Human Cornea Research Articles

Endogenous TSG-6 modulates corneal inflammation following chemical injury

PurposeTumor necrosis factor (TNF)-stimulated gene-6 (TSG-6) is upregulated in various pathophysiological contexts, where it has a diverse repertoire of immunoregulatory functions. Herein, we investigated the expression and function of TSG-6 during corneal homeostasis and after injury. MethodsHuman corneas, eyeballs from BALB/c (TSG-6+/+), TSG-6+/− and TSG-6−/− mice, human immortalized corneal epithelial cells and murine corneal epithelial progenitor cells were prepared for immunostaining and real time PCR analysis of endogenous expression of TSG-6. Mice were subjected to unilateral corneal debridement or alkali burn (AB) injuries and wound healing assessed over time using fluorescein stain, in vivo confocal microscopy and histology. ResultsTSG-6 is endogenously expressed in the human and mouse cornea and established corneal epithelial cell lines and is upregulated after injury. A loss of TSG-6 has no structural and functional effect in the cornea during homeostasis. No differences were noted in the rate of corneal epithelial wound closure between BALB/c, TSG-6+/− and TSG-6−/− mice. TSG-6−/− mice presented decreased inflammatory response within the first 24 h of injury and accelerated corneal wound healing following AB when compared to control mice. ConclusionTSG-6 is endogenously expressed in the cornea and upregulated after injury where it propagates the inflammatory response following chemical injury.

Novel strategy for multi-material 3D bioprinting of human stem cell based corneal stroma with heterogenous design

Three-dimensional (3D) bioprinting offers an automated, customizable solution to manufacture highly detailed 3D tissue constructs and holds great promise for regenerative medicine to solve the severe global shortage of donor tissues and organs. However, uni-material 3D bioprinting is not sufficient for manufacturing heterogenous 3D constructs with native-like microstructures and thus, innovative multi-material solutions are required. Here, we developed a novel multi-material 3D bioprinting strategy for bioprinting human corneal stroma. The human cornea is the transparent outer layer of your eye, and vision loss due to corneal blindness has serious effects on the quality of life of individuals. One of the main reasons for corneal blindness is the damage in the detailed organization of the corneal stroma where collagen fibrils are arranged in layers perpendicular to each other and the corneal stromal cells grow along the fibrils. Donor corneas for treating corneal blindness are scarce, and the current tissue engineering (TE) technologies cannot produce artificial corneas with the complex microstructure of native corneal stroma. To address this, we developed a novel multi-material 3D bioprinting strategy to mimic detailed organization of corneal stroma. These multi-material 3D structures with heterogenous design were bioprinted by using human adipose tissue -derived stem cells (hASCs) and hyaluronic acid (HA) -based bioinks with varying stiffnesses. In our novel design of 3D models, acellular stiffer HA-bioink and cell-laden softer HA-bioink were printed in alternating filaments, and the filaments were printed perpendicularly in alternating layers. The multi-material bioprinting strategy was applied for the first time in corneal stroma 3D bioprinting to mimic the native microstructure. As a result, the soft bioink promoted cellular growth and tissue formation of hASCs in the multi-material 3D bioprinted composites, whereas the stiff bioink provided mechanical support as well as guidance of cellular organization upon culture. Interestingly, cellular growth and tissue formation altered the mechanical properties of the bioprinted composite constructs significantly. Importantly, the bioprinted composite structures showed good integration to the host tissue in ex vivo cornea organ culture model. As a conclusion, the developed multi-material bioprinting strategy provides great potential as a biofabrication solution for manufacturing organized, heterogenous microstructures of native tissues. To the best of our knowledge, this multi-material bioprinting strategy has never been applied in corneal bioprinting. Therefore, our work advances the technological achievements in additive manufacturing and brings the field of corneal TE to a new level.

Effect of Anterior Chamber Air on Central Corneal Thickness in Human Donor Eyes.

The purpose of this study was to describe the effects of intracameral air on corneal edema. A laboratory investigation was performed on human donor corneas. Baseline pachymetry measurements through anterior segment optical coherence tomography and endothelial cell density were obtained for all corneas. Each pair of corneas was separated and randomly assigned to undergo air injection or Optisol-GS into a BIONIKO artificial anterior chamber for 5 minutes at physiologic intraocular pressure confirmed by digital palpation. Photographs were obtained immediately on connection of the cornea to the artificial anterior chamber and on completion of the 5 minutes of treatment, with anterior chamber air being exchanged for Optisol-GS. Pretreatment and posttreatment photographs were obtained. Immediately after treatment, pachymetry was again obtained on all corneas. Pachymetry data underwent statistical analysis. Corneal pachymetry improved from 690.5 ± 126.6 to 576.1 ± 87.2 μm, yielding a 114.4 ± 50.4 μm improvement of pachymetry in the group with air injected into the anterior chamber. This was a significant improvement of pachymetry when compared with the group with Optisol-GS injected into the anterior chamber, which showed an improvement from 662.3 ± 126.5 to 613.5 ± 108.0 μm, yielding an improvement of 48.8 ± 34.3 μm. Injection of air into the anterior chamber leads to a significant decrease in corneal pachymetry. We thereby propose that injecting air intracamerally is an effective intraoperative intervention when visualization is negatively affected by corneal edema.

Corneal topographer using null-screen patterned within a quadrangular acrylic prism

In this paper, the use of four flat-null-screens forming a quadrangular prism setup is employed as a target for an experimental corneal topographer. Topographic maps of three reflective calibration spheres with different radii of curvature: 7.70 mm, 9.42 mm, and 6.20 mm, and the corneal surface evaluation of a volunteer subject are presented. The experimental setup is based on the null-screen method. Zonal reconstruction of the surface shape of the human cornea is obtained by numerical integration of the normal vectors. The corresponding elevation map was found fitting an spherical model to the recovered data. The differences obtained in radius of curvature were 0.02% or less for the calibration spheres, and the cornea under test showed a difference of 1.39% (0.6 diopters) compared to the value obtained with ORBSCAN topographer.

Incisional surface quality of electron-beam irradiated cornea-extracted lenticule for stromal keratophakia: high nJ-energy vs. low nJ-energy femtosecond laser.

Corneal lenticules can be utilized as an additive material for stromal keratophakia. However, following extraction, they must be reimplanted almost immediately or cryopreserved in lenticule banks. Electron-beam (E-beam) irradiated corneas permit room-temperature storage for up to 2 years, enabling keratophakia to be performed on demand. This study aims to compare the performance of high nano Joule (nJ)-energy (VisuMax) and low nJ-energy (FEMTO LDV) femtosecond laser systems on the thickness consistency and surface quality and collagen morphology of lenticules produced from fresh and E-beamed corneas. A total of 24 lenticules with -6.00 dioptre power were cut in fresh human donor corneas and E-beamed corneas with VisuMax and FEMTO LDV. Before extraction, the thickness of the lenticules was measured with anterior segment-optical coherence tomography (AS-OCT). The incisional surface roughness of extracted lenticules was analyzed using atomic force microscopy (AFM) and scanning electron microscopy (SEM). Multiphoton microscopy was then used to assess the surface collagen morphometry. The E-beamed lenticules that were cut using FEMTO LDV were significantly thicker than the fresh specimens as opposed to those created with VisuMax, which had a similar thickness as the fresh lenticules. On the vertex, they were ∼11% thicker than the fresh lenticules. The surface roughness (Rq) of E-beamed lenticules incised with FEMTO LDV did not differ significantly from the fresh lenticules. This contrasted with the VisuMax-fashioned lenticules, which showed notably smoother surfaces (∼36 and ∼20% lower Rq on anterior and posterior surfaces, respectively) on the E-beamed than the fresh lenticules. The FEMTO LDV induced less cumulative changes to the collagen morphology on the surfaces of both fresh and E-beamed lenticules than the VisuMax. It has been previously demonstrated that the low nJ-energy FEMTO LDV produced a smoother cutting surface compared to high nJ-energy VisuMax in fresh lenticules. Here, we showed that this effect was also seen in the E-beamed lenticules. In addition, lower laser energy conferred fewer changes to the lenticular surface collagen morphology. The smaller disparity in surface cutting quality and collagen disturbances on the E-beamed lenticules could be beneficial for the early visual recovery of patients who undergo stromal keratophakia.

Simultaneous tensile and shear measurement of the human cornea in vivo using S0- and A0-wave optical coherence elastography

Understanding corneal stiffness is valuable for improving refractive surgery, detecting corneal abnormalities, and assessing intraocular pressure. However, accurately measuring the elastic properties, specifically the tensile and shear moduli that govern mechanical deformation, has been challenging. To tackle this issue, we have developed guided-wave optical coherence elastography that can simultaneously excite and analyze symmetric (S0) and anti-symmetric (A0) elastic waves in the cornea at around 10 kHz frequencies, enabling us to extract tensile and shear properties from measured wave dispersion curves. We verified the technique using elastomer phantoms and ex vivo porcine corneas and investigated the dependence on intraocular pressure using acoustoelastic theory that incorporates corneal tension and a nonlinear constitutive tissue model. In a pilot study involving six healthy human subjects aged 31 to 62, we measured shear moduli (Gzx) of 94±20 kPa (mean±standard deviation) and tensile moduli (Exx) of 4.0±1.1 MPa at central corneas. Our preliminary analysis of age-dependence revealed contrasting trends: -8.3±4.5 kPa/decade for shear and 0.30±0.21 MPa/decade for tensile modulus. This OCE technique has the potential to become a highly useful clinical tool for the quantitative biomechanical assessment of the cornea. Statement of SignificanceThis article reports an innovative elastography technique using two guided elastic waves, demonstrating the measurement of both tensile and shear moduli in human cornea in vivo with unprecedented precision. This technique paves the way for comprehensive investigations into corneal mechanics and holds clinical significance in various aspects of corneal health and disease management.

Road dust exposure and human corneal damage in a plateau high geological background provincial capital city: Spatial distribution, sources, bioaccessibility, and cytotoxicity of dust heavy metals

Ocular surface diseases are common in the plateau city, Kunming China, the continued daily exposure to heavy metals in dust may be an important inducement. In this study, the 150 road dust samples from five functional areas in Kunming were collected. The concentrations, distribution, possible sources, and bioaccessibility of heavy metals were analyzed. The adverse effects of dust extracts on human corneal epithelial cells and the underlying mechanisms were also assessed. The concentrations (mg·kg−1) of As (19.1), Cd (2.67), Cr (90.5), Cu (123), Pb (78.4), and Zn (389) in road dust were higher than the soil background, with commercial and residential areas showing the highest pollution. Their bioaccessibility in artificial tears was As (6.59 %) > Cu (5.11 %) > Ni (1.47 %) > Cr (1.17 %) > Mn (0.84 %) > Cd (0.76 %) > Zn (0.50 %) > Pb (0.31 %). The two main sources of heavy metals included tire and mechanical abrasion (24.5 %) and traffic exhaust (21.6 %). All dust extracts induced cytotoxicity, evidenced by stronger inhibition of cell viability, higher production of ROS, and altered mRNA expression of antioxidant enzymes and cell cycle-related genes, with commercial- areas-2 (CA2)-dust extract showing the greatest oxidative damage and cell cycle arrest. Our data may provide new evidence that dust exposure in high geological background cities could trigger human cornea damage.

Enhancing Ocular Drug Delivery: The Effect of Physicochemical Properties of Nanoparticles on the Mechanism of Their Uptake by Human Cornea Epithelial Cells.

This study investigates the effect of nanoparticle size and surface chemistry on interactions of the nanoparticles with human cornea epithelial cells (HCECs). Poly(lactic-co-glycolic) acid (PLGA) nanoparticles were synthesized using the emulsion-solvent evaporation method and surface modified with mucoadhesive (alginate [ALG] and chitosan [CHS]) and mucopenetrative (polyethylene glycol [PEG]) polymers. Particles were found to be monodisperse (polydispersity index (PDI) below 0.2), spherical, and with size and zeta potential ranging from 100 to 250 nm and from -25 to +15 mV, respectively. Evaluation of cytotoxicity with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay indicated that incubating cells with nanoparticles for 24 h at concentrations up to 100 μg/mL caused only mild toxicity (70-100% cell viability). Cellular uptake studies were conducted using an in vitro model developed with a monolayer of HCECs integrated with simulated mucosal solution. Evaluation of nanoparticle uptake revealed that energy-dependent endocytosis is the primary uptake mechanism. Among the different nanoparticles studied, 100 nm PLGA NPs and PEG-PLGA-150 NPs showed the highest levels of uptake by HCECs. Additionally, uptake studies in the presence of various inhibitors suggested that macropinocytosis and caveolae-mediated endocytosis are the dominant pathways. While clathrin-mediated endocytosis was found to also be partially responsible for nanoparticle uptake, phagocytosis did not play a role within the studied ranges of size and surface chemistries. These important findings could lead to improved nanoparticle-based formulations that could improve therapies for ocular diseases.

Intraoperative collagen imaging of sutured cornea: A way towards managing post-penetrating keratoplasty astigmatism

The birefringent nature of the human cornea plays an important role in comprehending its structural behavior in both diseased and surgical conditions. During corneal transplantation, irregular astigmatism is a common post-surgical complication that depends on the characteristics of suturing. Four human cadaver corneas are subjected to an in-vitro model of a typical full-thickness penetrating keratoplasty (PK) procedure using 16 simple interrupted 10–0 vicyrl sutures. The birefringence of these four corneas is analyzed using digital photoelasticity and compared with the control cornea (without PK). It is found that the sutures and their mutual interaction influence the morphology of the peripheral birefringence of the cornea. The findings of the present investigation are pertinent to intraoperative suture management during PK. Results suggest conserving the typical diamond-shaped morphology of peripheral birefringence would ensure uniform distribution of sutures. Therefore, birefringence imaging could be useful in suture management to ensure proper apposition of the graft-host junction, thus minimizing the risk of irregular astigmatism.

Hydrogels Based on Recombinant Spidroin Stimulate Proliferation and Migration of Human Corneal Cells.

The effect of recombinant spidroin (RS) hydrogel (HG) on anterior epithelial cells and keratocytes of the human cornea was studied in vitro. Corneal injuries are highly prevalent in developing countries according to the World Health Organization. Various technologies have recently been proposed to restore the damaged surface of the cornea. Use of biodegradable silk-based materials, including recombinant analogs of the spider silk protein spidroin, is an important avenue of research in the field of wound healing and corneal regeneration. Spidroins are well known for their optimal balance of strength and elasticity. Given their biological compatibility, lack of immunogenicity, and biodegradability, spidroins provide a biomaterial for tissue engineering and regenerative medicine. HGs based on RS rS2/12-RGDS were therefore tested for cytotoxicity toward isolated corneal epithelial cells and keratocytes with regard to possible changes in cell phenotype and migratory activity. A promising outlook and therapeutic potential were demonstrated for RS-based HGs.

The visibility of retinal amyloid deposits as a biomarker of Alzheimer’s disease when imaged in polarized light through the cornea

AbstractBackgroundWe previously reported a novel, dye‐free method to image amyloid deposits in the retina. Using polarized light interactions to create deposit contrast, the number of deposits in ex vivo retinas predicted the severity of brain amyloid found in association with Alzheimer’s disease (AD). Here we describe the effect of the potential range of corneal interactions with polarized light on the visibility of these retinal deposits when imaged in vivo.Method52 retinal amyloid deposits, positive for thioflavin (Fig 1) and with a range of visibility in polarized light were imaged ex vivo in retinas of 12 participants with a moderate (2) or high (10) likelihood of Alzheimer’s disease, based on brain pathology. New images of each deposit were created by combining the polarized light interactions of the deposit and surrounding retina with the known range of interactions of human corneas with polarized light. The varying contrasts of each deposit with 45 different sets of corneal properties (a total of 2340 deposit images) were compared to the initial ex vivo images.ResultAs a function of the modelled corneal properties, the root mean square (RMS) contrast of each deposit both increased and decreased from that without a cornea (Fig 2). On average, RMS contrast across deposits combined with a cornea decreased 12% from that without a cornea. For 3% of deposit images, there was a corneal property that reduced the deposit contrast by over 50% (Fig 2). However, even in these cases, because of the variation in interaction with polarized light across the deposit (Fig 3), the deposit remained visible against the background retina.ConclusionAlthough the cornea interaction with polarized light will affect images of amyloid deposits in the retina taken with our non‐invasive method, all cases investigated are predicted to remain visible against the background retina. In addition, during in vivo imaging, an established method of compensating for an individual’s corneal interaction with polarized light, would guarantee 100% visibility. Thus, in vivo, our retinal imaging method is predicted to give a biomarker of the severity of amyloid in the brain found in association with Alzheimer’s disease.

Preparation and evaluation of a suspension of human corneal endothelial cells isolated from the eyes of cadaveric donors for transplantation in an <em>ex vivo</em> experiment

Background. According to the World Health Organization, corneal diseases are one of the major causes of blindness globally. Endothelial dystrophy is one of the etiological factors leading to corneal diseases. The corneal endothelium is a monolayer of cells with virtually no mitotic activity. When the density of corneal endothelial cells falls below a critical threshold, the endothelium loses its ability to regulate corneal stromal hydration. This leads to corneal clouding and, consequently, to reduced visual acuity and quality of life of the patient. In this regard, various keratoplasty methods are widely used in clinical practice. Today, it is technically possible to transplant all corneal layers via penetrating keratoplasty, and to transplant the posterior epithelium via layer-bylayer keratoplasty. These surgical approaches are now widely used in everyday practice, but they require the use of scarce material – cadaveric donor corneas, from which grafts for the above-mentioned operations are formed in the conditions of an eye bank. In this regard, protocols for obtaining human corneal endothelial cell (HCEC) culture for subsequent transplantation have been proposed in recent years. However, the use of such approaches in Russia is limited by the law. The aim of this study was to experimentally justify the possibility of transplanting uncultured endothelial cells, isolated from cadaveric human corneas. Materials and methods. The first stage of the work consisted of obtaining a suspension of endothelial cells from cadaveric donor corneas and studying it; at the second stage, the transplantation effectiveness of the resulting cell suspension was assessed in an ex vivo experiment. Results. The cell phenotype after transplantation by the proposed method had high viability and preservation. Conclusions. The presented results suggest that phenotype and adhesion ability are preserved, and that the cell suspension has a high level of viability under adequate loss of endothelial cells during transplantation in the ex vivo experiment.

In Vivo Biomechanical Response of the Human Cornea to Acoustic Waves

The cornea is the optical window to the brain. Its optical and structural properties are responsible for optical transparency and vision. The shape, elasticity, rigidity, or stiffness are due to its biomechanical properties, whose stability results in ocular integrity and intraocular pressure dynamics. Here, we report in vivo observations of shape changes and biomechanical alterations in the human cornea induced by acoustic wave pressure within the frequency range of 50–350 Hz and the sound pressure level of 90 dB. The central corneal thickness (CCT) and eccentricity (e2) were measured using Scheimpflug imaging and biomechanical properties [corneal hysteresis (CH) and intraocular pressure (IOP)] were assessed with air-puff tonometry in six young, healthy volunteers. At the specific 150 Hz acoustic frequency, the variations in e2 and CCT were 0.058 and 7.33 µm, respectively. Biomechanical alterations were also observed in both the IOP (a decrease of 3.60 mmHg) and CH (an increase of 0.40 mmHg).

ST-ITEF: Spatio-Temporal Intraoperative Task Estimating Framework to recognize surgical phase and predict instrument path based on multi-object tracking in keratoplasty

Computer-assisted cognition guidance for surgical robotics by computer vision is a potential future outcome, which could facilitate the surgery for both operation accuracy and autonomy level. In this paper, multiple-object segmentation and feature extraction from this segmentation are combined to determine and predict surgical manipulation. A novel three-stage Spatio-Temporal Intraoperative Task Estimating Framework is proposed, with a quantitative expression derived from ophthalmologists’ visual information process and also with the multi-object tracking of surgical instruments and human corneas involved in keratoplasty. In the estimation of intraoperative workflow, quantifying the operation parameters is still an open challenge. This problem is tackled by extracting key geometric properties from multi-object segmentation and calculating the relative position among instruments and corneas. A decision framework is further proposed, based on prior geometric properties, to recognize the current surgical phase and predict the instrument path for each phase. Our framework is tested and evaluated by real human keratoplasty videos. The optimized DeepLabV3 with image filtration won the competitive class-IoU in the segmentation task and the mean phase jaccard reached 55.58 % for the phase recognition. Both the qualitative and quantitative results indicate that our framework can achieve accurate segmentation and surgical phase recognition under complex disturbance. The Intraoperative Task Estimating Framework would be highly potential to guide surgical robots in clinical practice.

Depth-dependent mechanical properties of the human cornea by uniaxial extension

The purpose of this study was to investigate the depth-dependent biomechanical properties of the human corneal stroma under uniaxial tensile loading. Human stroma samples were obtained after the removal of Descemet's membrane in the course of Descemet's membrane endothelial keratoplasty (DMEK) transplantation. Uniaxial tensile tests were performed at three different depths: anterior, central, and posterior on 2 × 6 × 0.15 mm strips taken from the central DMEK graft. The measured force-displacement data were used to calculate stress-strain curves and to derive the tangent modulus. The study showed that mechanical strength decreased significantly with depth. The anterior cornea appeared to be the stiffest, with a stiffness approximately 18% higher than that of the central cornea and approximately 38% higher than that of the posterior layer. Larger variations in mechanical response were observed in the posterior group, probably due to the higher degree of alignment of the collagen fibers in the posterior sections of the cornea. This study contributes to a better understanding of the biomechanical tensile properties of the cornea, which has important implications for the development of new treatment strategies for corneal diseases. Accurate quantification of tensile strength as a function of depth is critical information that is lacking in human corneal biomechanics to develop numerical models and new treatment methods.

A numerical model of the human cornea accounting for the fiber-distributed collagen microstructure

We present a fiber-distributed model of the reinforcing collagen of the human cornea. The model describes the basic connections between the components of the tissue by defining an elementary block (cell) and upscaling it to the physical size of the cornea. The cell is defined by two sets of collagen fibrils running in approximately orthogonal directions, characterized by a random distribution of the spatial orientation and connected by chemical bonds of two kinds. The bonds of the first kind describe the lamellar crosslinks, forming the ribbon-like lamellae; while the bonds of the second kind describe the stacking crosslinks, piling up the lamellae to form the structure of the stroma. The spatial replication of the cell produces a truss structure with a considerable number of degrees of freedom. The statistical characterization of the collagen fibrils leads to a mechanical model that reacts to the action of the deterministic intraocular pressure with a stochastic distribution of the displacements, here characterized by their mean value and variance. The strategy to address the solution of the heavy resulting numerical problem is to use the so-called stochastic finite element improved perturbation method combined with a fully explicit solver. The results demonstrate that the variability of the mechanical properties affects in a non-negligible manner the expected response of the structure to the physiological action.

An artificially-intelligent cornea with tactile sensation enables sensory expansion and interaction

We demonstrate an artificially-intelligent cornea that can assume the functions of the native human cornea such as protection, tactile perception, and light refraction, and possesses sensory expansion and interactive functions. These functions are realized by an artificial corneal reflex arc that is constructed to implement mechanical and light information coding, information processing, and the regulation of transmitted light. Digitally-aligned, long and continuous zinc tin oxide (ZTO) semiconductor fabric patterns were fabricated as the active channels of the artificial synapse, which are non-toxic, heavy-metal-free, low-cost, and ensure superior comprehensive optical properties (transmittance >99.89%, haze <0.36%). Precisely-tuned crystal-phase structures of the ZTO fibers enabled reconfigurable synaptic plasticity, which is applicable to encrypted communication and associative learning. This work suggests new strategies for the tuning of synaptic plasticity and the design of visual neuroprosthetics, and has important implications for the development of neuromorphic electronics and for visual restoration.

TGFβ1-driven SMAD2/3 phosphorylation and myofibroblast emergence are fully dependent on the TGFβ1 pre-activation of MAPKs and controlled by maternal leucine zipper kinase

Following wounding, endogenously secreted TGFβs drive resident and bone marrow-derived cells to convert into α-smooth actin (SMA)-rich, contractile myofibroblasts. The TGFβ effect is initiated by the phosphorylation of SMADs 2 and 3 (SMAD2/3). This event has been referred to as the canonical response to TGFβ. TGFβ also elicits other responses viewed as parallel events not directly connected to the SMAD activation, and thus referred to as noncanonical. A recognized response is the phosphorylation of the -activated kinase (TAK1/MAP3K), an upstream component of the mitogen-activated protein kinase (MAPK) cascade. We have now examined the relationship between these two effects of TGFβ1 at their earliest stages. The bulk of the studies were carried out with primary fibroblasts derived from the human cornea. The results' widespread relevance was confirmed in critical experiments with dermal-, and Tenon's capsule-derived fibroblasts. Cells were treated with kinase inhibitors or targeting siRNAs followed by induction by 2 ng/ml TGFβ1, and/or 10 ng/ml TNF-α. Cells were collected after 1 to 30 min for Western blot analysis and assayed for the accumulation of phosphorylated TAK1, ASK1, JNK1/2, p38, HPS27, MELK, SMAD2/3, and GAPDH. The effect of the kinase inhibitors on α-SMA expression and α-SMA stress fiber organization was also tested. For the immediate response to TGFβ1 we found that a) activation of the MAPK pathway was completed within 1 min after the addition of TGFβ1; b) phosphorylation of JNK1/2 was fully dependent on TAK1 and ASK1 activity, c) phosphorylation of MELK was fully dependent on JNK1/2 activity; d) phosphorylation of ASK1 depends on MELK activity, indicating the existence of an ASK1-MELK positive activation feedback loop; e) phosphorylation of SMAD2/3 started only after a 5 min period and reached a nadir after 10–15 min, f) the latter phosphorylation was fully blocked by inhibition of TAK1, ASK1, JNK1/2, and MELK, and siRNA-driven MELK downregulation; g) the inhibitors equally blocked the α-SMA protein expression, stress fiber development, and cell morphology changes at 72 h. These results demonstrate that the activation of the canonical pathway is fully subordinate to the activity of the MAPK pathway, challenging the concept of canonical and noncanonical TGFβ pathways and that SMAD2/3 activation is mediated by MELK, a kinase not previously associated with rapid pharmacological responses.

Human cornea-derived extracellular matrix hydrogel for prevention of post-traumatic corneal scarring: A translational approach.

Corneal scarring and opacification are a significant cause of blindness affecting millions worldwide. The current standard of care for corneal blindness is corneal transplantation, which suffers from several drawbacks. One alternative approach that has shown promise is the use of xenogeneic corneal extracellular matrix (ECM), but its clinical applicability is challenging due to safety concerns. This study reports the innovative use of human cornea-derived ECM to prevent post-traumatic corneal scarring. About 30 - 40% of corneas donated to the eye banks do not meet the standards defined for clinical use and are generally discarded, although they are completely screened for their safety. In this study, human cornea-derived decellularized ECM hydrogel was prepared from the non-transplantation grade human cadaveric corneas obtained from an accredited eye-bank. The prepared hydrogel was screened for its efficacy against corneal opacification following an injury in an animal model. Our in vivo study revealed that, the control collagen-treated group developed corneal opacification, while the prophylactic application of human cornea-derived hydrogel effectively prevented corneal scarring and opacification. The human hydrogel-treated corneas were indistinguishable from healthy corneas and comparable to those treated with the xenogeneic bovine corneal hydrogel. We also demonstrated that the application of the hydrogel retained the biological milieu including cell behavior, protein components, optical properties, curvature, and nerve regeneration by remodeling the corneal wound after injury. The hydrogel application is also sutureless, resulting in faster corneal healing. We envision that this human cornea-derived ECM-based hydrogel has potential clinical application in preventing scarring from corneal wounding. STATEMENT OF SIGNIFICANCE: There are significant challenges surrounding corneal regeneration after injury due to extensive scarring. Although there is substantial research on corneal regeneration, much of it uses synthetic materials with chemical cross-linking methods or xenogeneic tissue-based material devices which have to undergo exhaustive safety analysis before clinical trials. Herein, we demonstrate the potential application of a human corneal extracellular matrix hydrogel without any additional materials for scarless corneal tissue regeneration, and a method to reduce the wasting of donated allogenic corneal tissue from eye banks. We found no difference in efficacy between the usage of human tissues compared to xenogeneic sources. This may help ease clinical translation and can be used topically without sutures as an outpatient procedure.

A predictive model of UV-A-riboflavin crosslinking treatment on porcine corneas

The crosslinking technique (CXL) is an effective low-risk therapeutic treatment of keratoconus and other ectatic disorders of the human cornea. The effect of corneal CXL is to increase the stiffness of the stroma to prevent the progression of the cornea distortion. Several clinical and experimental studies have shown that the stiffening effects predominantly localize on the anterior portion of the stroma and that the in-depth stiffening distribution is highly dependent on the duration of treatment. Yet, how the stiffening effects distribute through the cornea thickness as a function of the treatment duration is an open question. Here, we propose an analytical model of the stiffening profile due to CXL treatment as a function of the irradiation time. We consider linear and nonlinear variations of the crosslinking effects across the thickness and implement them into a finite element model of the porcine cornea. We present a time-dependent in-depth stiffening profile that allows us to predict the post-operative cornea response to physiological intraocular pressure for different irradiation times. We anticipate that this predictive model will support the development of patient specific three-dimensional models that will allow clinicians to design customized CXL treatment, thus enhancing treatment outcomes.