Dynamic changes of endothelial and stromal cilia are required for the maintenance of corneal homeostasis.

Primary cilia are distributed extensively within the corneal epithelium and endothelium. However, the presence of cilia in the corneal stroma and the dynamic changes and roles of endothelial and stromal cilia in corneal homeostasis remain largely unknown. Here, we present compelling evidence for the presence of primary cilia in the corneal stroma, both in vivo and in vitro. We also demonstrate dynamic changes of both endothelial and stromal cilia during corneal development. In addition, our data show that cryoinjury triggers dramatic cilium formation in the corneal endothelium and stroma. Furthermore, depletion of cilia in mutant mice lacking intraflagellar transport protein 88 compromises the corneal endothelial capacity to establish the effective tissue barrier, leading to an upregulation of α-smooth muscle actin within the corneal stroma in response to cryoinjury. These observations underscore the essential involvement of corneal endothelial and stromal cilia in maintaining corneal homeostasis and provide an innovative strategy for the treatment of corneal injuries and diseases.

Acetylcholine decreases formation of myofibroblasts and excessive extracellular matrix production in an in vitro human corneal fibrosis model.

Acetylcholine (ACh) has been reported to play various physiological roles, including wound healing in the cornea. Here, we study the role of ACh in the transition of corneal fibroblasts into myofibroblasts, and in consequence its role in the onset of fibrosis, in an in vitro human corneal fibrosis model. Primary human keratocytes were obtained from healthy corneas. Vitamin C (VitC) and transforming growth factor-ß1 (TGF-ß1) were used to induce fibrosis in corneal fibroblasts. qRT-PCR and ELISA analyses showed that gene expression and production of collagen I, collagen III, collagen V, lumican, fibronectin (FN) and alpha-smooth muscle actin (α-SMA) were reduced by ACh in quiescent keratocytes. ACh treatment furthermore decreased gene expression and production of collagen I, collagen III, collagen V, lumican, FN and α-SMA during the transition of corneal fibroblasts into myofibroblasts, after induction of fibrotic process. ACh inhibited corneal fibroblasts from developing contractile activity during the process of fibrosis, as assessed with collagen gel contraction assay. Moreover, the effect of ACh was dependent on activation of muscarinic ACh receptors. These results show that ACh has an anti-fibrotic effect in an in vitro human corneal fibrosis model, as it negatively affects the transition of corneal fibroblasts into myofibroblasts. Therefore, ACh might play a role in the onset of fibrosis in the corneal stroma.

Development, structure, and bioengineering of the human corneal stroma: A review of collagen-based implants.

Bio-engineering technologies are currently used to produce biomimetic artificial corneas that should present structural, chemical, optical, and biomechanical properties close to the native tissue. These properties are mainly supported by the corneal stroma which accounts for 90% of corneal thickness and is mainly made of collagen type I. The stromal collagen fibrils are arranged in lamellae that have a plywood-like organization. The fibril diameter is between 25 and 35 nm and the interfibrillar space about 57 nm. The number of lamellae in the central stroma is estimated to be 300. In the anterior part, their size is 10-40 µm. They appear to be larger in the posterior part of the stroma with a size of 60-120 µm. Their thicknesses also vary from 0.2 to 2.5 µm. During development, the acellular corneal stroma, which features a complex pattern of organization, serves as a scaffold for mesenchymal cells that invade and further produce the cellular stroma. Several pathways including Bmp4, Wnt/ß-catenin, Notch, retinoic acid, and TGF-ß, in addition to EFTFs including the mastering gene Pax-6, are involved in corneal development. Besides, retinoic acid and TGF- ß seem to have a crucial role in the neural crest cell migration in the stroma. Several technologies can be used to produce artificial stroma. Taking advantage of the liquid-crystal properties of acid-soluble collagen, it is possible to produce transparent stroma-like matrices with native-like collagen I fibrils and plywood-like organization, where epithelial cells can adhere and proliferate. Other approaches include the use of recombinant collagen, cross-linkers, vitrification, plastically compressed collagen or magnetically aligned collagen, providing interesting optical and mechanical properties. These technologies can be classified according to collagen type and origin, presence of telopeptides and native-like fibrils, structure, and transparency. Collagen matrices feature transparency >80% for the appropriate 500-µm thickness. Non-collagenous matrices made of biopolymers including gelatin, silk, or fish scale have been developed which feature interesting properties but are less biomimetic. These bioengineered matrices still need to be colonized by stromal cells to fully reproduce the native stroma.

Reconstruction of a tissue-engineered cornea with porcine corneal acellular matrix as the scaffold.

Interest in developing tissue-engineered cornea has increased with the decrease in the supply of donor tissue. The aim of the present study was to investigate the feasibility and method of reconstructing corneal equivalents with porcine corneal acellular matrix as the scaffold in a dynamic culturing system. Applying the detergent Triton X-100 (1%) and a freeze-drying process, porcine corneas were decellularized and prepared as a scaffold, and hematoxylin-eosin staining and scanning electron microscopy showed no cells in the decellularized stroma. In order to measure the in vivo biocompatibility, part of the scaffold was transplanted into a pocket of rabbit corneal stroma and observed for 3 months. No sign of rejection were observed, and the acellular matrix gradually integrated in the rabbit cornea, indicating that the scaffold had good biocompatibility. To reconstruct a tissue-engineered cornea, cultured rabbit keratocytes were seeded into the scaffold. After 1 week of culture in a culturing vessel, rabbit epithelial and endothelial cells were seeded on both sides of the stroma, respectively. The reconstructed cornea consisted of three layers in histological structure: the epithelium, stoma and endothelium. Stratified epithelial cells formed on the surface, which were cytokeratin 3 positive in the cytoplasm; endothelial cell monolayers were located on the inner side, and pump-related aquaporin 1 was found in the cells. These results confirmed that the corneal acellular matrix can be used as a scaffold for tissue-engineered cornea, and a biological corneal equivalent can be reconstructed in a dynamic culturing system.

Evaluation of Megacell MEM as a storage medium for corneas destined for transplantation.

PURPOSE: Gibco's Minimum Essential Medium with Earle's salts and HEPES supplemented with glutamine, antibiotics (EB MEM) and 2% foetal calf serum (FCS) is used in European eye banks to store corneas. Although FCS is important to endothelial cell survival in this medium, it is a potential biohazard. Megacell MEM, formulated to reduce the FCS requirement of cells by a factor of 5, has therefore been evaluated as a corneal storage medium. METHODS: Corneal stromal and epithelial cells were incubated in Megacell MEM (serum-free or 2% FCS) to assess their viability in these media. Endothelial cell densities of paired corneas held in either EB MEM 2% FCS or Megacell MEM (serum-free or 2% FCS) were measured over 5 weeks. Discs subsequently punched from the centre of these corneas were weighed, dried and reweighed to determine hydration levels. RESULTS: Both corneal stromal and epithelial cells proliferated in Megacell MEM 2% FCS. Relative to EB MEM, 2% FCS Megacell MEM prolonged the viability of corneal endothelial cells and improved their morphological appearance, irrespective of whether it contained FCS or not. This was independent of corneal swelling. CONCLUSION: Serum-free Megacell MEM is a better storage medium than EB MEM 2% FCS for corneas destined for transplantation.

Femtosecond Laser-Assisted Surgery for Implantation of Bioengineered Corneal Stroma to Promote Corneal Regeneration.

The femtosecond laser has achieved widespread use in ophthalmology owing to its ability to deliver focused high energy that is rapidly dissipated and thereby does not damage surrounding tissue outside the precise focal region. Extremely accurate and smooth cuts can be made by the laser, enabling a range of applications in anterior segment surgery. Minimally invasive corneal surgical procedures can be performed using the femtosecond laser, and here we describe the application of such procedures to improve implantation of bioengineered materials into the cornea. Bioengineered corneal tissue, including the collagenous corneal stroma, promises to provide a virtually unlimited supply of biocompatible tissue for treating multiple causes of corneal blindness globally, thereby circumventing problems of donor tissue shortages and access to tissue banking infrastructure. Optimal implantation of bioengineered materials, however, is required, in order to facilitate postoperative wound healing for the maintenance of corneal transparency and avoidance of postoperative complications such as scarring, inflammation, and neovascularization. Moreover, the avoidance of a detrimental physiological physiological wound healing response is critical for facilitating the corneal stromal regeneration enabled by the bioengineered stroma. Without proper implantation, the tissue response will favor inflammation and pathologic processes instead of quiescent keratocyte migration and new collagen production. Here we describe several procedures for optimized biomaterial implantation into the corneal stroma, that facilitate rapid wound healing and regenerative restoration of corneal transparency without the use of human donor tissue. A step-by-step methodology is provided for the use of the femtosecond laser and associated techniques, to enable seamless integration of bioengineered materials into the corneal stroma.

Stereolithography 3D Bioprinting Method for Fabrication of Human Corneal Stroma Equivalent.

3D bioprinting technology is a promising approach for corneal stromal tissue regeneration. In this study, gelatin methacrylate (GelMA) mixed with corneal stromal cells was used as a bioink. The visible light-based stereolithography (SLA) 3D bioprinting method was utilized to print the anatomically similar dome-shaped structure of the human corneal stroma. Two different concentrations of GelMA macromer (7.5 and 12.5%) were tested for corneal stroma bioprinting. Due to high macromer concentrations, 12.5% GelMA was stiffer than 7.5% GelMA, which made it easier to handle. In terms of water content and optical transmittance of the bioprinted scaffolds, we observed that scaffold with 12.5% GelMA concentration was closer to the native corneal stroma tissue. Subsequently, cell proliferation, gene and protein expression of human corneal stromal cells encapsulated in the bioprinted scaffolds were investigated. Cytocompatibility in 12.5% GelMA scaffolds was observed to be 81.86 and 156.11% at day 1 and 7, respectively, which were significantly higher than those in 7.5% GelMA scaffolds. Elongated corneal stromal cells were observed in 12.5% GelMA samples after 7 days, indicating the cell attachment, growth, and integration within the scaffold. The gene expression of collagen type I, lumican and keratan sulfate increased over time for the cells cultured in 12.5% GelMA scaffolds as compared to those cultured on the plastic tissue culture plate. The expression of collagen type I and lumican were also visualized using immunohistochemistry after 28 days. These findings imply that the SLA 3D bioprinting method with GelMA hydrogel bioinks is a promising approach for corneal stroma tissue biofabrication.

Gene Editing for Corneal Stromal Regeneration.

CRISPR/Cas9 gene editing holds the promise of sequence-specific alteration of the genome to achieve therapeutic benefit in the treated tissue. Cas9 is an RNA-guided nuclease in which the sequence of the RNA can be altered to match the desired target. However, care must be taken in target choice and RNA guide design to ensure both maximum on-target and minimum off-target activity. The cornea is an ideal tissue for gene therapy due to its small surface area, accessibility, immune privilege, avascularity, and ease of visualization. Herein, we describe the design, testing, and delivery of Cas9 and guide RNAs to target genes expressed in the cornea.

The Self-assembly Approach as a Tool for the Tissue Engineering of a Bi-lamellar Human Cornea.

Tissue engineering is a flourishing field of regenerative medicine that allows the reconstruction of various tissues of our body, including the cornea. In addition to addressing the growing need for organ transplants, such tissue-engineered substitutes may also serve as good in vitro models for fundamental and preclinical studies. Recent progress in the field of corneal tissue engineering has led to the development of new technologies allowing the reconstruction of a human bi-lamellar cornea. One unique feature of this model is the complete absence of exogenous material. Indeed, these human corneal equivalents are exclusively composed of untransformed human corneal fibroblasts (hCFs) entangled in their own extracellular matrix, as well as untransformed human corneal epithelial cells (hCECs), both of which isolated from donor corneas. The reconstructed human bi-lamellar cornea thereby exhibits a well-organized stroma as well as a well-differentiated epithelium. This chapter describes the methods used for the isolation and culture of hCFs, the production and assembly of hCFs stromal sheets, the seeding of hCECs, and the maturation of the tissue-engineered cornea.

Formation of Corneal Stromal-Like Assemblies Using Human Corneal Fibroblasts and Macromolecular Crowding.

Tissue engineering by self-assembly allows for the formation of living tissue substitutes, using the cells' innate capability to produce and deposit tissue-specific extracellular matrix. However, in order to develop extracellular matrix-rich implantable devices, prolonged culture time is required in traditionally utilized dilute ex vivo microenvironments. Macromolecular crowding, by imitating the in vivo tissue density, dramatically accelerates biological processes, resulting in enhanced and accelerated extracellular matrix deposition. Herein, we describe the ex vivo formation of corneal stromal-like assemblies using human corneal fibroblasts and macromolecular crowding.

Development and Validation of a 3D In Vitro Model to Study the Chemotactic Behavior of Corneal Stromal Fibroblasts.

Chemotaxis plays a pivotal role in crucial biological phenomena including immune response, cancer metastasis, and wound healing. Although many chemotaxis assays have been developed to better understand these multicomplex biological mechanisms, most of them have serious limitations mainly due to the poor representation of native three-dimensional (3D) microenvironment. Here, we describe a method to develop and validate a novel 3D in vitro chemotaxis model to study the migration of corneal fibroblasts through a stromal equivalent. A hydrogel was used that contained gelatin microspheres loaded with platelet-derived growth factor-BB (PDGF-BB) in the inner section and corneal fibroblasts in the outer section. The cell migration toward the chemical stimuli over time can be monitored via confocal microscopy. The development of this in vitro model can be used for both qualitative and quantitative examinations of chemotaxis.

Morphometrics of corneal growth in chicks raised in constant light.

In this study we wish to augment our understanding of the effect of environment on corneal growth and morphology. To understand how corneal development of chicks raised in constant light differs from that of 'normal' eyes exposed to cyclic periods of light and dark, white Leghorn chicks were raised under either constant light (approximately 700 lux at cage top) or in 12 h light/12 h dark conditions for up to 12 weeks after hatching. To determine whether corneal expansion is uniform, some birds from each group received corneal tattoos for periodic photographic assessment. By 16 days of age, constant light corneas weighed less than light/dark regimen corneas [7.39 +/- 0.35 mg (SE) vs. 8.47 mg +/- 0.26 mg SE wet weight, P < or = 0.05], and corresponding differences were seen in corneal dry weights. Spatial expansion of the corneal surface was uniform in both groups, but the rate of expansion was slower in constant light chicks [0.0327 +/- 0.009 (SE) vs. 0.144 +/- 0.018 (SE) mm(2) day(-1) for normal chicks, P < or = 0.001]. At 1 day of age, there were 422 +/- 12.5 (SE) stromal cells 0.01 mm(-2) in the central cornea and 393 +/- 21.5 (SE) stromal cells 0.01 mm(-2 )peripherally. Although this difference is not statistically significant, the cell densities in the central cornea were always larger than those of the peripheral cornea in all eight measurements over a 10.5-week period, and this difference is significant (P < or = 0.008, binomial test). Light/dark regimen birds show no such consistent difference in cell densities between central and peripheral corneas. Thus, the density distribution of corneal stromal cells of chicks grown in constant light differs from that of normal chicks. Taken together, all these observations suggest that diurnal cycles of light and darkness are necessary for normal corneal growth.

Functional human corneal equivalents constructed from cell lines.

Human corneal equivalents comprising the three main layers of the cornea (epithelium, stroma, and endothelium) were constructed. Each cellular layer was fabricated from immortalized human corneal cells that were screened for use on the basis of morphological, biochemical, and electrophysiological similarity to their natural counterparts. The resulting corneal equivalents mimicked human corneas in key physical and physiological functions, including morphology, biochemical marker expression, transparency, ion and fluid transport, and gene expression. Morphological and functional equivalents to human corneas that can be produced in vitro have immediate applications in toxicity and drug efficacy testing, and form the basis for future development of implantable tissues.

Three-dimensional imaging of the extracellular matrix and cell interactions in the developing prenatal mouse cornea.

As the outer lens in the eye, the cornea needs to be strong and transparent. These properties are governed by the arrangement of the constituent collagen fibrils, but the mechanisms of how this develops in mammals is unknown. Using novel 3-dimensional scanning and conventional transmission electron microscopy, we investigated the developing mouse cornea, focusing on the invading cells, the extracellular matrix and the collagen types deposited at different stages. Unlike the well-studied chick, the mouse cornea had no acellular primary stroma. Collagen fibrils initially deposited at E13 from the presumptive corneal stromal cells, become organised into fibril bundles orthogonally arranged between cells. Extensive cell projections branched to adjacent stromal cells and interacted with the basal lamina and collagen fibrils. Types I, II and V collagen were expressed from E12 posterior to the surface ectoderm, and became widespread from E14. Type IX collagen localised to the corneal epithelium at E14. Type VII collagen, the main constituent of anchoring filaments, was localised posterior to the basal lamina. We conclude that the cells that develop the mouse cornea do not require a primary stroma for cell migration. The cells have an elaborate communication system which we hypothesise helps cells to align collagen fibrils.

Neonatal development of the corneal stroma in wild-type and lumican-null mice.

PURPOSE: Between days 8 and 14 of neonatal development, the corneal stroma of the mouse undergoes critical changes in tissue thickness, cell density, and light scattering. The authors investigate the stromal matrix structure in wild-type and lumican-deficient corneas in this developmental phase. METHODS: Wild-type (n = 44) and lumican-deficient (n = 42) mouse corneas at neonatal days 8, 10, 12, and 14 were investigated by synchrotron x-ray diffraction to establish the average collagen fibril spacing, average collagen fibril diameter, and level of fibrillar organization in the stromal matrix. RESULTS: Collagen interfibrillar spacing in the normal mouse cornea became more closely packed between days 8 and 14, though not significantly so. In lumican-null mice, interfibrillar spacing was significantly elevated at days 8, 10, and 12, but not day 14, compared with that in wild-type mice. At all stages investigated, collagen fibrils were, on average, marginally thinner than normal in lumican-null mutants, and the spatial distribution of the fibrils was less well organized. CONCLUSIONS: Transient thickening of the corneal stroma of the normal mouse at eye opening is probably not caused by widespread, homogeneous rearrangement of collagen fibrils but more likely by a temporary increase in cell or stromal "lake" volume. Lumican, structurally influential in adult mouse corneas, is also a key molecule in the neonatal development of the stromal matrix.

Neonatal corneal stromal development in the normal and lumican-deficient mouse.

PURPOSE: The purpose of this study was to characterize temporally stromal growth and transparency in lumican-deficient and normal neonatal mice. METHODS: Lumican-deficient mice and CD1 wild-type mice were evaluated by in vivo confocal microscopy through-focusing (CMTF) to quantify stromal and epithelial thickness and corneal light-scattering and by laser scanning CM to determine density of keratocytes from 1 day to 12 weeks after birth. RESULTS: CD1 corneas showed a rapid loss of light-scattering, decreasing by 50% from day 1 to day 12, that paralleled a 60% decrease in density of keratocytes. By contrast, the stroma demonstrated a marked swelling from day 8 to day 12, followed by thinning at day 14. Compared to corneas from CD1 mice, lumican-deficient corneas showed significantly increased (P < 0.05) light-scattering beginning at week 3 that remained elevated above wild-type levels for the duration of the study. Stromal development was also markedly altered, with thinning detected at week 3, followed by no detectable stromal growth for the duration of the study. Density of keratocytes was significantly increased, but the total cell number was similar compared with that in the wild-type cornea, suggesting no effect on keratocyte differentiation. CONCLUSIONS: Development of normal neonatal corneal transparency appears related to changes in density of keratocytes. The stroma, however, undergoes a marked swelling and thinning at the time of eyelid opening (days 8-14). In the lumican-deficient mouse, stromal swelling is abolished, indicating that this critical phase in stromal development is lumican dependent and essential for normal stromal growth and maintenance of stromal transparency.

Developmental changes in proteoglycans of rabbit corneal stroma.

Proteoglycans have been extracted from rabbit corneal stromas at developmental stages from fetal to adult. Ion exchange fractionation and gel chromatography show that proteodermatan sulfates decrease in sulfation and relative amount and proteokeratan sulfates increase in sulfation and relative amount during development. There are small increases in size of all of the proteoglycans up to 2 weeks after birth, and the final adult composition is achieved by 8 weeks.

Immunolocalization of type VI collagen in developing and healing rabbit cornea.

We have localized type VI collagen in normal developing and corneal scar tissue. Indirect immunofluorescence showed that type VI collagen was distributed throughout the normal stroma and most of the scar. No fluorescence was detected along the posterior margin of the scar and in a retrocorneal membrane continuous with the scar. Since the corneal endothelium in rabbits contributes to the formation of scar tissue and retrocorneal membrane, our observations suggest that the endothelium does not synthesize type VI collagen. Indirect immunoelectron microscopy showed that type VI collagen was located abundantly between collagen fibrils as fine filamentous structures containing beads with a periodicity of 100 nm, consistent with published observations of other tissues. Because these filaments are more prominent when stained with ruthenium red, and predigestion of tissue with Chondroitinase ABC enhances binding of monoclonal antibody to type VI collagen, proteoglycans probably are associated with this collagen in the cornea. Ultrastructural observations supported by previous biochemical analyses show that the proportion of type VI collagen to fibrillar collagen is smaller in scar tissue compared with fetal cornea. The abundance of type VI collagen and its distribution and association with proteoglycans in rabbit corneal tissues suggest that this macromolecule plays a role in the tensile strength and transparency of the stroma.

mRNA levels of alpha1(VI) collagen, alpha1(XII) collagen, and beta ig in rabbit cornea during normal development and healing.

PURPOSE: Type VI and XII collagens and beta ig, major components of the interfibrillar matrix, may maintain proper spacing among collagen fibrils, necessary for corneal transparency. During normal corneal stroma development and healing, changes in mRNA levels of these proteins were measured to determine whether differences in steady state levels are indicative of the unique structure produced by each corneal tissue. METHODS: A full-thickness excision wound was made in each cornea of six adult rabbits and allowed to heal for 1, 2, or 4 weeks. Scar tissue from two rabbits (four scars) were used from each time period and processed for RNA extraction. Total RNA from 23-day-old fetal rabbit corneas (equivalent to approximately 1 week of stromal development) and 8-day-old neonate corneas (equivalent to approximately 3.5 weeks of stromal development) was also extracted. Relative quantities of alpha1(VI) collagen, alpha1(XII) collagen, beta ig, and beta-actin mRNAs were determined by competitive reverse transcriptase-polymerase chain reaction. Glyceraldehyde-3-phosphate dehydrogenase was used as a housekeeping gene. RESULTS: Increased mRNA levels for alpha1(VI) and alpha1(XII) collagens, beta ig, and beta-actin were seen during the first 2 weeks of healing and were followed by a decrease in 4-week-old scars. Similar increases were seen in fetal corneas with a further increase in the neonate. Differences in the beta ig mRNA levels relative to that of alpha1(XII) collagen in fetal stroma and in comparison with 1-week-old wounds suggest a higher production of beta ig in early healing tissue. CONCLUSIONS: Alterations of mRNA levels during healing and development are consistent with the cellular events and deposition of extracellular matrices in these corneal tissues. Assuming that extracellular matrix protein production is regulated at the transcriptional level, relative changes in beta ig and collagen mRNA levels reflect differences in protein deposition in early fetal and healing tissues. This is consistent with differences in the organization of the interfibrillar matrices of these tissues and their transparency.