been investigated for regeneration of os, cartilage, and skin [176–178]. Chitosan-gelatin membranes with a 20 : 80 ratio supported the growth of LESCs that expressed CK3/12, CK15, and ABCG2 [179]. Again, the in vivo employ of this material has non been reported.
five.7. Silk Fibroin
Silk fibroin (SF), obtained from Bombyx mori (domesticated silkworm), can exist processed into sparse transparent membranes. It is nonimmunogenic, degradable, mechanically strong, and optically transparent and has been used as suture material and in bone and cartilage regeneration [180–182]. Cultivation of LESCs on nonporous SF films gives rise to a stratified corneal-similar epithelium [183–187]. Porous SF membranes tin be developed by mixing SF and poly(ethylene glycol) (PEG) and take supported LESC growth [183] although results accept varied [186]. It may be possible to coculture MSCs within pores to recreate the stromal microenvironment [186]. SF may likewise exist combined with chitosan (SF-CS) and the synthetic scaffolds have been investigated with some success [188, 189]. LESCs that were seeded on such lamellar corneas were comparable to native tissue, equally outgrown cells had physiological morphology and high levels of CK3/12 expression [189]. Furthermore, biocompatibility of SF and SF-CS films has been observed in rabbit corneas for upwards to vi months [183, 188]. Yet, membranes constructed from SF derived from Antheraea pernyi (wild silkworm) proved to exist more than decumbent to becoming opaque, displayed lower permeability, and were more brittle than conventional nonporous SF films [187].
5.eight. Man Anterior Lens Capsule
The Human Anterior Lens Sheathing (HaLC) is a dense membrane consisting of Collagen IV, laminin, and heparin sulphate proteoglycans. HaLC is characterized by a gradually increasing thickness (±0.35µgrand per yr) and simultaneous loss of mechanical strength (±i% each year) [190, 191]. LESCs have been successfully cultivated on HaLCs, with in vitro viability of >95%; jail cell density and cell morphology were like to LESCs cultivated on plastic [192]. LESCs, cultured under nonxenogenic conditions maintained their oligopotency, while some cells showed directional differentiation into corneal epithelium [193]. This promising culling scaffold needs further in vivo verification. Concern has been raised, however, that the bore of extracted HaLC may not be large enough for corneal treatments [192].
5.9. Keratin
Reichl et al. succeeded in fabricating a transparent membrane from keratin extracted from man hair [194]. LESC behavior on the films was similar to that on HAM and was not afflicted past prior plasma treatment sterilization of the cloth [195]. Unfortunately, suturing is impaired past a loftier charge per unit of suture tear-out [195].
five.10. Poly(lactide-co-glycolide)
Poly(lactide-co-glycolide) (PLGA) is an FDA-approved, biodegradable, and noncytotoxic material that has been used in products such equally dissolvable sutures [196]. Transparent electrospun PLGA scaffolds are easy to handle, store, and suture [197]; however when LESCs were cultivated on these carriers, the scaffolds began to disintegrate in vitro and were frail to handle. Additional research has shown that PLGA can be chemically altered to achieve predictable and slower breakdown, both in vitro and in vivo [198, 199]. Disintegration was now evident by two weeks after initiation of LESC cultivation, with complete breakdown occurring by half dozen weeks in vitro [199].
five.11. Polymethacrylate
Polymethacrylate has been used in ophthalmology to produce rigid intraocular lenses and contact lenses. Information technology can exist fabricated into transparent biocompatible hydrogels, which can support LESC proliferation [200, 201]. Augmenting the polymethacrylate with 1,4-diaminobutane has been shown to improve LESC adherence and proliferation [202].
5.12. Hydroxyethylmethacrylate
Hydroxyethylmethacrylate and poly-2-hydroxyethylmethacrylate have been used to manufacture soft contact lenses, the Chirila Kpro and the AlphaCor (Improver Technology Inc., Des Plaines, IL) [203, 204]. One study has investigated hydroxyethylmethacrylate in ocular surface reconstruction and ended that LESCs and fibroblasts could adhere and proliferate to hydroxyethylmethacrylate hydrogels that were surface modified with type I collagen and arginine-glycine-aspartic acid ligand [205].
5.13. Poly(ethylene glycol)
PEG is a biocompatible polymer used in pharmaceutical products (e.one thousand., capsules, tablet binders, ointments, and slow release medications). Transparent hydrogels based on PEG-diacrylate and PEG-diacrylamide have been used in vivo and showed favourable results for the latter as PEG-diacrylate implants showed inflammation, corneal haze, and corneal ulceration. Rabbits with PEG-diacrylamide implants, on the other hand, remained healthy and had clear corneas and noninflamed optics for up to 6 months after transplantation [206, 207]. In vitro experiments showed that photolithographical surface coating with collagen type I was necessary to allow LESC adhesion and proliferation [208]. PEG-diacrylate and PEG-diacrylamide hydrogels were intended for full thickness corneal regeneration; notwithstanding, thinner gels intended for anterior corneal regeneration are yet to be investigated. PEG has besides been combined with chitosan and silk fibroin to make even stronger and more transparent biomaterials [209].
v.14. Platelet Poor Plasma
Platelet-Poor Plasma (PPP) is claret plasma with very depression numbers of thrombocytes (</50), which are removed by centrifugation. Biodegradable, transparent PPP membranes can exist manufactured to function as a seeding scaffold in autologous and allogenic CLET. LESC allografts mounted on autologous PPP sheets in LSCD rabbits improved corneal transparency and resulted in a multilayered CK3/12+ epithelium [210, 211].
5.15. Poly(vinyl booze)
Poly(vinyl alcohol) is a transparent hydrogel with good mechanical strength. Poly(vinyl alcohol) shows low cell analogousness, but when incorporated with collagen type I information technology can back up a fully stratified corneal epithelium in vitro [212], but to support in vivo epithelialization poly(vinyl booze)-collagen requires the assistance of HAM [213].
6. Carrier-Free Transplantation
Nishida et al. reported a temperature-responsive polymer, that is, poly(N-isopropylacrylamide) (PIPAAm), that could release intact, transplantable epithelial sheets that retain stem cells and epithelial cells [214]. The copolymer PIPAAm-PEG is at present commercialized as Mebiol gel and is hydrophilic at temperatures below 20°C and hydrophobic at temperatures in a higher place. Experiments have shown that Mebiol supports LESC tillage in vitro and that autologous CLET in Mebiol restores the ocular epithelial surface in a LSCD rabbit model. The particular properties of Mebiol gel allow for easy graft transplantation. Drops of cooled Mebiol gel containing cultured LESCs can be practical to the ocular surface and a contact lens placed over information technology to keep it in place [215].
Furthermore, in vitro fibrin degradation, biodegradable type I collagen, and centrifugation proved to exist constructive techniques in fabricating carrier-free epithelial sheets. Cultured cells did proliferate and differentiate nether the respective conditions, and prison cell-survival in the subsequent carrier-gratuitous state was preserved [216–218].
seven. Alternative Cell Populations
LSCD frequently manifests equally a bilateral condition where no residual stem cells are bachelor for ex vivo culture. Allograft material from living related donors or cadavers may be used, but this is associated with an increased take chances of disease transmission, rejection, and neoplasia (associated with immunosuppressive agents). Culling jail cell populations could potentially supercede the utilise of allogenic fabric and within the last decade a number of approaches accept been explored with varying success [219].
seven.i. Oral Mucosal Epithelial Cells
In 2003, Nakamura et al. described Cultivated Oral Mucosal Epithelial Transplantation (COMET) in a rabbit animal model [220]. Oral Mucosal Epithelial Cells (OMECs) are cultured on a HAM until a stratified epithelium is attained and and so transplanted. The construct mimics the corneal epithelium as transplanted stem cells maintain their stemness at the ectopic site, and OMECs acquire corneal epithelial-like markers such as CK3, CK19, Ki-67, p63, p75, and cornea-specific PAX6 and CK12 [221–223]. COMET has been successful (i.e., regenerating a totally epithelized, stable, and avascular corneal surface) in patients with severe full LSCD [221, 223–232]. However, transplanted cultivated sheets are not completely identical to in vivo corneal epithelium, which leads to a variable degree of in vivo keratinization and stratification (upwardly to 12 cell layers) [221, 228]. Small case series favour CLET, as COMET is associated with college rates of peripheral corneal neovascularisation, junior best corrected visual improvement, and increased adventure of dry eye conditions postoperatively [221, 228].
7.2. Conjunctival Epithelial Cells
Human being conjunctival epithelial cells grown on HAM have been used to reconstruct the ocular surface in rabbits with LSCD [233]. The transplanted conjunctival call sheets formed a five- to six-layer epithelium that remained transparent, smoothen, avascular, and without epithelial defects [234]. Transplanted cells go on expressing both conjunctival (CK4) and corneal epithelial markers (CK3/12). Homo conjunctival epithelial cell transplantation has been used clinically [235] and in ane study in conjunction with a contact lens, which was removed at day 22 [43]. Well-nigh ii years after successful transplantation, a well-formed epithelium with five to 6 layers was present with rare PAS-positive cells, and positivity for CK3, CK19, P63, connexin 43, and MUC5AC [235]. Best corrected visual acuity significantly improved postoperatively, yet the result was rather modest compared to CLET. Hurting and photophobia were non beingness evaluated.
7.3. Hair Follicle Bulge-Derived Epithelial Stem Cells
Unlike OMECs, epithelial stem cells derived from the bulge region of the hair follicle are able to terminally differentiate into a corneal epithelial phenotype when transplanted onto the ocular surface [236]. The concept was proven in an animal study, in which hair follicle stem cells were cultured on a 3T3 feeder layer and transplanted into a LSCD mouse model [237]. The grafts were able to reconstruct the ocular surface in eighty% of transplanted animals [237].
7.iv. Amniotic Epithelial Cells
Man amniotic epithelial cells are characterized by their stalk prison cell properties, low immunogenicity, production of growth factors that promote epithelialization, and their ability of controlled transdifferentiation into other cell types [238–241]. Amniotic epithelial cells can differentiate into corneal epithelial cells when seeded on the superficial corneal stroma in rabbit LSCD models [238–240, 242]. The differentiated cells had a similar structure, morphology, and physiology as that of normal stratified corneal epithelium. However, i written report indicated that the stratified epithelial cells had no polarity with regard to divers superficial corneal epithelial cells, wing cells, or basal cells [238].
7.5. Human Embryonic Stem Cells
Man embryonic stem cells are pluripotent cells derived from the inner cell mass of the human embryo and can successfully differentiate into corneal epithelial-like cell [243, 244]. In a study from Zhu et al., human embryonic stalk cells were induced to grade LESC-like cells and were seeded on an acellular porcine corneal matrix [245]. Seeded cells formed stratified and closely arranged epithelioid cell sheets consisting of a basal layer of cuboid-shaped cells (p63a and ABCG2 positive) and suprabasal layers of elongated cells (CK3 positive). In rabbit LSCD models, the tissue engineered graft had the potential to reconstruct the ocular surface [245]. Embryonic stem cells also differentiate into corneal epithelial cells when in directly contact with the corneal stroma [246]. A major drawback to the use of human being embryonic stem cells is the immune response they elicit, and the upstanding controversy surrounding the origin of the stalk cells [244, 247].
seven.6. Induced Pluripotent Stalk Cells
Induced Pluripotent Stem Cells (iPSCs) are a type of stalk cells generated by manipulation of differentiated adult cells. In 2006, the iPSC technique was first described by Takahashi and Yamanaka and used 4 specific transcription factors to dedifferentiate adult cells into PSCs [248]. Hayashi et al.described a strategy to differentiate LESCs from homo iPSCs that were derived from human adult corneal limbal epithelial cells or human dermal fibroblasts [249]. The iPSCs derived from adult corneal limbal epithelial cells gave rise to more than corneal epithelial colonies and exhibited higher expression of specific corneal epithelial differentiation markers than iPSCs derived from fibroblasts [249, 250]. This may be due to the maintenance of epigenetic characteristics of the original adult prison cell during iPSC formation and subsequent differentiation [250, 251]. A significant drawback of the iPSC technique is that non all limbal epithelial cells preferentially differentiate into corneal epithelial cells [249]. Recently, a 2-step differentiation method was adult to differentiate human being iPSCs into a homogenous population of p63-positive epithelial cells with the power to differentiate into corneal epithelial-similar cells [252].
7.vii. Umbilical Cord Lining Epithelial Stem Cells and Wharton's Jelly Mesenchymal Stem Cells
In 2011, Reza et al. described umbilical mucin-expressing string lining epithelial stem cells as an alternative jail cell population in anterior corneal reconstruction [253]. These cells are nontumorigenic, highly proliferative, and ethically acceptable. The cells' low immunogenicity may obviate the postoperative use of immunosuppressants. In vivo verification in a rabbit model showed articulate corneal surface regeneration with phenotypical CK3/CK12 expression [253]. Wharton's Jelly Mesenchymal Stem Cells have also been proposed for anterior corneal tissue engineering. Garzón et al. demonstrated that these MSCs could transdifferentiate in vitro into corneal epithelial-like cells, with the expression of epithelial jail cell markers (CK3/CK12, PKG, ZO1, and Cnx43) [254].
vii.viii. Mesenchymal Stem Cells
In 2006, Ma et al. were the first to expand MSCs on HAM and afterwards transplant the construct onto the ocular surface of LSCD rats [255]. Although bone marrow-derived human being MSCs did not differentiate into epithelial-like cells, the transplanted MSCs successfully reconstructed the damaged corneal surface as a smooth and continuous epithelium, and avascular and transparent cornea were existence observed [255]. The therapeutic effect may be due to the MSCs' anti-inflammatory and antiangiogenic properties, rather than straight epithelial differentiation. Gu et al. afterwards succeeded in differentiating rabbit-derived bone marrow MSCs into corneal epithelial-like cells [256]. In vitro, differentiation was modulated by either (i) coculturing rabbit LESCs with MSCs or (ii) adding a LESC-derived supernatant to the MSCs [256]. Several other methods of inducing MSC differentiation have since been described [257–259]. In a LSCD rat model, corneal epithelial-like differentiation was modulated by cytokines, produced by rat Corneal Stromal Cells [257]. In 2011, Reinshagen et al. injected enriched MSCs under an AMT in LSCD rabbits [258]. Data indicated that injected MSCs may maintain their stalk jail cell grapheme or may differentiate into epithelial progenitor cells. More recently, it has been discovered that os marrow-derived MSCs are capable of differentiating into corneal epithelial-like cells, when cultured in specialized DMEM-medium [259]. Adipose tissue-derived MSCs and limbal MSCs besides tin differentiate into corneal epithelial-similar cells when exposed to (i) secreted factors of differentiated human corneal epithelial cells or (2) DMEM-medium, respectively [260–263].
7.9. Man Young Dental Pulp Stem Cells
Human immature dental pulp stem cells express both MSC and embryonic stem jail cell markers and have the capacity to differentiate into derivatives of the three germinal layers in vitro. In a LSCD rabbit experiment, transplanted human young dental lurid stalk cells were capable of reconstructing the ocular surface with a well-formed corneal epithelium that expresses LESC markers in the basal prison cell layer and EC markers in suprabasal cell layers [74, 264].
8. Conclusion
Over the past few years, dandy advances in LESC identification and label and ocular surface reconstruction accept been made. With the introduction of CLET and SLET, a safe and successful treatment selection for LSCD has been introduced [22–threescore, 94–97, 119, 120]. In particular, the trend towards (i) standardized nonxenogenic GMP protocols in scaffold manufacturing and cell cultivation and (ii) "no impact graft surgery" is expected to improve success rates in future CLET trials [52, 55, 58, 59]. SLET seems to exist very promising [94–97, 119, 120]; however, large accomplice inclusion, allogenic transplantation, and long-term follow-up have still to be performed. Further elaboration of "tear sampling" every bit a tool to place factors that may be involved in the development and/or maintenance of corneal neovascularization in humans has been described [265]. This technique may assist in monitoring the inflammatory state of the LSCD eye and further improve preoperative direction and postoperative result of patients. However, specific identification of the LESCs remains a hurdle and characterization is nevertheless based on a combination of phenotypic expression patterns [266]. Despite the successes and evolving techniques in LESC transplantation, detailed interaction and signaling pathways between LESCs, niche cells, and surrounding extracellular matrix are not fully understood. Research and noesis within these domains will help understand (i) physiological LESC maintenance, (ii) in vitro and in vivo microenvironment simulation, and (iii) long-term effectiveness of LESC transplantation. Such cognition may potentiate the evolution of new pharmacological solutions (east.g., center drops that contain LESC growth factors) that stimulate remaining dormant LESCs of the diseased middle. These alternatives would be of bully value in cases of extensive ocular inflammation, as these patients are non good candidates for surgical intervention.
Meliorate in vitro and in vivo replication of the niche may also pb to more efficient cultivation and transplantation of LESCs and alternative cell populations. Of the investigated alternative seeding membranes, simply HAM, fibrin, Siloxane Hydrogen contact lens, and collagen membranes accept been used in patients [22–60, 94–97, 119, 120, 149, 151]. In item, the conditional approving of Holoclar (Chiesi, Italy) is a huge step frontward in the accessibility of LSCD treatment in daily practice. Furthermore, RHC membranes seem to be very promising for tissue engineering, the collagen being of nonxenogenic origin and the addition of MPC addressing many shortcomings of conventional collagen hydrogels. Other alternative scaffolds are notwithstanding in an experimental phase and accept nevertheless to be validated in humans. COMET and human conjunctival epithelial cell transplantation have both been successfully performed in selected patients [43, 221, 223–232, 235]. Withal, as iPSCs get widespread attention in many medical disciplines, it is believed that this autologous cell population will play a prominent role in LSCD treatment in the coming years.
In conclusion, it tin be sure that better and more user-friendly treatment options for LSCD patients will emerge in the near future. New treatment options will target optical transparency, biocompatibility, intraoperative treatment, physicochemical strength, and cost-effectiveness. The important focus on sterility, reproducibility, and minimal mutagenicity and cytotoxicity is further stimulated by the widespread introduction of GMP guidelines.
Conflict of Interests
The authors declare that there are no competing financial interests for whatsoever of them.
Acknowledgments
This research was funded by "The Research Foundation-Flanders" (FWO) and EuroNanoMed2.
Copyright
Copyright © 2016 Michel Haagdorens et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted utilise, distribution, and reproduction in any medium, provided the original piece of work is properly cited.
Source: https://www.hindawi.com/journals/sci/2016/9798374/
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