Human Cornea Research Articles

Morphological characterization of the human corneal epithelium by in vivo confocal laser scanning microscopy.

Regarding the growing interest and importance of understanding the cellular changes of the cornea in diseases, a quantitative cellular characterization of the epithelium is becoming increasingly important. Towards this, the latest research offers considerable improvements in imaging of the cornea by confocal laser scanning microscopy (CLSM). This study presents a pipeline to generate normative morphological data of epithelial cell layers of healthy human corneas. 3D in vivo CLSM was performed on the eyes of volunteers (n=25) with a Heidelberg Retina Tomograph II equipped with an in-house modified version of the Rostock Cornea Module implementing two dedicated piezo actuators and a concave contact cap. Image data were acquired with nearly isotropic voxel resolution. After image registration, stacks of en-face sections were used to generate full-thickness volume data sets of the epithelium. Beyond that, an image analysis algorithm quantified en-face sections of epithelial cells regarding the depth-dependent mean of cell density, area, diameter, aggregation (Clark and Evans index of aggregation), neighbor count and polygonality. Imaging and cell segmentation were successfully performed in all subjects. Thereby intermediated cells were efficiently recognized by the segmentation algorithm while efficiency for superficial and basal cells was reduced. Morphological parameters showed an increased mean cell density, decreased mean cell area and mean diameter from anterior to posterior (5,197.02 to 8,190.39 cells/mm2; 160.51 to 90.29 µm2; 15.9 to 12.3 µm respectively). Aggregation gradually increased from anterior to posterior ranging from 1.45 to 1.53. Average neighbor count increased from 5.50 to a maximum of 5.66 followed by a gradual decrease to 5.45 within the normalized depth from anterior to posterior. Polygonality gradually decreased ranging from 4.93 to 4.64 sides of cells. The neighbor count and polygonality parameters exhibited profound depth-dependent changes. This in vivo study demonstrates the successful implementation of a CLSM-based imaging pipeline for cellular characterization of the human corneal epithelium. The dedicated hardware in combination with an adapted image registration method to correct the remaining motion-induced image distortions followed by a dedicated algorithm to calculate characteristic quantities of different epithelial cell layers enabled the generation of normative data. Further significant effort is necessary to improve the algorithm for superficial and basal cell segmentation.

Chemical Cross-Linking of Corneal Tissue to Reduce Progression of Loss of Sight in Patients With Keratoconus.

PurposeWe aimed to develop a novel chemical cross-linker treatment for keratoconus by reacting dicarboxylic acid spacer molecules and amine functional groups on protein structure of the tissue using carbodi-imide chemistry. We propose this as an alternative to conventional cross-linking treatment for keratoconus.MethodsThe study involved optimization of the cross-linker formulation. Mechanical stiffness of ex vivo porcine and human corneas after application of the cross-linker was measured. Histochemical analysis was performed to record changes in gross morphology after cross-linker treatment on ex vivo porcine and human and in vivo rabbit corneas. Terminal deoxynucleotidyl transferase–mediated dUTP-X nick-end-labeling (TUNEL) staining was performed to study apoptotic effects of cross-linker. Cytotoxicity potential of cross-linker was evaluated by studying explant cultures for cellular outgrowth and immunostaining assays on porcine and human corneas after treatment.ResultsWe demonstrated a clinically relevant increase in stiffness in ex vivo experiments using porcine and human cornea without removal of corneal epithelium. Histological analysis showed no change in gross morphology of cornea and no evidence of apoptosis. In vivo treatment of rabbit eyes demonstrated initial thinning of corneal epithelium that recovered after seven days although with abnormal regularity of cells. Cellular outgrowth from corneal explant cultures after treatment further confirmed cell survival after treatment.ConclusionsThis chemical cross-linking of corneal tissue has potential advantages over current therapeutic options including lower cytotoxicity to stromal cells than ultraviolet A treatment.Translational RelevanceThe cross-linker has potential to become a treatment for keratoconus because it overcomes the need for procedures using specialized equipment and ensures accessibility to large populations.

Terahertz spectroscopy analysis of human corneal sublayers.

.Significance: Corneal diseases is a major cause of reversible blindness in the world. Monitoring the progression of human corneal edema or corneal scarring to prevent the disease entering into the end stage is crucial.Aim: We present a method for sensing human corneal composition at different depths, namely focused on the epithelium and stromal layer, using high-sensitivity terahertz (THz) broadband spectroscopy.Approach: From the proposed methodology, the THz temporal and absorption spectra of human corneas at different edema stages have been studied. THz wave signals were collected from the direct reflection and four other collection points along the THz wave propagation direction as reviewed from the simulation THz electrical field.Result: Our results show that the epithelium layer acts as a good barrier to maintain hydration level of the stroma, and the quality of the epithelium can be used to predict the level of corneal swelling in corneal edema. At the detection points near to the incident point, the THz frequency spectra demonstrated interference oscillation behavior. At the final edema observing time, results showed that the epithelium lose its barrier properties. The intactness of the epithelium can be used to predict the edema severity in the final stage. When the detection points are further away from the incident point, the THz spectra are believed to contain information from stromal layer. Stromal absorption spectra demonstrated correlation with optical coherence tomography thickness results.Conclusion: The hydration concentration from stromal layer was further quantitatively calculated. At the end of the experiment, all the corneal hydration levels reach to the same value which shows that the edema hydration has reached maximum saturation. The information of individual sublayers of the cornea is obtained by characterizing noninvasively with the use of THz spectroscopy. To our knowledge, this is the first report of using THz for noninvasive characterization of sublayers of the cornea.

Tumor necrosis factor-alpha and interferon-gamma induce inflammasome-mediated corneal endothelial cell death

PurposeChronic corneal endothelial cell (CEC) loss results in corneal edema and vision loss in conditions such as pseudophakic bullous keratopathy (PBK), Fuchs’ dystrophy, and corneal graft failure. Low CEC density has been associated with an elevation of intraocular pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α and interferon (INF)-γ. These cytokines are capable of triggering pyroptosis, a programmed cell death mechanism mediated by the inflammasome, prompting the activation of the pro-inflammatory cytokine interleukin (IL)-1β, the perpetuation of inflammation, and subsequent damage of corneal endothelial tissue. Therefore, the purpose of this study was to determine the deleterious contribution of the inflammasome and pyroptosis to CEC loss. MethodsCECs from human donor corneas were treated ex vivo with TNF-α and IFN-γ for 48 h. Levels of caspase-1 and IL-1β were then assayed by ELISA, and the expression of caspase-1 and gasdermin-D (GSDM-D) were confirmed by immunofluorescence. Endothelial cell damage was analyzed by a lactate dehydrogenase (LDH) release assay, and oxidative stress was determined by measuring the levels of reactive oxygen species (ROS) in the culture media. ResultsInflammasome activation and oxidative stress were elevated in CECs following exposure to TNF-α and IFN-γ, which resulted in cell death by pyroptosis as determined by LDH release which was inhibited by the caspase-1 inhibitor Ac-YVAD-cmk. ConclusionCEC death is induced by the pro-inflammatory cytokines TNF-α and IFN-γ, which contribute to inflammasome activation. Moreover, the inflammasome is a promising therapeutic target for the treatment of chronic CEC loss.

A single cell atlas of human cornea that defines its development, limbal progenitor cells and their interactions with the immune cells

PurposeSingle cell (sc) analyses of key embryonic, fetal and adult stages were performed to generate a comprehensive single cell atlas of all the corneal and adjacent conjunctival cell types from development to adulthood. MethodsFour human adult and seventeen embryonic and fetal corneas from 10 to 21 post conception week (PCW) specimens were dissociated to single cells and subjected to scRNA- and/or ATAC-Seq using the 10x Genomics platform. These were embedded using Uniform Manifold Approximation and Projection (UMAP) and clustered using Seurat graph-based clustering. Cluster identification was performed based on marker gene expression, bioinformatic data mining and immunofluorescence (IF) analysis. RNA interference, IF, colony forming efficiency and clonal assays were performed on cultured limbal epithelial cells (LECs). ResultsscRNA-Seq analysis of 21,343 cells from four adult human corneas and adjacent conjunctivas revealed the presence of 21 cell clusters, representing the progenitor and differentiated cells in all layers of cornea and conjunctiva as well as immune cells, melanocytes, fibroblasts, and blood/lymphatic vessels. A small cell cluster with high expression of limbal progenitor cell (LPC) markers was identified and shown via pseudotime analysis to give rise to five other cell types representing all the subtypes of differentiated limbal and corneal epithelial cells. A novel putative LPCs surface marker, GPHA2, expressed on the surface of 0.41% ± 0.21 of the cultured LECs, was identified, based on predominant expression in the limbal crypts of adult and developing cornea and RNAi validation in cultured LECs. Combining scRNA- and ATAC-Seq analyses, we identified multiple upstream regulators for LPCs and demonstrated a close interaction between the immune cells and limbal progenitor cells. RNA-Seq analysis indicated the loss of GPHA2 expression and acquisition of proliferative limbal basal epithelial cell markers during ex vivo LEC expansion, independently of the culture method used. Extending the single cell analyses to keratoconus, we were able to reveal activation of collagenase in the corneal stroma and a reduced pool of limbal suprabasal cells as two key changes underlying the disease phenotype. Single cell RNA-Seq of 89,897 cells obtained from embryonic and fetal cornea indicated that during development, the conjunctival epithelium is the first to be specified from the ocular surface epithelium, followed by the corneal epithelium and the establishment of LPCs, which predate the formation of limbal niche by a few weeks. ConclusionsOur scRNA-and ATAC-Seq data of developing and adult cornea in steady state and disease conditions provide a unique resource for defining genes/pathways that can lead to improvement in ex vivo LPCs expansion, stem cell differentiation methods and better understanding and treatment of ocular surface disorders.

A novel 3D culture model of fungal keratitis to explore host-pathogen interactions within the stromal environment

Fungal keratitis (FK) pathology is driven by both fungal growth and inflammation within the corneal stroma. Standard in vitro infection models ̶ involving co-culture of the pathogen and the corneal cells in tissue culture medium ̶ are sufficient to probe host responses to the fungus; however, they lack the physiological structure and nutrient composition of the stroma to accurately study fungal invasiveness and metabolic processes. We therefore sought to develop a culture model of FK that would allow for both host and fungal cell biology to be evaluated in parallel. Towards this end, we employed a previously described system in which primary human cornea fibroblasts (HCFs) are cultured on transwell membranes, whereupon they secrete a three-dimensional (3D) collagen matrix that resembles the human stroma. We demonstrated that two common mold agents of FK, Fusarium petroliphilum and Aspergillus fumigatus, penetrated into these constructs and caused a disruption of the collagen matrix that is characteristic of infection. HCF morphology appeared altered in the presence of fungus and electron microscopy revealed a clear internalization of fungal spores into these cells. Consistent with this apparent phagocyte-like activity of the HCFs, mRNA and protein levels for several pro-inflammatory cytokines/chemokines (including TNFα, IL-1β, IL-6, and IL-8) were significantly upregulated compared to uninfected samples. We similarly found an upregulation of several HCF metalloproteases (MMPs), which are enzymes that breakdown collagen during wound healing and may further activate pro-inflammatory signaling molecules. Finally, several fungal collagenase genes were upregulated during growth in the constructs relative to growth in tissue culture media alone, suggesting a fungal metabolic shift towards protein catabolism. Taken together, our results indicate that this 3D-stromal model provides a physiologically relevant system to study host and fungal cell pathobiology during FK.

Human Cadaveric Donor Cornea Derived Extra Cellular Matrix Microparticles for Minimally Invasive Healing/Regeneration of Corneal Wounds.

Biological materials derived from extracellular matrix (ECM) proteins have garnered interest as their composition is very similar to that of native tissue. Herein, we report the use of human cornea derived decellularized ECM (dECM) microparticles dispersed in human fibrin sealant as an accessible therapeutic alternative for corneal anterior stromal reconstruction. dECM microparticles had good particle size distribution (≤10 µm) and retained the majority of corneal ECM components found in native tissue. Fibrin–dECM hydrogels exhibited compressive modulus of 70.83 ± 9.17 kPa matching that of native tissue, maximum burst pressure of 34.3 ± 3.7 kPa, and demonstrated a short crosslinking time of ~17 min. The fibrin–dECM hydrogels were found to be biodegradable, cytocompatible, non-mutagenic, non-sensitive, non-irritant, and supported the growth and maintained the phenotype of encapsulated human corneal stem cells (hCSCs) in vitro. In a rabbit model of anterior lamellar keratectomy, fibrin–dECM bio-adhesives promoted corneal re-epithelialization within 14 days, induced stromal tissue repair, and displayed integration with corneal tissues in vivo. Overall, our results suggest that the incorporation of cornea tissue-derived ECM microparticles in fibrin hydrogels is non-toxic, safe, and shows tremendous promise as a minimally invasive therapeutic approach for the treatment of superficial corneal epithelial wounds and anterior stromal injuries.

Interferometric Ex Vivo Evaluation of the Spatial Changes to Corneal Biomechanics Introduced by Topographic CXL: A Pilot Study.

To determine the efficacy of interferometry for examining the spatial changes to the corneal biomechanical response to simulated intraocular pressure (IOP) fluctuations that occur after corneal cross-linking (CXL) applied in different topographic locations. Displacement speckle pattern interferometry (DSPI) was used to measure the total anterior surface displacement of human and porcine corneas in response to pressure variations up to 1 mm Hg from a baseline pressure of 16.5 mm Hg, both before and after CXL treatment, which was applied in isolated topographic locations (10-minute riboflavin soak [VibeX-Xtra; Avedro, Inc], 8-minute ultraviolet-A exposure at 15 mW/cm2). Alterations to biomechanics were evaluated by directly comparing the responses before and after treatment for each cornea. Before CXL, the corneal response to loading indicated spatial variability in mechanical properties. CXL treatments had a variable effect on the corneal response to loading dependent on the location of treatment, with reductions in regional displacement of up to 80% in response to a given pressure increase. Selectively cross-linking in different topographic locations introduces position-specific changes to mechanical properties that could potentially be used to alter the refractive power of the cornea. Changes to the biomechanics of the cornea after CXL are complex and appear to vary significantly depending on treatment location and initial biomechanics. Hence, further investigations are required on a larger number of corneas to allow the development of customized treatment protocols. In this study, laser interferometry was demonstrated to be an effective and valuable tool to achieve this. [J Refract Surg. 2021;37(4):263-273.].

A novel human donor cornea preservation cocktail incorporating a thermo-reversible gelation polymer (TGP), enhancing the corneal endothelial cell density maintenance and explant culture of corneal limbal cells

PurposeMcCarey-Kaufman’s (MK) medium and Optisol-GS medium are the most commonly employed media for human donor corneal preservation. In this study, we evaluated the preservation efficacy of discarded human donor corneas using a Thermo-reversible gelation polymer (TGP) added to these two media.MethodsThirteen human corneal buttons collected from deceased donors, which were otherwise discarded due to low endothelial cell density (ECD) were used. They were stored in four groups: MK medium, MK medium with TGP, Optisol-GS and Optisol-GS with TGP at 4 °C for 96 h. Slit lamp examination and specular microscopy were performed. Corneal limbal tissues from these corneas were then cultured using explant methodology one with and the other without TGP scaffold, for 21 days.ResultsMK + TGP and Optisol-GS + TGP preserved corneas better than without TGP, which was observed by maintenance of ECD which was significantly higher in Optisol-GS + TGP than MK + TGP (p-value = 0.000478) and corneal thickness remaining the same for 96 h. Viable corneal epithelial cells could be grown from the corneas stored only in MK + TGP and Optisol-GS + TGP. During culture, the TGP scaffold helped maintain the native epithelial phenotype and progenitor/stem cell growth was confirmed by RT-PCR characterization.ConclusionTGP reconstituted with MK and Optisol—GS media yields better preservation of human corneal buttons in terms of relatively higher ECD maintenance and better in vitro culture outcome of corneal limbal tissue. This method has the potential to become a standard donor corneal transportation-preservation methodology and it can also be extended to other tissue or organ transportation upon further validation.

Three-Dimensional Bioprinting for Tissue Engineering and Regenerative Medicine in Down Under: 2020 Australian Workshop Summary.

Three-Dimensional Bioprinting for Tissue Engineering and Regenerative Medicine in Down Under: 2020 Australian Workshop Summary.

How Implementing a Quality Management System at the LIONS Eye Bank Saar-Lor-Lux, Trier/Western Palatinate from 2006 to 2016 Impacted the Rate and Reasons for Discarding Human Organ-Cultured Corneas

The aim of this study was to explore how the implementation of the quality management system (QM; according to DIN EN ISO 9001 : 2008) at the LIONS Eye Bank impacted the rate and reasons for discarding donated corneas. The QM system was introduced in November 2010. The rate of and reasons for discarding cornea tissues at the LIONS Eye Bank Saar-Lor-Lux,Trier/Western Palatinate from 2006 to 2016 were analysed retrospectively. 4,140 corneas from 2,084 donors were analysed. 1,640 corneas were processed before the QM system had been introduced and 2,500 corneas thereafter. Main reasons for discarding cornea tissues were endothelial quality, contamination of the medium and a positive conjunctival swab. The data was extracted from clinical files and data filing systems, entered into a Microsoft Access database and analysed statistically with SPSS. Following the implementation of a QM system, the discard rate decreased significantly from 50.1% to 39.7% (p < 0.0001). Prior to the implementation of the QM system, 493 (30.1%) cornea tissues had been discarded due to endothelial quality, while this figure improved significantly to 604 (24.2%) after the QM system had been implemented (p < 0.0001). Contamination was the reason for discarding tissue in 173 (10.5%) cases before implementation of the QM system, and in 124 (5.0%) cases thereafter (p < 0.0001). Positive conjunctival swab was the reason for discarding tissue in 10 (0.6%) cases before the QM system had been implemented, and in 53 (2.1%) cases thereafter (p < 0.0001). Implementation of a QM system significantly reduced the rate of discarded donor tissue in the LIONS Eye Bank. Better management of contamination as a reason for discarding cornea tissues can be attributed to improved standards, protocols and training that are part of this QM system.

Comparative analysis of the regeneratory potential of blood derivatives on a cell model of corneal epithelium damage

The problem of the restoration of the epithelial layer after various modifications of keratoplasty is of great fundamental interest. In this regard, new methods of induction of regeneration are being developed; one of the promising approaches in this area is the use of autologous blood derivatives with a high regenerative potential. Objective: to compare the effect of 3 blood derivatives serum, platelet-rich plasma and plasma rich growth factors on the culture of corneal epithelial cells. The study was carried out on cells of the epithelium of the human cornea of passage 3. To confirm corneal affiliation, cells were typed for characteristic cytokeratins. The dynamics of migration was assessed in the test for wound healing of the monolayer. Proliferation was assessed by the results of the formazan test. Plasma rich growth factors had the greatest stimulating effect on cell proliferation. There were no significant differences between groups in the rate of wound healing of the monolayer. It was found that, in comparison with the control, all stimulants shift the morphological phenotype of cells to a more mature side. As a result of the study, it was shown that all 3 types of tested blood derivatives are promoters of corneal re-epithelialization. The use of drugs obtained from blood can positively influence the processes of epithelialization in persistent epithelial corneal defects, which requires further study.

Multimode fiber enables control of spatial coherence in Fourier-domain full-field optical coherence tomography for in vivo corneal imaging.

Fourier-domain full-field optical coherence tomography (FD-FF-OCT) has recently emerged as a fast alternative to point-scanning confocal OCT in eye imaging. However, when imaging the cornea with FD-FF-OCT, a spatially coherent laser can focus down on the retina to a spot that exceeds the maximum permissible exposure level. Here we demonstrate that a long multimode fiber with a small core can be used to reduce the spatial coherence of the laser and, thus, enable ultrafast in vivo volumetric imaging of the human cornea without causing risk to the retina.

All-printed stretchable corneal sensor on soft contact lenses for noninvasive and painless ocular electrodiagnosis

Electroretinogram examinations serve as routine clinical procedures in ophthalmology for the diagnosis and management of many ocular diseases. However, the rigid form factor of current corneal sensors produces a mismatch with the soft, curvilinear, and exceptionally sensitive human cornea, which typically requires the use of topical anesthesia and a speculum for pain management and safety. Here we report a design of an all-printed stretchable corneal sensor built on commercially-available disposable soft contact lenses that can intimately and non-invasively interface with the corneal surface of human eyes. The corneal sensor is integrated with soft contact lenses via an electrochemical anchoring mechanism in a seamless manner that ensures its mechanical and chemical reliability. Thus, the resulting device enables the high-fidelity recording of full-field electroretinogram signals in human eyes without the need of topical anesthesia or a speculum. The device, superior to clinical standards in terms of signal quality and comfortability, is expected to address unmet clinical needs in the field of ocular electrodiagnosis.

Toward electron-beam sterilization of a pre-assembled Boston keratoprosthesis

PurposeTo evaluate the effects of electron-beam (E-beam) irradiation on the human cornea and the potential for E-beam sterilization of Boston keratoprosthesis (BK) devices when pre-assembled with a donor cornea prior to sterilization. MethodsHuman donor corneas and corneas pre-assembled in BK devices were immersed in recombinant human serum albumin (rHSA) media and E-beam irradiated at 25 kGy. Mechanical (tensile strength and modulus, and compression modulus), chemical, optical, structural, and degradation properties of the corneal tissue after irradiation and after 6 months of preservation were evaluated. ResultsThe mechanical evaluation showed that E-beam irradiation enhanced the tensile and compression moduli of human donor corneas, with no impact on their tensile strength. By chemical and mechanical analysis, E-beam irradiation caused a minor degree of crosslinking between collagen fibrils. No ultrastructural changes due to E-beam irradiation were observed. E-beam irradiation slightly increased the stability of the cornea against collagenase-induced degradation and had no impact on glucose diffusion. The optical evaluation showed transparency of the cornea was maintained. E-beam irradiated corneal tissues and BK-cornea pre-assembled devices were stable for 6 months after room-temperature preservation. ConclusionsE-beam irradiation generated no detrimental effects on the corneal tissues or BK-cornea pre-assembled devices and improved native properties of the corneal tissue, enabling prolonged preservation at room temperature. The pre-assembly of BK in a donor cornea, followed by E-beam irradiation, offers the potential for an off-the-shelf, ready to implant keratoprosthesis device.

Diffusion-based degeneration of the collagen reinforcement in the pathologic human cornea

We describe a multiphysics model of the collagen structure of the cornea undergoing a progressive localized reduction of the stiffness, preluding to the development of ectasia and keratoconus. The architecture of the stromal collagen is assumed to follow the simplified two-family model proposed in Pandolfi et al. (A microstructural model of cross-link interaction between collagen fibrils in the human cornea. Philos Trans R Soc A 377:20180079, 2019), where the mechanical stiffness of the structure is supplied by transversal bonds within the fibrils of the same family (inter-crosslink bonds) and across the fibrils of the two families (intra-crosslink bonds). In Pandolfi et al. (A microstructural model of cross-link interaction between collagen fibrils in the human cornea. Philos Trans R Soc A 377:20180079, 2019), it was shown that the loss of the spherical shape due to the protrusion of a cone can be ascribed to the mechanical weakening of the intra-crosslink bonds in the central region of the collagen structure. In the present study, the reduction of bond stiffness is coupled to an evolutive pathologic phenomenon, modeled as a reaction–diffusion process of a normalized scalar field. We assume that the scalar field is a concentration-like measure of the degeneration of the chemical bonds stabilizing the structural collagen. We follow the evolution of the mechanical response of the system in terms of shape change, according to the propagation of the degeneration field, and identify the critical loss of mechanical stability resulting in the typical bulging of keratoconus corneas.

Safety and efficacy of combination of suberoylamilide hydroxyamic acid and mitomycin C in reducing pro-fibrotic changes in human corneal epithelial cells

Corneal haze post refractive surgery is prevented by mitomycin c (MMC) treatment though it can lead to corneal endothelial damage, persistent epithelial defects and necrosis of cells. Suberanilohydroxamic acid (SAHA) however has been proposed to prevent corneal haze without any adverse effects. For clinical application we have investigated the short and long term outcome of cells exposed to SAHA. Human donor cornea, cultured limbal epithelial cells, corneal rims and lenticules were incubated with SAHA and MMC. The cells/tissue was then analyzed by RT-qPCR, immunofluorescence and western blot for markers of apoptosis and fibrosis. The results reveal that short term exposure of SAHA and SAHA + MMC reduced apoptosis levels and increased αSMA expression compared to those treated with MMC. Epithelial cells derived from cultured corneal rim that were incubated with the MMC, SAHA or MMC + SAHA revealed enhanced apoptosis, reduced levels of CK3/CK12, ∆NP63 and COL4A compared to other treatments. In SAHA treated lenticules TGFβ induced fibrosis was reduced. The results imply that MMC treatment for corneal haze has both short term and long term adverse effects on cells and the cellular properties. However, a combinatorial treatment of SAHA + MMC prevents expression of corneal fibrotic markers without causing any adverse effect on cellular properties.

Development of a terahertz time-domain scanner for topographic imaging of spherical targets.

Topographical abnormality in corneal tissue is a common diagnostic marker for many eye diseases and injuries. Using an asynchronous optical sampling terahertz time-domain spectroscopy setup, we developed a non-contact and normal-incidence imaging system to measure topographic changes along the surface of spherical samples. We obtained orthogonal 1D scans of calibration spheres to evaluate the minimum axial resolution of our system. We determined the axial and spatial resolution of the scanner using 3D-printed spherical cross and Boehler star targets. Furthermore, we characterized the asymmetrical performance of the scanner due to the use of an off-axis parabolic mirror. Finally, we developed an edge-detection filter to aid with improving the topographic scans. We showed that when imaging samples were comparable in size to the human cornea, the axial and spherical spatial resolutions were limited to about 15 µm (∼λ/67) and 1 mm, respectively.

Epithelial Regeneration in Human Corneas Preserved in an Active Storage Machine.

PurposeTo characterize the corneal epithelium (CE) and limbal epithelium (LE) of human corneas stored in an innovative active storage machine (ASM) after a period of organ culture (OC).MethodsCorneas unsuitable for graft and stored in a standard commercial OC medium for 2 to 5 weeks were transferred into our ASM for 14 days. The ASM actively maintained an overpressure on the endothelial side (20 mm Hg) while ensuring medium renewal. We compared three modalities of storage in the ASM's epithelial chamber: (1) alternating exposure to a supplemental hormonal epithelial medium (SHEM) and air (air-lifting), (2) continuous immersion in SHEM, and (3) continuous immersion in OC medium. Passive immersion of the whole cornea in OC medium or of the CE in SHEM with or without airlifting served as controls. Paired corneas were used for better comparability. Histology, differentiation (by immunolabeling), and ultrastructure were analyzed at the end.ResultsThe ASM with air-lifting was most effective in regenerating a pluristratified and differentiated CE (apical ZO-1 and MUC16 staining and regeneration of the glycocalyx). In addition, the LE was stratified with preserved expression of ABCB5. The ASM with immersion in SHEM or OC medium gave a less stratified and differentiated CE. In the three control groups, the epithelia, when present, were paucistratified and less differentiated.ConclusionsIn human corneas previously stored in OC, the ASM regenerates a CE with differentiation characteristics close to normal.Translational RelevanceRegeneration of the epithelium of human corneas discarded by eye banks will increase tissue availability for research.

Evaluation of Marine Agarose Biomaterials for Tissue Engineering Applications.

Five agarose types (D1LE, D2LE, LM, MS8 and D5) were evaluated in tissue engineering and compared for the first time using an array of analysis methods. Acellular and cellular constructs were generated from 0.3–3%, and their biomechanical properties, in vivo biocompatibility (as determined by LIVE/DEAD, WST-1 and DNA release, with n = 6 per sample) and in vivo biocompatibility (by hematological and biochemical analyses and histology, with n = 4 animals per agarose type) were analyzed. Results revealed that the biomechanical properties of each hydrogel were related to the agarose concentration (p < 0.001). Regarding the agarose type, the highest (p < 0.001) Young modulus, stress at fracture and break load were D1LE, D2LE and D5, whereas the strain at fracture was higher in D5 and MS8 at 3% (p < 0.05). All agaroses showed high biocompatibility on human skin cells, especially in indirect contact, with a correlation with agarose concentration (p = 0.0074 for LIVE/DEAD and p = 0.0014 for WST-1) and type, although cell function tended to decrease in direct contact with highly concentrated agaroses. All agaroses were safe in vivo, with no systemic effects as determined by hematological and biochemical analysis and histology of major organs. Locally, implants were partially encapsulated and a pro-regenerative response with abundant M2-type macrophages was found. In summary, we may state that all these agarose types can be safely used in tissue engineering and that the biomechanical properties and biocompatibility were strongly associated to the agarose concentration in the hydrogel and partially associated to the agarose type. These results open the door to the generation of specific agarose-based hydrogels for definite clinical applications such as the human skin, cornea or oral mucosa.