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Purpose. To study the survival of keratocytes and endothelial cells of a human donor cornea storage in the standard and the new media which was specifically designed for optimized cornea hydration. Material and methods. 2D cell cultures of keratocytes and endothelial cells obtained from the Eye tissue bank were used for culture in improved storage media over a period of 14 and 7 days subsequently. To confirm phenotype characteristics, the cells were stained by the following markers: for keratocytes – Lumikan, Keratocan, and α-smooth muscle actin; for endothelial cells – ZO-1 and Na/K-ATPase. The onset of apoptosis in cell culture of keratocytes were detected with Cytochrome C, BAX, and Caspase 3 and 8. Viability of cell cultures after the cultivation was carried out using a commercial set of "Live and Dead". Morphology of the endothelial cells was assessed using an electron scanning microscope. Results. It was shown that the 2D keratocyte culture cultured in the improved storage media expressed specific markers: Lumican, Keratocan, and did not express α-smooth muscle actin. There were no markers of apoptosis in the cell culture of keratocytes after 14 days of cultivation. Corneal endothelium cultured in the improved storage media expresses ZO-1, Na/K-ATPase and presented hexagonal cell shape morphology according to electron microscopy. Conclusion. The improved storage media allow to preserve the unique phenotype of keratocytes, with a slight decrease in proliferative cells activity during 14 days. The media maintain a viable and functional corneal endothelium for at least seven days of cultivation. Key words: cell culture; corneal endothelium; keratocyte; posterior lamellar graft, corneal storage media.

Propagating large amounts of human corneal stromal cells (hCSCs) in vitro while maintaining the physiological quality of their phenotypes is necessary for their application in cell therapy. Here, a novel medium to propagate hCSCs obtained from small incision lenticule extraction (SMILE)‐derived lenticules was investigated and the feasibility of intrastromal injection of these hCSCs was assessed. Primary hCSCs were cultured in porcine corneal stroma extract (pCSE) with RIFA medium including ROCK inhibitor Y27632, insulin‐transferrin‐selenium, fibroblast growth factor 2, L‐ascorbate 2‐phosphate and 0.5% FBS (RIFA medium + pCSE). Protein profiling of the pCSE was identified using nanoscale liquid chromatography coupled to tandem mass spectrometry (nano LC‐MS/MS). After subculturing in RIFA medium + pCSE or 10% FBS normal medium (NM), hCSCs at P4 were transplanted into mouse corneal stroma. Compared with NM, ALDH3A1, keratocan and lumican were significantly more expressed in the RIFA medium + pCSE. ALDH3A1 was also more expressed in the RIFA medium + pCSE than in the RIFA medium. Fibronectin and α‐SMA were less expressed in the RIFA medium + pCSE than in the NM. Using Metascape analysis, the pCSE with its anti‐fibrosis, pro‐proliferation and anti‐apoptosis activities, was beneficial for hCSC cultivation. The intrastromally implanted hCSCs in the RIFA medium + pCSE had positive CD34 expression but negative CD45 expression 35 days after injection. We provide a valuable new medium that is advantageous for the proliferation of hCSCs with the properties of physiological keratocytes. Intrastromal injection of hCSCs in RIFA medium + pCSE has the potential for clinical cell therapy.

Purpose To evaluate the effects of the vascular endothelial growth factor inhibitor conbercept (KH902) on corneal neovascularization and wound healing following penetrating keratoplasty in rabbits. Methods Conbercept was administered to New Zealand white rabbits through topical and subconjunctival routes. Corneal neovascularization and wound healing were examined by slit-lamp photography and histological analyses. The expressions of vascular endothelial growth factor inhibitor, α-smooth muscle actin, and keratocan in the corneal grafts were measured by real-time quantitative polymerase chain reaction (RT-qPCR). Results The anterior segment photographs demonstrated that corneal neovascularization started in the 2nd week. In the 4th week, histologically, the superficial corneal stroma layer showed disordered arrangement, and there were large numbers of dense inflammatory cells and blood vessels in the stroma layer. Vascular endothelial growth factor in the experimental groups was significantly decreased at all time points compared with the control group (both P = 0.001). Expression of α-smooth muscle actin in corneal grafts demonstrated an increase in time even it was lower in experimental groups, but the difference was not statistically significant (P equaled to 0.507 and 0.723, respectively). There were no significant differences with the expression of keratocan in all groups except that it significantly declined at the 4th week as to the second week in all groups and P values were 0.022, 0.020 and 0.014 in control (C), topical (E1), and subconjunctival (E2) group, respectively. Conclusion The study found that conbercept inhibited the formation of corneal neovascularization without affecting keratocan-mediated corneal wound healing and there were no significant differences between topical administration of different doses of conbercept on the rabbit corneal neovascularization after penetrating keratoplasty in this study.

Pacinian corpuscles are onion bulb-like multilayered mechanoreceptors that consist of a complicated structure of axon terminals, Schwann related cells (inner core), endoneural related cells (intermediate layer) and perineurial related cells (outer core-capsule). The cells forming those compartments are continuous and share the properties of that covering the nerve fibers. Small leucine-rich proteoglycans are major proteoglycans of the extracellular matrix and regulate collagen fibrillogenesis, cell signalling pathways and extracellular matrix assembly. Here we used immunohistochemistry to investigate the distribution of class I (biglycan, decorin, asporin, ECM2 and ECMX) and class II (fibromodulin, lumican, prolargin, keratocan and osteoadherin) small leucine-rich proteoglycans in human cutaneous Pacinian corpuscles. The distribution of these compounds was: the inner core express decorin, biglycan, lumican, fibromodulin, osteoadherin; the intermediate layer display immunoreactivity for osteoadherin; the outer core biglycan, decorin, lumican, fibromodulin and osteoadherin; and the capsule contains biglycan, decorin, fibromodulin, and lumican. Asporin, prolargin and keratocan were undetectable. These results complement our knowledge about the distribution of small leucine-rich proteoglycans in human Pacinian corpuscles, and help to understand the composition of the extracellular matrix in these sensory formations.

Autosomal recessive cornea plana is a very rare hereditary ocular disease, characterized by a flattened corneal curvature, marked hyperopia due to low refractive power and frequently consequent accommodative esotropia. Other features include various cornea anterior segment abnormalities, without systemic problems. The purpose of the present study was to investigate the clinical and molecular alterations in a Chinese family with cornea plana. Full ophthalmic examinations of the patients were performed, including slit‑lamp examination, fundus examination and ocular ultrasound. Whole‑exome sequencing data were screened for pathological variants in the proband, which were confirmed by Sanger sequencing. One novel missense mutation, c.242A>G (p.N81S) and another novel 7 base‑pair deletion mutation, c.772‑779del (p.G258Cfs*30), were detected in the keratocan (KERA) gene; two affected siblings inherited these variations in a compound heterozygous state, which were derived from the clinically unaffected heterozygous father (c.772_779del) and mother (c.242A>G), respectively. Neither mutation was observed in unrelated healthy controls (n=200). Multiple computer software predictions supported the pathogenicity of the two variants. Furthermore, protein modeling prediction was performed to better understand the molecular basis of cornea plana, particularly the importance of the leucine‑rich repeat domain. This study presents the 14th pathogenic KERA mutations identified worldwide and the first in East Asia so far, to the best of our knowledge. These findings guided prenatal diagnosis for the family in question and expand on the variant spectrum of KERA, therefore facilitating genetic counseling.

Corneal transparency relies on the precise arrangement and orientation of collagen fibrils, made of mostly Type I and V collagen fibrils and proteoglycans (PGs). PGs are essential for correct collagen fibrillogenesis and maintaining corneal homeostasis. We investigated the spatial and temporal distribution of glycosaminoglycans (GAGs) and PGs after a chemical injury. The chemical composition of chondroitin sulfate (CS)/dermatan sulfate (DS) and heparan sulfate (HS) were characterized in mouse corneas 5 and 14 days after alkali burn (AB), and compared to uninjured corneas. The expression profile and corneal distribution of CS/DSPGs and keratan sulfate (KS) PGs were also analyzed. We found a significant overall increase in CS after AB, with an increase in sulfated forms of CS and a decrease in lesser sulfated forms of CS. Expression of the CSPGs biglycan and versican was increased after AB, while decorin expression was decreased. We also found an increase in KS expression 14 days after AB, with an increase in lumican and mimecan expression, and a decrease in keratocan expression. No significant changes in HS composition were noted after AB. Taken together, our study reveals significant changes in the composition of the extracellular matrix following a corneal chemical injury.

The shortage of human donor corneas has raised important concerns about engineering of corneal endothelial cells (CECs) for clinical use. However, due to the limited proliferative capacity of human CECs, driving them into proliferation and regeneration may be difficult. Unlike human CECs, rabbit CECs have a marked proliferative capacity. To clarify the potential reason for this difference, we analysed the proteomes of four human corneal endothelium samples and four rabbit corneal endothelium samples with quantitative label-free proteomics and downstream analysis. We discovered that vitamin and selenocompound metabolism and some signaling pathways such as NF-kappa B signaling pathway differed between the samples. Moreover, TGFβ, PITX2 and keratocan were distinctively expressed in rabbit samples, which might be associated with active proliferation in rabbit CECs. This study illustrates the proteomic differences between human and rabbit CECs and might promote CEC engineering strategies.

Decellularized porcine corneal scaffolds are a potential alternative to human cornea for keratoplasty. Although clinical trials have reported promising results, there can be corneal haze or scar tissue. Here, we examined if recellularizing the scaffolds with human keratocytes would result in a better outcome. Scaffolds were prepared that retained little DNA (14.89 ± 5.56 ng/mg) and demonstrated a lack of cytotoxicity by in vitro. The scaffolds were recellularized using human corneal stromal cells and cultured for between 14 in serum-supplemented media followed by a further 14 days in either serum free or serum-supplemented media. All groups showed full-depth cell penetration after 14 days. When serum was present, staining for ALDH3A1 remained weak but after serum-free culture, staining was brighter and the keratocytes adopted a native dendritic morphology with an increase (p < 0.05) of keratocan, decorin, lumican and CD34 gene expression. A rabbit anterior lamellar keratoplasty model was used to compare implanting a 250 μm thick decellularized lenticule against one that had been recellularized with human stromal cells after serum-free culture. In both groups, host rabbit epithelium covered the implants, but transparency was not restored after 3 months. Post-mortem histology showed under the epithelium, a less-compact collagen layer, which appeared to be a regenerating zone with some α-SMA staining, indicating fibrotic cells. In the posterior scaffold, ALDH1A1 staining was present in all the acellular scaffold, but in only one of the recellularized lenticules. Since there was little difference between acellular and cell-seeded scaffolds in our in vivo study, future scaffold development should use acellular controls to determine if cells are necessary.

The purpose of this study was to investigate the expression and localization of transforming growth factor (TGF) β1 and TGFβ2 in rabbit corneas that healed with and without stromal fibrosis, and to further study defective perlecan incorporation in the epithelial basement membrane (EBM) in corneas with scarring fibrosis. A total of 120 female rabbits had no surgery, −4.5D PRK, or -9D PRK. Immunohistochemistry (IHC) was performed at time points from unwounded to eight weeks after surgery, with four corneas at each time point in each group. Multiplex IHC was performed for TGFβ1 or TGFβ2, with Image-J quantitation, and keratocan, vimentin, alpha-smooth muscle actin (SMA), perlecan, laminin-alpha 5, nidogen-1 or CD11b. Corneas at the four-week peak for myofibroblast and fibrosis development were evaluated using Imaris 3D analysis. Delayed regeneration of both an apical epithelial growth factor barrier and EBM barrier function, including defective EBM perlecan incorporation, was greater in high injury -9D PRK corneas compared to −4.5D PRK corneas without fibrosis. Defective apical epithelial growth factor barrier and EBM allowed epithelial and tear TGFβ1 and tear TGFβ2 to enter the corneal stroma to drive myofibroblast generation in the anterior stroma from vimentin-positive corneal fibroblasts, and likely fibrocytes. Vimentin-positive cells and unidentified vimentin-negative, CD11b-negative cells also produce TGFβ1 and/or TGFβ2 in the stroma in some corneas. TGFβ1 and TGFβ2 were at higher levels in the anterior stroma in the weeks preceding myofibroblast development in the -9D group. All -9D corneas (beginning two to three weeks after surgery), and four −4.5D PRK corneas developed significant SMA + myofibroblasts and stromal fibrosis. Both the apical epithelial growth factor barrier and/or EBM barrier functions tended to regenerate weeks earlier in −4.5D PRK corneas without fibrosis, compared to −4.5D or -9D PRK corneas with fibrosis. SMA-positive myofibroblasts were markedly reduced in most corneas by eight weeks after surgery. The apical epithelial growth factor barrier and EBM barrier limit TGFβ1 and TGFβ2 entry into the corneal stroma to modulate corneal fibroblast and myofibroblast development associated with scarring stromal fibrosis. Delayed regeneration of these barriers in corneas with more severe injuries promotes myofibroblast development, prolongs myofibroblast viability and triggers stromal scarring fibrosis.

Purpose This study is to investigate the corneal anomaly caused by excess transforming growth factor-α (TGF-α) during mouse development. Methods Bitransgenic KeraRT/TGF-α mice, generated via cross-mating tetO-TGF-α and KeraRT mice, were induced to overexpress TGF-α by doxycycline commencing at embryonic day 0 or postnatal day 0 to different developmental stages. Bitransgenic mice with doxycycline induction were defined as TGF-αECK mice (TGF-α excess expression by corneal keratocytes). Mouse eyes were examined by hematoxylin and eosin staining, immunofluorescent staining and transmission electron microscopy. Protein and RNA from mouse cornea were subjected to western blotting and real-time quantitative polymerase chain reaction. Results In TGF-αECK mice, TGF-α overexpression resulted in corneal opacity. Excess TGF-α initially caused corneal epithelial hyperplasia and subsequent epithelium degeneration as the mouse developed, which was accompanied by gradually diminished K12 expression from the periphery of corneal epithelium and increased K13 expression toward the corneal center. Interestingly, K14 was detected in all layers of corneal epithelium of TGF-αECK mice, whereas it was limited at basal layer of controls. Transmission electron microscopy showed desmosome loss between corneal epithelial cells of TGF-αECK mice. In TGF-αECK mice, keratocan expression was abolished; α-SMA expression was increased while expression of Col1a1, Col1a2, and Col5a1 was diminished. Cell proliferation increased in the corneal epithelium and stroma, but not in the endothelium of TGF-αECK mice. Conclusions Excess TGF-α had detrimental effects on corneal morphogenesis during mouse development in that it changed the cell fate of corneal epithelial cells to assume conjunctival phenotypic expression of K13, and keratocytes to myofibroblast phenotype.

No other tissue in the body depends more on the composition and organization of the extracellular matrix (ECM) for normal structure and function than the corneal stroma. The precise arrangement and orientation of collagen fibrils, lamellae and keratocytes that occurs during development and is needed in adults to maintain stromal function is dependent on the regulated interaction of multiple ECM components that contribute to attain the unique properties of the cornea: transparency, shape, mechanical strength, and avascularity. This review summarizes the contribution of different ECM components, their structure, regulation and function in modulating the properties of the corneal stroma. Fibril forming collagens (I, III, V), fibril associated collagens with interrupted triple helices (XII and XIV), network forming collagens (IV, VI and VIII) as well as small leucine-rich proteoglycans (SLRP) expressed in the stroma: decorin, biglycan, lumican, keratocan, and fibromodulin are some of the ECM components reviewed in this manuscript. There are spatial and temporal differences in the expression of these ECM components, as well as interactions among them that contribute to stromal function. Unique regions within the stroma like Bowman's layer and Descemet's layer are discussed. To define the complexity of corneal stroma composition and structure as well as the relationship to function is a daunting task. Our knowledge is expanding, and we expect that this review provides a comprehensive overview of current knowledge, definition of gaps and suggests future research directions.

Substrate topographic patterning is a powerful tool that can be used to manipulate cell shape and orientation. To gain a better understanding of the relationship between surface topography and keratocyte behavior, surface patterns consisting of linear aligned or orthogonally aligned microchannels were used. Photolithography and polymer molding techniques were used to fabricate micropatterns on the surface of polydimethylsiloxane (PDMS). Cells on linear aligned substrates were elongated and aligned in the channel direction, while cells on orthogonal substrates had a more spread morphology. Both linear and orthogonal topographies induced chromatin condensation and resulted in higher expressions of keratocyte specific genes and sulfated glycosaminoglycans (sGAG), compared with non-patterned substrates. However, despite differences in cell morphology and focal adhesions, many genes associated with a native keratocyte phenotype, such as keratocan and ALDH3A1, remain unchanged on the different patterned substrates. This information could be used to optimize substrates for keratocyte culture and to develop scaffolds for corneal regeneration.

The purpose of this investigation was to study Descemet's membrane and corneal endothelial regeneration, myofibroblast generation and disappearance, and TGF beta-1 localization after Descemet's membrane-endothelial excision (Descemetorhexis) in rabbits. Thirty-six rabbits had 8 mm Descemetorhexis and standardized slit lamp photos at 1, 2 and 4 days, 1, 2 and 4 weeks, and 2, 4 and 6 months, as well as multiplex IHC for stromal cell markers keratocan, vimentin, and alpha-smooth muscle actin (SMA); basement membrane (BM) components perlecan, nidogen-1, laminin alpha-5, and collagen type IV; and corneal endothelial marker Na,K-ATPase β1, and TGF beta-1, with ImageJ quantitation. Stromal transparency increased from the periphery beginning at two months after injury and progressed into the central cornea by six months. At six months, central transparency was primarily limited by persistent mid-stromal neovascularization. Stromal myofibroblast zone thickness in the posterior stroma peaked at one month after injury, and then progressively decreased until to six months when few myofibroblasts remained. The regeneration of a laminin alpha-5 and nidogen-1 Descemet's membrane “railroad track” structure was accompanied by corneal endothelial closure and stromal cell production of BM components in corneas from four to six months after injury. TGF beta-1 deposition at the posterior corneal surface from the aqueous humor peaked at one day after Descemetorhexis and diminished even before regeneration of the endothelium and Descemet's membrane. This decrease was associated with collagen type IV protein production by corneal fibroblasts, and possibly myofibroblasts, in the posterior stroma. Descemet's membrane and the corneal endothelium regenerated in the rabbit cornea by six months after eight mm Descemetorhexis. Real-time quantitative RT-PCR experiments in vitro with marker-verified rabbit corneal cells found that 5 ng/ml or 10 ng/ml TGF beta-1 upregulated col4a1 or col4a2 mRNA expression after 6 hours or 12 hours of exposure in corneal fibroblasts, but not in myofibroblasts. Stromal cells produced large amounts of collagen type IV that likely decreased TGF beta-1 penetration into the stroma and facilitated the resolution of myofibroblast-generated fibrosis.

Purpose To determine whether (1) the in vitro expression of epithelial basement membrane components nidogen-1, nidogen-2, and perlecan by keratocytes, corneal fibroblasts, and myofibroblasts is modulated by cytokines/growth factors, and (2) perlecan protein is produced by stromal cells after photorefractive keratectomy. Methods Marker-verified rabbit keratocytes, corneal fibroblasts, myofibroblasts were stimulated with TGF-β1, IL-1α, IL-1β, TGF-β3, platelet-derived growth factor (PDGF)-AA, or PDGF-AB. Real-time quantitative RT-PCR was used to detect expression of nidogen-1, nidogen-2, and perlecan mRNAs. Western blotting evaluated changes in protein expression. Immunohistochemistry was performed on rabbit corneas for perlecan, alpha-smooth muscle actin, keratocan, vimentin, and CD45 at time points from 1 day to 1 month after photorefractive keratectomy (PRK). Results IL-1α or -1β significantly upregulated perlecan mRNA expression in keratocytes. TGF-β1 or -β3 markedly downregulated nidogen-1 or -2 mRNA expression in keratocytes. None of these cytokines had significant effects on nidogen-1, -2, or perlecan mRNA expression in corneal fibroblasts or myofibroblasts. IL-1α significantly upregulated, while TGF-β1 significantly downregulated, perlecan protein expression in keratocytes. Perlecan protein expression was upregulated in anterior stromal cells at 1 and 2 days after -4.5 or -9 diopters (D) PRK, but the subepithelial localization of perlecan became disrupted at 7 days and later time points in -9-D PRK corneas when myofibroblasts populated the anterior stroma. Conclusions IL-1 and TGF-β1 have opposing effects on perlecan and nidogen expression by keratocytes in vitro. Proximate participation of keratocytes is likely needed to regenerate normal epithelial basement membrane after corneal injury.

Blinding corneal scarring is predominately treated with allogeneic graft tissue; however, there is a worldwide shortage of donor tissue leaving millions in need of therapy. Human corneal stromal stem cells (CSSC) have been shown produce corneal tissue when cultured on nanofibre scaffolding, but this tissue cannot be readily separated from the scaffold. In this study, scaffold-free tissue engineering methods were used to generate biomimetic corneal stromal tissue constructs that can be transplanted in vivo without introducing the additional variables associated with exogenous scaffolding. CSSC were cultured on substrates with aligned microgrooves, which directed parallel cell alignment and matrix organization, similar to the organization of native corneal stromal lamella. CSSC produced sufficient matrix to allow manual separation of a tissue sheet from the grooved substrate. These constructs were cellular and collagenous tissue sheets, approximately 4 μm thick and contained extracellular matrix molecules typical of corneal tissue including collagen types I and V and keratocan. Similar to the native corneal stroma, the engineered corneal tissues contained long parallel collagen fibrils with uniform diameter. After being transplanted into mouse corneal stromal pockets, the engineered corneal stromal tissues became transparent, and the human CSSCs continued to express human corneal stromal matrix molecules. Both in vitro and in vivo, these scaffold-free engineered constructs emulated stromal lamellae of native corneal stromal tissues. Scaffold-free engineered corneal stromal constructs represent a novel, potentially autologous, cell-generated, biomaterial with the potential for treating corneal blindness. Copyright © 2016 John Wiley & Sons, Ltd.

Keratocytes of the corneal limbal stroma can derive populations of mesenchymal stem cells (MSC) when expanded in vitro. However, once a corneal MSC (cMSC) phenotype is achieved, regaining the keratocyte phenotype can be challenging, and there is no standardised differentiation medium. Here, we investigated the transition of keratocytes to cMSC and compared different supplements in their ability to return cMSC to a keratocyte phenotype. Immunofluorescence and RT-qPCR demonstrated in vivo keratocyte expression of ALDH3A1, CD34 and keratocan, but not any of the typical MSC markers (CD73, CD90, CD105). As the keratocytes were expanded in vitro, the phenotypic profile reversed and the cells expressed MSC markers but not keratocyte markers. Differentiating the cMSC back to a keratocyte phenotype using non-supplemented, serum-free medium restored keratocyte markers but did not maintain cell viability or support corneal extracellular matrix (ECM) production. Supplementing the differentiation medium with combinations of fibroblast growth factor-2 (FGF-2), transforming growth factor-β3 (TGF-β3) and retinoic acid (RA) maintained viability, restored expression of CD34, ALDH3A1 and keratocan, and facilitated production of abundant ECM as shown by immunofluorescent staining for collagen-I and lumican, alongside quantitative assays for collagen and glycosaminoglycan production. However, no differentiation medium was able to downregulate the expression of MSC markers in the 21-day culture period. This study shows that the keratocyte to MSC transition can be partially reversed using serum-free media and supplementation with RA, FGF-2 and TGF-β3 can enhance this effect. This is relevant for development of corneal regenerative strategies that require the production of a keratocyte phenotype.

The objective of this study was to determine whether corneal stromal cells (CSCs) from the limbal and central corneal stroma in dogs have multipotent mesenchymal stem/stromal cell (MSC) properties, and whether this cell population can be differentiated into keratocyte-like cells (KDCs). Normal, donated, mesocephalic dog corneas were used to isolate CSC in vitro. Immunohistochemistry demonstrated a distinct population of CD90 expressing cells in the anterior stroma throughout the limbal and central cornea. CSC could be cultured from both the limbal and central cornea and the culture kinetics showed a progenitor cell profile. The CSC expressed stem cell markers CD90, CD73, CD105, N-cadherin, and Pax6, while CD34 was negative. Limbal and central CSC differentiated into osteoblasts, chondrocytes, and adipocytes confirming their multipotency. Coculturing allogeneic peripheral blood mononuclear cells (PBMCs) with limbal CSCs did not affect baseline PBMC proliferation indicating a degree of innate immune privilege. Limbal CSC could be differentiated into KDCs that expressed Keratocan, Lumican, and ALDH1A3 and downregulated Pax6 and N-cadherin. In conclusion, canine CSCs have multipotent MSC properties similarly described in humans and could serve as a source of cells for cell therapy and studying corneal diseases.

Corneal injury due to ocular trauma or infection is one of the most challenging vision impairing pathologies that exists. Many studies focus on the pro-inflammatory and pro-angiogenic effects of interleukin-1 beta(IL-1 beta) on corneal wound healing. However, the effect of IL-1 beta on keratocyte phenotype and corneal repair, as well as the underlying mechanisms, is not clear. This study reports, for the first time, that IL-1 beta induces phenotype changes of keratocytes in vitro, by significantly down-regulating the gene and protein expression levels of keratocyte markers (Keratocan, Lumican, Aldh3a1 and CD34). Furthermore, it is found that the NF-kappa B pathway is involved in the IL-1 beta-induced changes of keratocyte phenotype, and that the selective IKK beta inhibitor TPCA-1, which inhibits NF-kappa B, can preserve keratocyte phenotype under IL-1 beta simulated pathological conditions in vitro. By using a murine model of corneal injury, it is shown that sustained release of TPCA-1 from degradable silk fibroin hydrogels accelerates corneal wound healing, improves corneal transparency, enhances the expression of keratocyte markers, and supports the regeneration of well-organized epithelium and stroma. These findings provide insights not only into the pathophysiological mechanisms of corneal wound healing, but also into the potential development of new treatments for patients with corneal injuries.

Purpose: To investigate the impact of tissue derived biological particles on enzyme-mediated weakened corneas. Methods: Rabbit corneas were treated with enzymes to create an ex vivo ectatic model that simulated representative characteristics of keratoconus (KC). Porcine cornea, cartilage, and lymph node tissues were processed to remove most cellular components and cryomilled into microparticles. The KC corneas were cultured in medium containing the tissue-derived biological particles (TDP) overnight. The mechanical, thermal, ultrastructural changes, and gene expressions of corneal stromal cells were characterized to evaluate the effects of the TDP treatment. Results: The enzyme treatment significantly reduced corneal mechanics and thermal stability, and also disrupted the extracellular matrix ultrastructure. After culturing with TDP medium, the Young&rsquo, s modulus of the modeled KC corneas increased by ~50%, comparable to normal cornea controls. Similarly, the thermal denaturation temperature of the corneas was restored. These findings also corresponded to a significant increase in collagen fibril density after TDP treatment. Furthermore, corneas cultured in TDP medium significantly downregulated expression of the pro-inflammatory gene Tnf&alpha, and restored the expression of the key keratocyte markers Aldh, keratocan, and biglycan. Conclusions: Tissue-derived biological particles reinforce mechanical and thermal properties of corneal tissue in an ex vivo model of KC. Through this study, we demonstrate and characterize the previously unexplored impact of tissue-derived biological scaffolds on corneal biomechanics, thermal stability, and gene expression, presenting a potential new therapy for ocular disease.

We studied the early protein profile in the ocular tissue extracted after LASIK and SMILE surgery. SMILE and LASIK was performed in contralateral eyes and stromal tissue samples were collected from 10 eyes of 5 donors. The stromal tissue samples were analyzed using label free quantification approach and ITRAQ labelling approach in LC-MS/MS. Combined functional analysis revealed many differentially expressed proteins which were involved in important biological processes. About 117 unique differentially expressed proteins were identified using two different proteomic approaches. Collagens, proteoglycans, corneal crystallins were enriched and showed differential expression in SMILE and LASIK as compared to the non-surgical control. Apart from these, 14-3-3 class of proteins, Lysozyme (LYZ), Macrophage Migratory Inhibitory Factor protein (MIF), Pigment Epithelial Derived Factor (PEDF) were differentially expressed when compared between LASIK and SMILE. Peroxiredoxin 1 (PRDX1) expression was found to be reduced in LASIK as compared to SMILE. The expression of Lysozyme C and Macrophage Migratory Inhibitory Factor inflammatory response was found to be less in SMILE as compared to LASIK. Western blot validation of specific markers such as Collagen IV (COL4), Keratocan (KERA), Lumican (LUM), Aldehyde dehydrogenase 3 A1 (ALDH3A1), Lysozyme C (LYZC) confirmed the differences in the protein levels observed in SMILE and LASIK operated tissues as compared to non-surgical controls. In conclusion, this study revealed the early molecular changes occurring in the cornea resulting from these two surgical procedures which may have implications on managing post-operative complications.

Purpose The purpose of this study was to evaluate the effect of removal of Descemet's basement membrane and endothelium compared with removal of the endothelium alone on posterior corneal fibrosis. Methods Twelve New Zealand White rabbits were included in the study. Six eyes had removal of the Descemet's membrane-endothelial complex over the central 8 mm of the cornea. Six eyes had endothelial removal with an olive-tipped cannula over the central 8 mm of the cornea. All corneas developed stromal edema. Corneas in both groups were cryofixed in optimum cutting temperature (OCT) formula at 1 month after surgery. Immunohistochemistry (IHC) was performed for α-smooth muscle actin (SMA), keratocan, CD45, nidogen-1, vimentin, and Ki-67, and a TUNEL assay was performed to detect apoptosis. Results Six of six corneas that had Descemet's membrane-endothelial removal developed posterior stromal fibrosis populated with SMA+ myofibroblasts, whereas zero of six corneas that had endothelial removal alone developed fibrosis or SMA+ myofibroblasts (P < 0.01). Myofibroblasts in the fibrotic zone of corneas that had Descemet's membrane-endothelial removal were undergoing both mitosis and apoptosis at 1 month after surgery. A zone between keratocan+ keratocytes and SMA+ myofibroblasts contained keratocan-SMA-vimentin+ cells that were likely CD45- corneal fibroblasts and CD45+ fibrocytes. Conclusions Descemet's basement membrane has an important role in modulating posterior corneal fibrosis after injury that is analogous to the role of the epithelial basement membrane in modulating anterior corneal fibrosis after injury. Fibrotic areas had myofibroblasts undergoing mitosis and apoptosis, indicating that fibrosis is in dynamic flux.

In order to expand cells quickly and in high numbers for corneal tissue engineering applications corneal stromal cells, or keratocytes, are often cultured in the presence of serum. However, keratocytes become fibroblastic when exposed to serum leading to a downregulation of corneal stromal specific markers. The purpose of this current study was to determine if corneal stromal cells, made fibroblastic by serum, could display native quiescent keratocyte characteristics when cultured under serum-free conditions supplemented by different growth factors. Markers specific to a native keratocyte phenotype such as keratocan and aldehyde dehydrogenase 3A1 (ALDH3A1) and those specific to a fibrotic phenotype such as α-smooth muscle actin (αSMA) and collagen type III were examined. Cells were cultured in monolayer, self-assembled pellets or collagen hydrogels. Growth factors known to modulate keratocyte phenotype were chosen to supplement the serum free media, specifically insulin-like growth factor 1 (IGF-1) and transforming growth factor beta 1 and 3 (Tβ1 and Tβ3). The effects of serum-free media, growth factors and culture system on cell proliferation and morphology and extracellular matrix (ECM) synthesis were evaluated. The expression of keratocyte markers was evaluated by real-time PCR, immunofluorescent staining and western blotting. In addition, cell migration was tested using scratch assays. When serum was removed from the cells they displayed a reduction in proliferation and ECM synthesis (not significant), in addition to a significant decrease in migratory capacity (p < 0.05). Serum-free media promoted increased expression of keratocan (130.68 ± 47.44-fold increase; p < 0.05) and collagen type I (15.58 ± 9.49-fold increase; p < 0.05). However, there was no significant change in ALDH3A1 and αSMA expression, while collagen type III expression was significantly increased (44.66 ± 25.61-fold increase; p < 0.05). In addition, cells retained an elongated fibroblastic morphology. In monolayer, the addition of Tβ1 and Tβ3 to serum free media resulted in reduced expression of keratocan, ALDH3A1 and collagen type I and III, increased expression of αSMA (p < 0.05) and an increase in cell proliferation and ECM synthesis. Pellet cultured cells demonstrated a significant increase in ALDH3A1 and collagen type I over 14 days relative to day 5 (p < 0.05), however the expression of fibrotic markers was also enhanced. Cells in collagen hydrogels did not increase expression of keratocyte markers in serum free conditions and underwent contraction in Tβ1 and Tβ3 supplemented media. These results demonstrate that corneal fibroblasts only partially express the phenotypic characteristics of keratocytes when cultured in serum-free medium. While growth factors did not significantly enhance this phenotype, it appears that pellet or self-assembled culture could be more beneficial to promoting a keratocyte phenotype.

The cornea is a transparent, well-organized connective tissue that provides two-thirds of the eye’s total refractive power. The centermost layer of the cornea is the stroma that makes up approximately 90% of the corneal volume. The corneal stromal limbus contains anatomical features, named the palisades of Vogt, that form a niche for both limbal epithelial stem cells (LESCs) and corneal stromal stem cells (CSSC). CSSC are derived from the neural crest as they express genes typical for descendants of the neural ectoderm such as PAX6, Six2, Six3, and Notch1. The adult stem cell marker ABCG2 is well expressed by CSSC. Moreover, CSSC express mesenchymal stem cell (MSC) markers such as CD73, CD90, and CD166. They exhibit clonal growth, self-renewal properties, and a potential for differentiation into multiple distinct tissue types. Upon the incubation in a keratocyte differentiation medium, CSSC showed the potential to differentiate into a keratocyte by producing extracellular matrix components that in adults are uniquely expressed in the corneal stroma such as keratocan, keratan sulfate, and aldehyde dehydrogenase 3A1. Several studies and the anatomical proximity of LESC and CSSC populations in the limbal niche suggest that the principal role of CSSC in vivo is the homeostatic maintenance of the LESC. Human CSSC do not trigger a xenogeneic T-cell-mediated immune response reaction in vivo and suppress T-cell proliferation in vitro. These findings support an immunomodulatory function for CSSC and the potential use of allogeneic CSSC in different cell-based or tissue-engineered therapeutic applications.

Numerous animal species have been proposed as sources of corneal tissue for obtaining decellularized xenografts. The selection of an appropriate animal model must take into consideration the differences in the composition and structure of corneal proteins between humans and other animal species in order to minimize immune response and improve outcome of the xenotransplant. Here, we compared the amino-acid sequences of 16 proteins present in the corneal stromal matrix of 14 different animal species using Basic Local Alignment Search Tool, and calculated a similarity score compared to the respective human sequence. Primary amino acid structures, isoelectric point and grand average of hydropathy (GRAVY) values of the 7 most abundant proteins (i.e. collagen ?-1 (I), ?-1 (VI), ?-2 (I) and ?-3 (VI), as well as decorin, lumican, and keratocan) were also extracted and compared to those of human. The pig had the highest similarity score (91.8%). All species showed a lower proline content compared to human. Isoelectric point of pig (7.1) was the closest to the human. Most species have higher GRAVY values compared to human except horse. Our results suggest that porcine cornea has a higher relative suitability for corneal transplantation into humans compared to other studied species. © 2019, The Author(s). This paper was supported by Boston Keratoprosthesis research fund.

Producción Científica, The extracellular matrix (ECM) confers transparency to the cornea because of the precise organization of collagen fibrils and a wide variety of proteoglycans. We monitored the corneal wound healing process after alkali burns in rabbits. We analyzed the location and expression of collagens and proteoglycans, the clinical impact, and the recovery of optical transparency. After the animals received both general and ocular topical anesthesia, the central cornea of the left eye was burned by placing an 8-mm diameter filter paper soaked in 0.5 N NaOH for 60 s. The eyes were evaluated under a surgical microscope at 1, 3, and 6 months after burning. At each time point, the clinical conditions of the burned and control corneas were observed. The arrangement of collagen fibers in the corneal stroma was visualized by Picrosirius-red staining, Gomori's silver impregnation and transmission electronic microscopy. Corneal light transmittance was also measured. Myofibroblasts presence was analyzed by immunohistochemistry. mRNA expression levels of collagen types I and III, lumican, decorin, keratocan and alpha-smooth muscle actin were determined by quantitative real-time polymerase chain reaction. One month after alkali burn, the ECM was disorganized and filled with lacunae containing different types of cells and collagen type III fibers in the wound area. Corneal opacities were present with attendant loss of light transmittance. Collagen and proteoglycan mRNA expression levels were up-regulated. After three months, wound healing progress was indicated by reduced corneal opacity, increased light transmittance, reorganization of collagen fibers and only collagen type I expression levels were at control levels. After six months, the wound area ECM morphology was similar to controls, but transmittance values remained low, denoting incomplete restoration of the stromal architecture. This multidisciplinary study of the stromal wound healing process revealed changes in corneal transmittance, collagen organization, myofibroblasts presence and ECM composition at 1, 3, and 6 months after alkali burning. Documenting wound resolution during the six-month period provided reliable information that can be used to test new therapies., Instituto de Salud Carlos III (Health Research Fund PI15/01906 ), Ministerio de Economía, Industria y Competitividad ( project BFU2016-75360-R), Junta de Castilla y León (Project VA114P17)

To identify biochemical cues that could promote a keratocyte-like phenotype in human corneal stromal cells that had become fibroblastic when expanded in serum-supplemented media while also examining the effect on cell proliferation and migration.Proliferation was assessed by PrestoBlueAscorbic Acid (AA), Retinoic Acid (RA), Insulin-Transferrin-Selenium (ITS), Insulin-like Growth Factor 1 (IGF-1) and 3-isobutyl-1-methylxanthine (IBMX) promoted a dendritic morphology, increased the expression of keratocyte markers, such as keratocan, aldehyde dehydrogenase 3 family member A1 (ALDH3A1) and CD34, and prevented myofibroblast differentiation, while in some cases increasing proliferation. Transforming Growth Factor beta 1 (TGF-β1) and 3 (TGF-β3) promoted the differentiation toward myofibroblasts, with increased expression of α-SMA. Fibroblast Growth Factor 2 (FGF-2) supported a fibroblastic phenotype while Platelet-Derived Growth Factor Homodimer B (PDGF-BB) induced a pro-migratory fibroblastic phenotype. A combination of all the pro-keratocyte factors was also compared to the serum-free only, which significantly increased CD34 and keratocan expression.Partially recovery towards a quiescent keratocyte-like phenotype was achieved by the removal of serum and the addition of AA, IGF-1, RA, ITS and IBMX to a basal medium. These findings can be used to develop cell-based corneal therapies and to study corneal diseases in vitro.

The transformation of keratocytes and fibroblasts to myofibroblasts is important to corneal wound healing as well as formation of stromal haze. The purpose of this study was to determine the effect of latrunculin B, an actin cytoskeleton disruptor in conjunction with a fundamental biophysical cue, substrate stiffness, on myofibroblast transformation in vitro and in vivo. Rabbit corneal fibroblasts were cultured on substrates of differing compliance (1.5, 22, and 71 kPa) and tissue culture plastic (TCP; > 1 GPa) in media containing 0 or 10 ng/ml TGFβ1 for 72 h. Cells were treated with 0.4 μM Lat-B or DMSO for 30 min every 24 h for 72 h. RNA was collected from cells and expression of alpha-smooth muscle actin (α-SMA), keratocan, and ALDH1A1 determined using qPCR; immunocytochemistry was used to assess α-SMA protein expression. A rabbit phototherapeutic keratectomy (PTK) model was used to assess the impact of 0.1% Lat-B (n = 3) or 25% DMSO (vehicle control, n = 3) on corneal wound healing by assessment of epithelial wound size with fluorescein stain and semi-quantitative stromal haze scoring by an observer masked to treatment group as well as Fourier-domain optical coherence tomography (FD-OCT) at set time points. Statistical analysis was completed using one-way or two-way analysis of variance. Treatment with Lat-B versus DMSO resulted in significantly less αSMA mRNA (P ≤ 0.007) for RCF cells grown on 22 and 71 kPa substrates as well as TCP without or with TGFβ1, and significantly decreased α-SMA protein expression in RCFs cultured on the intermediate (22 kPa) stiffness in the absence (P = 0.028) or presence (P = 0.018) of TGFβ1. Treatment with Lat-B versus DMSO but did not significantly alter expression of keratocan or ALDH1A1 mRNA in RCFs (P > 0.05) in the absence or presence of TGFβ1, but RCFs grown on stiff hydrogels (71 kPa) had significantly more keratocan mRNA expression versus the 22 kPa hydrogel or TCP (P

Retinoic acid has recently been shown to control the phenotype and extracellular matrix composition of corneal stromal cells cultured in vitro as monolayers. This study set out to investigate the effects of retinoic acid on human corneal keratocytes within a 3D environment. Human corneal keratocytes were encapsulated in collagen gels, which were subsequently compressed under load, and cultured in serum-free media supplemented with 10 µM retinoic acid or DMSO vehicle for 30 days. Cell proliferation was quantified on selected days, while the expression of several important keratocytes markers was evaluated at day 30 using RT-PCR and immunoblotting. The weight and size of the collagen constructs were measured before and after hydration and contraction analyses. Retinoic acid enhanced keratocyte proliferation until day 30, whereas cells in control culture conditions showed reduced numbers after day 21. Both gene and protein expressions of keratocyte-characteristic proteoglycans (keratocan, lumican and decorin), corneal crystallins and collagen type I and V were significantly increased following retinoic acid supplementation. Retinoic acid also significantly reduced the expression of matrix metalloproteases 1, 3 and 9 while not increasing α-smooth muscle actin and fibronectin expression. Furthermore, these effects were also correlated with the ability of retinoic acid to significantly inhibit the contractility of keratocytes while allowing the build-up of corneal stromal extracellular matrix within the 3D constructs. Thus, retinoic acid supplementation represents a promising strategy to improve the phenotype of 3D-cultured keratocytes, and their usefulness as a model of corneal stroma for corneal biology and regenerative medicine applications.

Corneal blindness afflicts millions of individuals worldwide and is currently treated by grafting with cadaveric tissues; however, there are worldwide donor tissue shortages, and many allogeneic grafts are eventually rejected. Autologous stem cells present a prospect for personalized regenerative medicine and an alternative to cadaveric tissue grafts. Dental pulp contains a population of adult stem cells and, similar to corneal stroma, develops embryonically from the cranial neural crest. We report that adult dental pulp cells (DPCs) isolated from third molars have the capability to differentiate into keratocytes, cells of the corneal stoma. After inducing differentiation in vitro, DPCs expressed molecules characteristic of keratocytes, keratocan, and keratan sulfate proteoglycans at both the gene and the protein levels. DPCs cultured on aligned nanofiber substrates generated tissue-engineered, corneal stromal-like constructs, recapitulating the tightly packed, aligned, parallel fibrillar collagen of native stromal tissue. After injection in vivo into mouse corneal stroma, human DPCs produced corneal stromal extracellular matrix containing human type I collagen and keratocan and did not affect corneal transparency or induce immunological rejection. These findings demonstrate a potential for the clinical application of DPCs in cellular or tissue engineering therapies for corneal stromal blindness.