Collagen XII Regulates Corneal Stromal Structure by Modulating Transforming Growth Factor-ß Activity.

Collagen XII is a regulator of corneal stroma structure and function. The current study examined the role of collagen XII in regulating corneal stromal transforming growth factor (TGF)-ß activation and latency. Specifically, with the use of conventional collagen XII null mouse model, the role of collagen XII in the regulation of TGF-ß latency and activity in vivo was investigated. Functional quantification of latent TGF-ß in stromal matrix was performed by using transformed mink lung reporter cells that produce luciferase as a function of active TGF-ß. Col12a1 knockdown with shRNA was used to test the role of collagen XII in TGF-ß activation. Col12a1-/- hypertrophic stromata were observed with keratocyte hyperplasia. Increased collagen fibril forward signal was found by second harmonic generation microscopy in the absence of collagen XII. Collagen XII regulated mRNA synthesis of Serpine1, Col1a1, and Col5a1 and deposition of collagens in the extracellular matrix. A functional plasminogen activator inhibitor luciferase assay showed that collagen XII is necessary for latent TGF-ß storage in the extracellular matrix and that collagen XII down-regulates active TGF-ß. Collagen XII dictates stromal structure and function by regulating TGF-ß activity. A hypertrophic phenotype in Col12a1-/- corneal tissue can be explained by abnormal up-regulation of TGF-ß activation and decreased latent storage.

Developmental Changes in Patterns of Distribution of Fibronectin and Tenascin-C in the Chicken Cornea: Evidence for Distinct and Independent Functions during Corneal Development and Morphogenesis.

The cornea forms the tough and transparent anterior part of the eye and by accurate shaping forms the major refractive element for vision. Its largest component is the stroma, a dense collagenous connective tissue positioned between the epithelium and the endothelium. In chicken embryos, the stroma initially develops as the primary stroma secreted by the epithelium, which is then invaded by migratory neural crest cells. These cells secrete an organised multi-lamellar collagenous extracellular matrix (ECM), becoming keratocytes. Within individual lamellae, collagen fibrils are parallel and orientated approximately orthogonally in adjacent lamellae. In addition to collagens and associated small proteoglycans, the ECM contains the multifunctional adhesive glycoproteins fibronectin and tenascin-C. We show in embryonic chicken corneas that fibronectin is present but is essentially unstructured in the primary stroma before cell migration and develops as strands linking migrating cells as they enter, maintaining their relative positions as they populate the stroma. Fibronectin also becomes prominent in the epithelial basement membrane, from which fibronectin strings penetrate into the stromal lamellar ECM at right angles. These are present throughout embryonic development but are absent in adults. Stromal cells associate with the strings. Since the epithelial basement membrane is the anterior stromal boundary, strings may be used by stromal cells to determine their relative anterior-posterior positions. Tenascin-C is organised differently, initially as an amorphous layer above the endothelium and subsequently extending anteriorly and organising into a 3D mesh when the stromal cells arrive, enclosing them. It continues to shift anteriorly in development, disappearing posteriorly, and finally becoming prominent in Bowman's layer beneath the epithelium. The similarity of tenascin-C and collagen organisation suggests that it may link cells to collagen, allowing cells to control and organise the developing ECM architecture. Fibronectin and tenascin-C have complementary roles in cell migration, with the former being adhesive and the latter being antiadhesive and able to displace cells from their adhesion to fibronectin. Thus, in addition to the potential for associations between cells and the ECM, the two could be involved in controlling migration and adhesion and subsequent keratocyte differentiation. Despite the similarities in structure and binding capabilities of the two glycoproteins and the fact that they occupy similar regions of the developing stroma, there is little colocalisation, demonstrating their distinctive roles.

Chondroitin Sulphate/Dermatan Sulphate Proteoglycans: Potential Regulators of Corneal Stem/Progenitor Cell Phenotype In Vitro.

Chondroitin sulphate (CS) proteoglycans with variable sulphation-motifs along their glycosaminoglycan (GAG) chains are closely associated with the stem cell niche of articular cartilage, where they are believed to influence the characteristics of the resident stem cells. Here, we investigated the immunohistochemical distribution of hybrid CS/dermatan sulphate (DS) GAGs in the periphery of the adult chicken cornea, which is the location of the cornea's stem cell niche in a number of species, using a monoclonal antibody, 6C3, that recognises a sulphation motif-specific CS/DS GAG epitope. This revealed positive labelling that was restricted to the subepithelial corneal stroma, as well as nearby bony structures within the sclera, called ossicles. When cultivated on cell culture dishes coated with 6C3-rich CS/DS, corneal stromal cells (keratocytes) that had been isolated from embryonic chicken corneas formed circular colonies, which took several days to reach confluency. A flow cytometric analysis of these keratocytes revealed changes in their expression levels of the indicative stem cell markers, Connexin 43 (Cx43), Paired Box 6 (PAX6), B-lymphoma Moloney murine leukemia virus insertion region-1 (Bmi-1), and C-X-C Chemokine Receptor 4 (CXCR4) suggestive of a less-differentiated phenotype compared with expression levels in cells not exposed to CS/DS. These findings support the view that CS/DS promotes the retention of a stem cell phenotype in corneal cells, much as it has been proposed to do in other connective tissues.

A Pilot Study to Evaluate Genipin in Staphylococcus aureus and Pseudomonas aeruginosa Keratitis Models: Modulation of Pro-Inflammatory Cytokines and Matrix Metalloproteinases.

Infectious keratitis is a vision-threatening microbial infection. The increasing antimicrobial resistance and the fact that severe cases often evolve into corneal perforation necessitate the development of alternative therapeutics for effective medical management. Genipin, a natural crosslinker, was recently shown to exert antimicrobial effects in an ex vivo model of microbial keratitis, highlighting its potential to serve as a novel treatment for infectious keratitis. This study aimed to evaluate the antimicrobial and anti-inflammatory effects of genipin in an in vivo model of Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) keratitis. Clinical scores, confocal microscopy, plate count, and histology were carried out to evaluate the severity of keratitis. To assess the effect of genipin on inflammation, the gene expression of pro- and anti-inflammatory factors, including matrix metalloproteinases (MMPs), were evaluated. Genipin treatment alleviated the severity of bacterial keratitis by reducing bacterial load and repressing neutrophil infiltration. The expression of interleukin 1B (IL1B), interleukin 6 (IL6), interleukin 8 (IL8), interleukin 15 (IL15), tumor necrosis factor-α (TNF-α), and interferon γ (IFNγ), as well as MMP2 and MMP9, were significantly reduced in genipin-treated corneas. Genipin promoted corneal proteolysis and host resistance to S. aureus and P. aeruginosa infection by suppressing inflammatory cell infiltration, regulating inflammatory mediators, and downregulating the expression of MMP2 and MMP9.

Ultrastructural variability of the juxtacanalicular tissue along the inner wall of Schlemm's canal.

Purpose: Increased resistance of aqueous humor drainage from the eye through Schlemm's canal (SC) is the basis for elevated intraocular pressure in glaucoma. Experimental evidence suggests that the bulk of outflow resistance lies in the vicinity of the inner wall endothelial lining of SC and the adjacent juxtacanalicular tissue (JCT). However, there is little understanding of how this resistance is generated, and a detailed understanding of the structure-function relationship of the outflow pathway has not been established yet. In the present study, regional variations in the ultrastructure of the JCT and the inner wall of SC were investigated in three dimensions. Methods: With the use of serial block face scanning electron microscopy (SBF-SEM), the volume occupied by the electron lucent spaces of the JCT compared to that occupied by the cellular and extracellular matrix was investigated and quantified. The distribution of giant vacuoles (GVs) and pores in the inner wall endothelium of SC was further examined. Results: With increasing distance from the inner wall of SC, the volume of the electron lucent spaces increased above 30%. In contrast, the volume of these spaces in immediate contact with the inner wall endothelium was minimal (<10%). Circumferential variability in the type and distribution of GVs was observed, and the percentage of GVs with pores varied between 3% and 27%. Conclusions: These studies provide a detailed quantitative analysis of the ultrastructure of JCT and the distribution of GVs along the circumference of SC in three dimensions, supporting the non-uniform or segmental aqueous outflow.

Cell-independent matrix configuration in early corneal development.

Mechanisms controlling the spatial configuration of the remarkably ordered collagen-rich extracellular matrix of the transparent cornea remain incompletely understood. We previously described the assembly of the emerging corneal matrix in the mid and late stages of embryogenesis and concluded that collagen fibril organisation was driven by cell-directed mechanisms. Here, the early stages of corneal morphogenesis were examined by serial block face scanning electron microscopy of embryonic chick corneas starting at embryonic day three (E3), followed by a Fourier transform analysis of three-dimensional datasets and theoretical considerations of factors that influence matrix formation. Eyes developing normally and eyes that had the lens surgically removed at E3 were studied. Uniformly thin collagen fibrils are deposited by surface ectoderm-derived corneal epithelium in the primary stroma of the developing chick cornea and form an acellular matrix with a striking micro-lamellar orthogonal arrangement. Fourier transform analysis supported this observation and indicated that adjacent micro-lamellae display a clockwise rotation of fibril orientation, depth-wise below the epithelium. We present a model which attempts to explain how, in the absence of cells in the primary stroma, collagen organisation might be influenced by cell-independent, intrinsic mechanisms, such as fibril axial charge derived from associated proteoglycans. On a supra-lamellar scale, fine cords of non-collagenous filamentous matrix were detected over large tissue volumes. These extend into the developing cornea from the epithelial basal lamina and appear to associate with the neural crest cells that migrate inwardly to form, first the corneal endothelium and then keratocytes which synthesise the mature, secondary corneal stroma. In a small number of experimental specimens, matrix cords were present even when periocular neural crest cell migration and corneal morphogenesis had been perturbed following removal of the lens at E3.

Three-dimensional architecture of collagen type VI in the human trabecular meshwork.

PURPOSE: Type VI collagen is a primary component of the extracellular matrix of many connective tissues. It can form distinct aggregates depending on tissue structure, chemical environment, and physiology. In the current study we examine the ultrastructure and mode of aggregation of type VI collagen molecules in the human trabecular meshwork. METHODS: Trabecular meshwork was dissected from donor human eyes, and three-dimensional transmission electron microscopy of type VI collagen aggregates was performed. RESULTS: Electron-dense collagen structures were detected in the human trabecular meshwork and identified as collagen type VI assemblies based on the three-dimensional spatial arrangement of the type VI collagen molecules, the 105-nm axial periodicity of the assemblies themselves, and their characteristic double bands, which arose from the globular domains of the type VI collagen molecules. Sulfated proteoglycans were also seen to associate with the assemblies either with the globular domain or the inner rod-like segments of the tetramers. CONCLUSIONS: No extended structural regularity in the organization of type VI collagen assemblies within the trabecular meshwork was evident, and the lateral separation of the tetramers forming the assemblies varied, as did the angle formed by the main axes of adjacent tetramers. This is potentially reflective of the specific nature of the trabecular meshwork environment, which facilitates aqueous outflow from the eye, and we speculate that extracellular matrix ions and proteins might prevent a more tight packing of type VI collagen tetramers that form the assemblies.

X-ray absorption near-edge structure (XANES) spectroscopy identifies differential sulfur speciation in corneal tissue.

The chemical composition of tissues can influence their form and function. As a prime example, the lattice-like arrangement of collagen fibrils required for corneal transparency is controlled, in part, by sulfated proteoglycans, which, via core proteins, bind to the collagen at specific locations along the fibril axis. However, to date, no studies have been able to directly identify and characterize sulfur (S) in the cornea as a function of tissue location. In this study, X-ray absorption near-edge structure spectroscopy and micro-beam X-ray fluorescence (µ-XRF) chemical contrast imaging were employed to probe the nature of the mature (bovine) cornea as a function of position from the anterior sub-epithelial region into the deep stroma. Data indicate an inhomogeneity in the composition of S species in the first ≈50 µm of stromal depth. In µ-XRF chemical contrast imaging, S did not co-localize with phosphorous (P) in the deep stroma where sulfates are prominent. Rather, P is present only as isolated micrometric spots, presumably identifiable as keratocytes. This study lends novel insights into the elemental physiology of mature cornea, especially in relation to its S distribution; future studies could be applied to human tissues. Moreover, it defines an analytical protocol for the interrogation of S species in biological tissues with micrometric resolution.

Cell-Cell and Cell-Matrix Interactions at the Presumptive Stem Cell Niche of the Chick Corneal Limbus.

(1) Background: Owing to its ready availability and ease of acquisition, developing chick corneal tissue has long been used for research purposes. Here, we seek to ascertain the three-dimensional microanatomy and spatiotemporal interrelationships of the cells (epithelial and stromal), extracellular matrix, and vasculature at the corneo-scleral limbus as the site of the corneal stem cell niche of the chicken eye. (2) Methods: The limbus of developing (i.e., embryonic days (E) 16 and 18, just prior to hatch) and mature chicken eyes was imaged using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and the volume electron microscopy technique, serial-block face SEM (SBF-SEM), the latter technique allowing us to generate three-dimensional reconstructions from data sets of up to 1000 serial images; (3) Results: Data revealed that miniature limbal undulations of the embryonic basement membrane, akin to Palisades of Vogt (PoV), matured into distinct invaginations of epithelial cells that extended proximally into a vascularized limbal stroma. Basal limbal epithelial cells, moreover, occasionally exhibited a high nuclear:cytoplasmic ratio, which is a characteristic feature of stem cells. SBF-SEM identified direct cell-cell associations between corneal epithelial and stromal cells at the base of structures akin to limbal crypts (LCs), with cord-like projections of extracellular matrix extending from the basal epithelial lamina into the subjacent stroma, where they made direct contact with stomal cells in the immature limbus. (4) Conclusion: Similarities with human tissue suggest that the corneal limbus of the mature chicken eye is likely the site of a corneal stem cell niche. The ability to study embryonic corneas pre-hatch, where we see characteristic niche-like features emerge, thus provides an opportunity to chart the development of the limbal stem cell niche of the cornea.

Chemical composition and sulfur speciation in bulk tissue by x-ray spectroscopy and x-ray microscopy: corneal development during embryogenesis.

The chemical composition and sulfur (S) speciation of developing chick corneas at embryonic days 12, 14, and 16 were investigated using synchrotron scanning x-ray fluorescence microscopy and x-ray absorption near-edge structure spectroscopy. The aim was to develop techniques for the analysis of bulk tissue and identify critical physicochemical variations that correlate with changes in corneal structure-function relationships. Derived data were subjected to principal component analysis and linear discriminant analysis, which highlighted differences in the elemental and S species composition at different stages of embryonic growth. Notably, distinct elemental compositions of chlorine, potassium, calcium, phosphorus, and S altered with development during the transition of the immature opaque cornea to a mature transparent tissue. S structure spectroscopy revealed developmentally regulated alterations in thiols, organic monosulfides, ester sulfate, and inorganic sulfate species. The transient molecular structures and compositional changes reported here provide a deeper understanding of the underlying basis of corneal development during the acquisition of transparency. The experimental and analytical approach is new, to our knowledge, and has wide potential applicability in the life sciences.

Genipin Delays Corneal Stromal Enzymatic Digestion.

Purpose: To evaluate the use of genipin in delaying enzymatic digestion of corneal stroma. Methods: Human corneal stromal tissue was treated with genipin, a known chemical crosslinker, and then along with control tissue was subjected to enzymatic digestion with collagenase. The effects of genipin treatment in retarding stromal digestion were analyzed with phase contrast microscopy, a protein quantification assay, second harmonic generation imaging, and transmission electron microscopy. Results: Genipin increased stromal resistance to enzymatic digestion when compared with untreated stroma. A morphologic analysis and protein quantification showed increased stromal resistance to enzymatic digestion once stromal tissue was treated with genipin. Second harmonic generation imaging revealed persistent fibrillar collagen signaling in genipin-treated tissue in contrast with untreated tissue suggesting that genipin retards collagenolysis. Conclusions: Genipin increases stromal resistance to enzymatic digestion in controlled experiments as demonstrated by protein quantification studies and through morphologic imaging. Translational Relevance: This study explores the novel use of genipin in delaying enzymatic stromal digestion. Delaying stromal melting in the setting of corneal infectious or autoimmune keratitis can potentially decrease clinical morbidity.

Response to Letter to Editor "Comments on 'Cell regulation of collagen fibril macrostructure during corneal morphogenesis' by Koudouna et al."

STATEMENT OF SIGNIFICANCE:  .

Genipin in an Ex Vivo Corneal Model of Bacterial and Fungal Keratitis.

Purpose: To determine whether genipin (a natural crosslinker) could reduce the colonization and proliferation of bacteria and fungi in an ex vivo model of corneal infection. Methods: This study, using an ex vivo model of bacterial and fungal keratitis, investigated the antimicrobial efficacy of genipin crosslinking. Excised corneoscleral buttons were wounded by scalpel incision and subsequently infected with Staphylococcus aureus, Pseudomonas aeruginosa, or Candida albicans. After inoculation, corneas were treated with genipin for 24 hours at 37°C. Histologic examinations were carried out, and the number of viable colony-forming units (CFU)/cornea was determined. Results: Genipin exerts bactericidal action against S. aureus and P. aeruginosa, as well as fungicidal action against C. albicans and significantly reduced the CFU compared to contralateral eyes that received saline treatment (P < 0.05). Conclusions: These data identify genipin as a novel ocular antimicrobial agent that has the potential to be incorporated into the therapeutic armamentarium against microbial keratitis. Translational Relevance: This study provided evidence for the antimicrobial and antifungal properties of genipin as an alternative crosslinker that could be used in the management of infectious keratitis.

Recapitulation of normal collagen architecture in embryonic wounded corneas.

Wound healing is characterized by cell and extracellular matrix changes mediating cell migration, fibrosis, remodeling and regeneration. We previously demonstrated that chick fetal wound healing shows a regenerative phenotype regarding the cellular and molecular organization of the cornea. However, the chick corneal stromal structure is remarkably complex in the collagen fiber/lamellar organization, involving branching and anastomosing of collagen bundles. It is unknown whether the chick fetal wound healing is capable of recapitulating this developmentally regulated organization pattern. The purpose of this study was to examine the three-dimensional collagen architecture of wounded embryonic corneas, whilst identifying temporal and spatial changes in collagen organization during wound healing. Linear corneal wounds that traversed the epithelial layer, Bowman´s layer, and anterior stroma were generated in chick corneas on embryonic day 7. Irregular thin collagen fibers are present in the wounded cornea during the early phases of wound healing. As wound healing progresses, the collagen organization dramatically changes, acquiring an orthogonal arrangement. Fourier transform analysis affirmed this observation and revealed that adjacent collagen lamellae display an angular displacement progressing from the epithelium layer towards the endothelium. These data indicate that the collagen organization of the wounded embryonic cornea recapitulate the native macrostructure.

Chondroitin Sulfate as a Potential Modulator of the Stem Cell Niche in Cornea.

Chondroitin sulfate (CS) is an important component of the extracellular matrix in multiple biological tissues. In cornea, the CS glycosaminoglycan (GAG) exists in hybrid form, whereby some of the repeating disaccharides are dermatan sulfate (DS). These CS/DS GAGs in cornea, through their presence on the proteoglycans, decorin and biglycan, help control collagen fibrillogenesis and organization. CS also acts as a regulatory ligand for a spectrum of signaling molecules, including morphogens, cytokines, chemokines, and enzymes during corneal growth and development. There is a growing body of evidence that precise expression of CS or CS/DS with specific sulfation motifs helps define the local extracellular compartment that contributes to maintenance of the stem cell phenotype. Indeed, recent evidence shows that CS sulfation motifs recognized by antibodies 4C3, 7D4, and 3B3 identify stem cell populations and their niches, along with activated progenitor cells and transitional areas of tissue development in the fetal human elbow. Various sulfation motifs identified by some CS antibodies are also specifically located in the limbal region at the edge of the mature cornea, which is widely accepted to represent the corneal epithelial stem cell niche. Emerging data also implicate developmental changes in the distribution of CS during corneal morphogenesis. This article will reflect upon the potential roles of CS and CS/DS in maintenance of the stem cell niche in cornea, and will contemplate the possible involvement of CS in the generation of eye-like tissues from human iPS (induced pluripotent stem) cells.

Cell regulation of collagen fibril macrostructure during corneal morphogenesis.

While tissue form and function is highly dependent upon tissue-specific collagen composition and organization, little is known of the mechanisms controlling the bundling of collagen fibrils into fibers and larger structural designs that lead to the formation of bones, tendons and other tissues. Using the cornea as a model system, our previous 3 dimensional mapping of collagen fiber organization has demonstrated that macrostructural organization of collagen fibers involving interweaving, branching and anastomosing plays a critical role in controlling mechanical stiffness, corneal shape and refractive power. In this work, the cellular and mechanical mechanisms regulating critical events in the assembly of collagen macrostructure are analysed in the developing chicken cornea. We elucidated the temporal events leading to adult corneal structure and determined the effects of intraocular pressure (IOP) on the organization of the collagen macrostructure. Our findings indicate that the complex adult collagen organization begins to appear on embryonic day 10 (E10) after deposition of the primary stroma and full invasion of keratocytes. Importantly, organizational changes in keratocytes appearing at E9 preceded and predicted later changes in collagen organization. Corneal collagen organization remained unaffected when the development of IOP was blocked at E4. These findings support a primary role for keratocytes in controlling stromal organization, mechanical stiffness and corneal shape that are not regulated by the IOP. Our findings also suggest that the avian cornea represents an excellent experimental model for elucidating key regulatory steps and mechanisms controlling the collagen fiber organization that is critical to determining tissue form and function. STATEMENT OF SIGNIFICANCE: This work by using an ex ovo model system, begins to investigate the potential mechanisms controlling collagen fibril macrostructure. In particular, this work highlights a convergent role for the corneal keratocytes in organizing the complex collagen macrostructure, necessary to support high visual acuity. Our data supports that the intraocular pressure does not influence collagen fibril macrostructure and suggest that the avian cornea represents an excellent experimental model for elucidating key regulatory steps and mechanisms controlling the collagen fiber organization that is critical to determining tissue form and function. Clearly understanding the cellular and molecular mechanisms that underlie collagen fibril macrostructure will be highly beneficial for future tissue engineering and regenerative medicine applications.

Evolution of the vertebrate corneal stroma.

Although the cornea is the major refractive element of the eye, the mechanisms controlling corneal shape and hence visual acuity remain unknown. To begin to address this question we have used multiphoton, non-linear optical microscopy to image second harmonic generated signals (SHG) from collagen to characterize the evolutionary and structural changes that occur in the collagen architecture of the corneal stroma. Our studies show that there is a progression in complexity of the stromal collagen organization from lower (fish and amphibians) to higher (birds and mammals) vertebrates, leading to increasing tissue stiffness that may control shape. In boney and cartilaginous fish, the cornea is composed of orthogonally arranged, rotating collagen sheets that extend from limbus to limbus with little or no interaction between adjacent sheets, a structural paradigm analogous to 'plywood'. In amphibians and reptiles, these sheets are broken down into broader lamellae that begin to show branching and anastomosing with adjacent lamellae, albeit maintaining their orthogonal, rotational organization. This paradigm is most complex in birds, which show the highest degree of lamellar branching and anastomosing, forming a 'chicken wire' like pattern most prominent in the midstroma. Mammals, on the other hand, diverged from the orthogonal, rotational organization and developed a random lamellar pattern with branching and anastomosing appearing highest in the anterior stroma, associated with higher mechanical stiffness compared to the posterior stroma.

Light transmission/absorption characteristics of the meibomian gland.

PURPOSE: While meibography has proven useful in identifying structural changes in the meibomian gland (MG), little is known regarding the MG spectral transmission and absorption characteristics. The purpose of this study was to measure the transmission/absorption spectra of the MG compared to other eyelid tissues. METHODS: Human and rabbit eyelids were fixed in paraformaldehyde, serial sectioned (50 µm) using a cryotome and imaged by brightfield and reflectance microscopy. Eyelid regions (MG, muscle, tarsus and dermis) were then illuminated by a 100 µm spot using a infrared enhanced white light source. Transmission spectra over a 550-950 nm range were then measured using a spectrometer and differences compared using two-way analysis of variance. RESULTS: Brightfield microscopy of both human and rabbit eyelid tissue showed a marked decrease in light transmission for MG acini compared to other eyelid tissues. In rabbit, the dermis showed 5× and the muscle showed 2× more light transmission compared to MG (P < .001 and P < .001, respectively). For human, the muscle showed 14× and the tarsus showed 84× more light transmission compared to MG (P < .01 and P < .001, respectively). No specific spectral region of light absorption could be detected in either rabbit or human MG. Loss of light transmission in MG was localized to acini containing small lipid droplets, averaging 2.7 ±â€¯0.8 µm in diameter. CONCLUSIONS: The data suggest that light transmission is dramatically reduced in the acini due to light scattering by small lipid droplets, suggesting that Meibography detects active lipid synthesis in differentiating meibocytes.

Template Curvature Influences Cell Alignment to Create Improved Human Corneal Tissue Equivalents.

To accurately create corneal stromal equivalents with native-like structure and composition, a new biofunctionalized, curved template is developed that allows the precise orientation of cells and of their extracellular matrix. This template is the first demonstration that curvature alone is sufficient to induce the alignment of human corneal stromal cells, which in turn are able to biofabricate stromal tissue equivalents with cornea-like shape and composition. Specifically, tissues self-released from curved templates show a highly organized nanostructure, comprised of aligned collagen fibrils, significantly higher expression of corneal stroma-characteristic markers keratocan, lumican, decorin, ALDH3, and CHST6 (p = 0.012, 0.033, 0.029, 0.003, and 0.02, respectively), as well as significantly higher elastic modulus (p = 0.0001) compared with their planar counterparts. Moreover, curved tissues are shown to support the growth, stratification, and differentiation of human corneal epithelial cells in vitro, while maintaining their structural integrity and shape without any supporting carriers, scaffolds, or crosslinking agents. Together, these results demonstrate that corneal stromal cells can align and create highly organized, purposeful tissues by the influence of substrate curvature alone, and without the need of additional topographical cues. These findings can be important to further understand the mechanisms of corneal biosynthesis both in vitro and in vivo.

Immune Cells on the Corneal Endothelium of an Allogeneic Corneal Transplantation Rabbit Model.

Purpose: Corneal endothelial cell density undergoes a progressive decrease for many years after transplantation, eventually threatening patients with late endothelial failure. The purpose of this study was to investigate the possibility of an immunologic response in successfully grafted corneal endothelium. Methods: The corneal endothelium of patients who had undergone corneal transplantation was evaluated by specular microscopy. Rabbit models were subjected to penetrating keratoplasty (PK) with either syngeneic or allogeneic corneal transplants and Descemet's stripping endothelial keratoplasty (DSEK) with allogeneic corneal transplants. The presence of immune cells and expression of proinflammatory cytokines were determined by immunostaining. The corneal endothelium and immune cells were also evaluated by scanning electron microscopy. Results: Scanning slit contact specular microscopy of patients with no features of graft rejection revealed cell-like white dots on the grafted corneal endothelium. The corneal endothelium of the allogeneic PK and DSEK rabbit models displayed the presence of immune cells, including CD4+ T-helper cells, CD8+ cytotoxic T cells, CD20+ B lymphocytes, CD68+ macrophages, and neutrophils, but these immune cells were rarely observed in the syngeneic PK model. These immune cells also produced proinflammatory cytokines. Notably, some of the corneal endothelial cells situated near these immune cells exhibited features of apoptosis. Conclusions: T lymphocytes, B lymphocytes, macrophages, and neutrophils are present on the grafted corneal endothelium in both PK and DSEK allogeneic rabbit models. The potential involvement of immune cells as an underlying pathophysiology for late endothelial failure deserves further examination.