Fabrication of Micropatterns of Aligned Collagen Fibrils.

Many tissues in vivo contain aligned structures such as filaments, fibrils, and fibers, which expose cells to anisotropic structural and topographical cues that range from the nanometer to micrometer scales. Understanding how cell behavior is regulated by these cues during physiological and pathological processes (e.g., wound healing, cancer invasion) requires substrates that can expose cells to anisotropic cues over several length scales. In this study, we developed a novel method of fabricating micropatterns of aligned collagen fibrils of different geometry onto PDMS-coated glass coverslips that allowed us to investigate the roles of topography and confinement on corneal cell behavior. When corneal cells were cultured on micropatterns of aligned collagen fibrils in the absence of confinement, the degree of cell alignment increased from 40 ± 14 to 82 ± 5% as the size of the micropattern width decreased from 750 to 50 µm. Although the cell area (∼2500 µm2), cell length (∼160 µm), and projected nuclear area (∼175 µm2) were relatively constant on the different micropattern widths, cells displayed an increased aspect ratio as the width of the aligned collagen fibril micropatterns decreased. We also observed that the morphology of cells adhering to the surrounding uncoated PDMS was dependent upon both the size of the aligned collagen fibril micropattern and the distance from the micropatterns. When corneal cells were confined to the micropatterns of aligned collagen fibrils by a Pluronic coating to passivate the surrounding area, a similar trend in increasing cell alignment was observed (35 ± 10 to 89 ± 2%). However, the projected nuclear area decreased significantly (∼210 to 130 µm2) as the micropattern width decreased from 750 to 50 µm. The development of this method allows for the deposition of aligned collagen fibril micropatterns of different geometries on a transparent and elastic substrate and provides an excellent model system to investigate the role of anisotropic cues in cell behavior.

Cell motility and mechanics in three-dimensional collagen matrices.

Fibrous connective tissues provide mechanical support and frameworks for other tissues of the body and play an integral role in normal tissue physiology and pathology. Three-dimensional collagen matrices exhibit mechanical and structural features that resemble fibrous connective tissue and have become an important model system to study cell behavior in a tissue-like environment. This review focuses on motile and mechanical interactions between cells—especially fibroblasts—and collagen matrices. We describe several matrix contraction models, the interactions between fibroblasts and collagen fibrils at global and subcellular levels, unique features of mechanical feedback between cells and the matrix, and the impact of the cell-matrix tension state on cell morphology and mechanical behavior. We develop a conceptual framework to explain the balance between cell migration and collagen translocation including the concept of promigratory and procontractile growth factor environments. Finally, we review the significance of these concepts for the physiology of wound repair.

The impact of UV cross-linking on corneal stromal cell migration, differentiation and patterning.

Previous studies have demonstrated that UV cross-linking (CXL) increases stromal stiffness and produces alterations in extracellular matrix (ECM) microstructure. In order to investigate how CXL impacts both keratocyte differentiation and patterning within the stroma, and fibroblast migration and myofibroblast differentiation on top of the stroma, we combined CXL with superficial phototherapeutic keratectomy (PTK) in a rabbit model. Twenty-six rabbits underwent a 6 mm diameter, 70 µm deep phototherapeutic keratectomy (PTK) with an excimer laser to remove the epithelium and anterior basement membrane. In 14 rabbits, standard CXL was performed in the same eye immediately after PTK. Contralateral eyes served as controls. In vivo confocal microscopy through focusing (CMTF) was used to analyze corneal epithelial and stromal thickness, as well as stromal keratocyte activation and corneal haze. CMTF scans were collected pre-operatively, and from 7 to 120 days after the procedure. A subset of rabbits was sacrificed at each time point, and corneas were fixed and labeled in situ for multiphoton fluorescence microscopy and second harmonic generation imaging. In vivo and in situ imaging demonstrated that haze after PTK was primarily derived from a layer of myofibroblasts that formed on top of the native stroma. Over time, this fibrotic layer was remodeled into more transparent stromal lamellae, and quiescent cells replaced myofibroblasts. Migrating cells within the native stroma underneath the photoablated area were elongated, co-aligned with collagen, and lacked stress fibers. In contrast, following PTK + CXL, haze was derived primarily from highly reflective necrotic "ghost cells" in the anterior stroma, and fibrosis on top of the photoablated stroma was not observed at any time point evaluated. Cells formed clusters as they migrated into the cross-linked stromal tissue and expressed stress fibers; some cells at the edge of the CXL area also expressed α-SM actin, suggesting myofibroblast transformation. Stromal thickness increased significantly between 21 and 90 days after PTK + CXL (P < 0.001) and was over 35 µm higher than baseline at Day 90 (P < 0.05). Overall, these data suggest that cross-linking inhibits interlamellar cell movement, and that these changes lead to a disruption of normal keratocyte patterning and increased activation during stromal repopulation. Interestingly, CXL also prevents PTK-induced fibrosis on top of the stroma, and results in long term increases in stromal thickness in the rabbit model.

ECM stiffness modulates the proliferation but not the motility of primary corneal keratocytes in response to PDGF-BB.

During corneal wound healing, keratocytes present within the corneal stroma become activated into a repair phenotype upon the release of growth factors, such as transforming growth factor-beta 1 (TGF-ß1) and platelet-derived growth factor-BB (PDGF-BB). The process of injury and repair can lead to changes in the mechanical properties of the tissue, and previous work has shown that the TGF-ß1-mediated myofibroblast differentiation of corneal keratocytes depends on substratum stiffness. It is still unclear, however, if changes in stiffness can modulate keratocyte behavior in response to other growth factors, such as PDGF-BB. Here, we used a polyacrylamide (PA) gel system to determine whether changes in stiffness influence the proliferation and motility of primary corneal keratocytes treated with PDGF-BB. In the presence of PDGF-BB, cells on stiffer substrata exhibited a more elongated morphology and had higher rates of proliferation than cells in a more compliant microenvironment. Using a freeze-injury to assay cell motility, however, we did not observe any stiffness-dependent differences in the migration of keratocytes treated with PDGF-BB. Taken together, these data highlight the importance of biophysical cues during corneal wound healing and suggest that keratocytes respond differently to changes in ECM stiffness in the presence of different growth factors.

ECM Stiffness Controls the Activation and Contractility of Corneal Keratocytes in Response to TGF-ß1.

After surgery or traumatic injury, corneal wound healing can cause a scarring response that stiffens the tissue and impairs ocular function. This fibrosis is caused in part by the activation of corneal keratocytes from a native mechanically quiescent state to an activated myofibroblastic state. This transformation is tied to signaling downstream of transforming growth factor-ß1 (TGF-ß1). Here, to better understand how biochemical and biophysical cues interact to regulate keratocyte activation and contractility, we cultured primary rabbit corneal keratocytes on flexible substrata of varying stiffness in the presence (or absence) of TGF-ß1. Time-lapse fluorescence microscopy was used to assess changes in keratocyte morphology, as well as to quantify the dynamic traction stresses exerted by cells under different experimental conditions. In other experiments, keratocytes were fixed after 5 days of culture and stained for markers of both contractility and myofibroblastic activation. Treatment with TGF-ß1 elicited distinct phenotypes on substrata of different stiffnesses. Cells on soft (1 kPa) gels formed fewer stress fibers and retained a more dendritic morphology, indicative of a quiescent keratocyte phenotype. Keratocytes cultured on stiff (10 kPa) gels or collagen-coated glass coverslips, however, had broad morphologies, formed abundant stress fibers, exhibited greater levels of α-smooth muscle actin (α-SMA) expression, and exerted larger traction forces. Confocal images of phospho-myosin light chain (pMLC) immunofluorescence, moreover, revealed stiffness-dependent differences in the subcellular distribution of actomyosin contractility, with pMLC localized at the tips of thin cellular processes in mechanically quiescent cells. Importantly, keratocytes cultured in the absence of TGF-ß1 showed no stiffness-dependent differences in α-SMA immunofluorescence, suggesting that a stiff microenvironment alone is insufficient to induce myofibroblastic activation. Taken together, these data suggest that changes in ECM stiffness can modulate the morphology, cytoskeletal organization, and subcellular pattern of force generation in corneal keratocytes treated with TGF-ß1.

Keratocyte mechanobiology.

In vivo, corneal keratocytes reside within a complex 3D extracellular matrix (ECM) consisting of highly aligned collagen lamellae, growth factors, and other extracellular matrix components, and are subjected to various mechanical stimuli during developmental morphogenesis, fluctuations in intraocular pressure, and wound healing. The process by which keratocytes convert changes in mechanical stimuli (e.g. local topography, applied force, ECM stiffness) into biochemical signaling is known as mechanotransduction. Activation of the various mechanotransductive pathways can produce changes in cell migration, proliferation, and differentiation. Here we review how corneal keratocytes respond to and integrate different biochemical and biophysical factors. We first highlight how growth factors and other cytokines regulate the activity of Rho GTPases, cytoskeletal remodeling, and ultimately the mechanical phenotype of keratocytes. We then discuss how changes in the mechanical properties of the ECM have been shown to regulate keratocyte behavior in sophisticated 2D and 3D experimental models of the corneal microenvironment. Finally, we discuss how ECM topography and protein composition can modulate cell phenotypes, and review the different methods of fabricating in vitro mimics of corneal ECM topography, novel approaches for examining topographical effects in vivo, and the impact of different ECM glycoproteins and proteoglycans on keratocyte behavior.

Positive toxicology and reactive serology in tissue donors: a retrospective study over a 3-year period.

Assessment of donor suitability and criteria development for tissue donation evaluation which appropriately addresses the risk factors for disease transmission, especially high risk for Hepatitis B or C, HIV or other transmissible diseases as defined by the Food and Drug Administration, FDA, is a continuing concern for tissue banks. The relationship of drug use, especially IV drugs, has been determined to be associated with an increased possibility of reactive serology (Centers for Disease Control and Prevention (USCDC) in Division of Viral Hepatitis, National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention. Hepatitis C questions and answers for health professionals. https://www.cdc.gov/hepatitis/hcv/hcvfaq.htm ; Centers for Disease Control and Prevention (USCDC) in infectious diseases, opioids and injection drug use, 2018. https://www.cdc.gov/pwid/opioid-use.html ; HIH National Institute on Drug Abuse in Health Consequences of Drug Misuse, 2017. https://www.drugabuse.gov/related-topics/health-consequences-drug-misuse ). Therefore, prior drug use determined by medical social history screening frequently results in deferral of a potential donor even when the route of drug administration has not been determined to be intravenous. Because of the association of drug use in numerous cases, which come under Medical Examiner jurisdiction, a possible rule out of a number of otherwise suitable medical examiner cases could occur. This retrospective review of medical examiner cases, tissue bank referrals and tissue donors in a 3-year period examines the relationship, if any, between reactive serology and positive toxicology results. These results would appear to indicate assessment of donor medical social history screening is effective in reducing recovery of high-risk donors.

A high-throughput microfluidic method for fabricating aligned collagen fibrils to study Keratocyte behavior.

In vivo, keratocytes are surrounded by aligned type I collagen fibrils that are organized into lamellae. A growing body of literature suggests that the unique topography of the corneal stroma is an important regulator of keratocyte behavior. In this study we describe a microfluidic method to deposit aligned fibrils of type I collagen onto glass coverslips. This high-throughput method allowed for the simultaneous coating of up to eight substrates with aligned collagen fibrils. When these substrates were integrated into a PDMS microwell culture system they provided a platform for high-resolution imaging of keratocyte behavior. Through the use of wide-field fluorescence and differential interference contrast microscopy, we observed that the density of collagen fibrils deposited was dependent upon both the perfusion shear rate of collagen and the time of perfusion. In contrast, a similar degree of fibril alignment was observed over a range of shear rates. When primary normal rabbit keratocytes (NRK) were seeded on substrates with a high density of aligned collagen fibrils and cultured in the presence of platelet derived growth factor (PDGF) the keratocytes displayed an elongated cell body that was co-aligned with the underlying collagen fibrils. In contrast, when NRK were cultured on substrates with a low density of aligned collagen fibrils, the cells showed no preferential orientation. These results suggest that this simple and inexpensive method can provide a general platform to study how simultaneous exposure to topographical and soluble cues influence cell behavior.

Temporal and spatial analysis of stromal cell and extracellular matrix patterning following lamellar keratectomy.

Extracellular matrix (ECM) supplies both physical and chemical signals to keratocytes which can impact their differentiation to fibroblasts and/or myofibroblasts. It also provides a substrate through which they migrate during wound repair. We have previously shown that following transcorneal freeze injury (FI), migrating corneal fibroblasts align parallel to the stromal lamellae during wound repopulation. In this study, we compare cell and ECM patterning both within and on top of the stroma at different time points following lamellar keratectomy (LK) in the rabbit. Twelve rabbits received LK in one eye. Rabbits were monitored using in vivo confocal microscopy at 3, 7, 21 and 60 days after injury. A subset of animals was sacrificed at each time point to further investigate cell and matrix patterning. Tissue was fixed and labeled in situ with Alexa Fluor 488 phalloidin (for F-actin), and imaged using multiphoton fluorescence and second harmonic generation (SHG) imaging (for collagen). Immediately following LK, cell death occurred in the corneal stroma directly beneath the injury. At 7 and 21 days after LK, analysis of fluorescence (F-actin) and SHG results (collagen) indicated that fibroblasts were co-aligned with the collagen lamellae within this region. In contrast, stromal cells accumulating on top of the stromal wound bed were randomly arranged, contained more prominent stress fibers, and expressed alpha smooth muscle actin (α-SMA) and fibronectin. At 60 days, cells and matrix in this region had become co-aligned into lamellar-like structures; cells were elongated but did not express stress fibers. Corneal haze measured using in vivo confocal microscopy peaked at 21 days after LK, and was significantly reduced by 60 days. Cell morphology and patterning observed in vivo was similar to that observed in situ. Our results suggest that the topography and alignment of the collagen lamellae direct fibroblast patterning during repopulation of the native stroma after LK injury in the rabbit. In contrast, stromal cells accumulating on top of the stromal wound bed initially align randomly and produce a fibrotic ECM. Remarkably, over time, these cells appear to remodel the ECM to produce a lamellar structure that is similar to the native corneal stroma.

The impact of type 1 diabetes mellitus on corneal epithelial nerve morphology and the corneal epithelium.

Diabetic corneal neuropathy can result in chronic, sight-threatening corneal pathology. Although the exact etiology is unknown, it is believed that a reduction in corneal sensitivity and loss of neurotrophic support contributes to corneal disease. Information regarding the relationship between nerve loss and effects on the corneal epithelium is limited. We investigated changes in the corneal epithelium and nerve morphology using three-dimensional imaging in vivo and in situ in a streptozotocin-induced diabetic mouse model. Streptozotocin-treated mice showed increased levels of serum glucose and growth retardation consistent with a severe diabetic state. A reduction in the length of the subbasal nerve plexus was evident after 6 weeks of disease. Loss of the subbasal nerve plexus was associated with corneal epithelial thinning and a reduction in basal epithelial cell density. In contrast, loss of the terminal epithelial nerves was associated with animal age. Importantly, this is the first rodent model of type 1 diabetes that shows characteristics of corneal epithelial thinning and a reduction in basal epithelial cell density, both previously have been documented in humans with diabetic corneal neuropathy. These findings indicate that in type 1 diabetes, nerve fiber damage is evident in the subbasal nerve plexus before terminal epithelial nerve loss and that neurotrophic support from both the subbasal nerve plexus and terminal epithelial nerves is essential for the maintenance of corneal epithelial homeostasis.

Effect of HDAC Inhibitors on Corneal Keratocyte Mechanical Phenotypes in 3-D Collagen Matrices.

PURPOSE: Histone deacetylase inhibitors (HDAC) have been shown to inhibit the TGFß-induced myofibroblast transformation of corneal fibroblasts in 2-D culture. However, the effect of HDAC inhibitors on keratocyte spreading, contraction, and matrix remodeling in 3-D culture has not been directly assessed. The goal of this study was to investigate the effects of the HDAC inhibitors Trichostatin A (TSA) and Vorinostat (SAHA) on corneal keratocyte mechanical phenotypes in 3-D culture using defined serum-free culture conditions. METHODS: Rabbit corneal keratocytes were plated within standard rat tail type I collagen matrices (2.5 mg/ml) or compressed collagen matrices (~100 mg/ml) and cultured for up to 4 days in serum-free media, PDGF BB, TGFß1, and either 50 nM TSA, 10 µM SAHA, or vehicle (DMSO). F-actin, α-SM-actin, and collagen fibrils were imaged using confocal microscopy. Cell morphology and global matrix contraction were quantified digitally. The expression of α-SM-actin was assessed using western blotting. RESULTS: Corneal keratocytes in 3-D matrices had a quiescent mechanical phenotype, as indicated by a dendritic morphology, a lack of stress fibers, and minimal cell-induced matrix remodeling. This phenotype was generally maintained following the addition of TSA or SAHA. TGFß1 induced a contractile phenotype, as indicated by a loss of dendritic cell processes, the development of stress fibers, and significant matrix compaction. In contrast, cells cultured in TGFß1 plus TSA or SAHA remained dendritic and did not form stress fibers or induce ECM compaction. Western blotting showed that the expression of α-SM actin after treatment with TGFß1 was inhibited by TSA and SAHA. PDGF BB stimulated the elongation of keratocytes and the extension of dendritic processes within 3-D matrices without inducing stress fiber formation or collagen reorganization. This spreading response was maintained in the presence of TSA or SAHA. CONCLUSIONS: Overall, HDAC inhibitors appear to mitigate the effects of TGFß1 on the transformation of corneal keratocytes to a contractile, myofibroblast phenotype in both compliant and rigid 3-D matrices while preserving normal cell spreading and their ability to respond to the pro-migratory growth factor PDGF.

Mechanical interactions and crosstalk between corneal keratocytes and the extracellular matrix.

The generation of cellular forces and the application of these physical forces to the ECM play a central role in mediating matrix patterning and remodeling during fundamental processes such as developmental morphogenesis and wound healing. In addition to growth factors and other biochemical factors that can modulate the keratocyte mechanical phenotype, another key player in the regulation of cell-induced ECM patterning is the mechanical state of the ECM itself. In this review we provide an overview of the biochemical and biophysical factors regulating the mechanical interactions between corneal keratocytes and the stromal ECM at the cellular level. We first provide an overview of how Rho GTPases regulate the sub-cellular pattern of force generation by corneal keratocytes, and the impact these forces have on the surrounding ECM. We next review how feedback from local matrix structural and mechanical properties can modulate keratocyte phenotype and mechanical activity. Throughout this review, we provide examples of how these biophysical interactions may contribute to clinical outcomes, with a focus on corneal wound healing.

Impact of Donor Age on Corneal Endothelium-Descemet Membrane Layer Scroll Formation.

OBJECTIVE: To correlate corneal endothelium-Descemet membrane (EDM) layer parameters of scroll tightness with donor age, endothelial cell density (ECD), and history of diabetes. METHODS: Endothelium-Descemet membrane layer scrolls were harvested from 26 corneoscleral buttons using the SCUBA technique by a cornea-fellowship trained ophthalmologist masked to donor age. Two independent outcome parameters were used to characterize the scrolling severity of successfully harvested tissue: scroll width and tendency for EDM scroll formation (referred to as scroll rating on a 1-4 scale: incomplete scroll formation to tightly scrolled). RESULTS: Mean donor age was 59 ± 17 (15-69) years. Mean ECD of EDM scroll was 2,451 ± 626 (range: 1,307-3,195) cells per square millimeter. Using stepwise linear regression, a significant correlation was found between scroll width and donor age (R=0.497, P<0.05). Additionally, a significant inverse correlation was found between scroll width and ECD (R=-0.605, P<0.05). There was no statistically significant correlation between a donor history of diabetes and the parameters of scrolling tendency. CONCLUSIONS: Our data suggest that using older donors reduces EDM scroll tightness.

MMP regulation of corneal keratocyte motility and mechanics in 3-D collagen matrices.

Previous studies have shown that platelet derived growth factor (PDGF) can stimulate corneal keratocyte spreading and migration within 3-D collagen matrices, without inducing transformation to a contractile, fibroblastic phenotype. The goal of this study was to investigate the role of matrix metalloproteinases (MMPs) in regulating PDGF-induced changes in keratocyte motility and mechanical differentiation. Rabbit corneal keratocytes were isolated and cultured in serum-free media (S-) to maintain their quiescent phenotype. A nested collagen matrix construct was used to assess 3-D cell migration, and a standard collagen matrix model was used to assess cell morphology and cell-mediated matrix contraction. In both cases constructs were cultured in S- supplemented with PDGF, with or without the broad spectrum MMP inhibitors GM6001 or BB-94. After 4 days, f-actin, nuclei and collagen fibrils were imaged using confocal microscopy. To assess sub-cellular mechanical activity (extension and retraction of cell processes), time-lapse DIC imaging was also performed. MT1-MMP expression and MMP-mediated collagen degradation were also examined. Results demonstrated that neither GM6001 nor BB-94 affected corneal keratocyte viability or proliferation in 3-D culture. PDGF stimulated elongation and migration of corneal keratocytes within type I collagen matrices, without causing a loss of their dendritic morphology or inducing formation of intracellular stress fibers. Treatment with GM6001 and BB-94 inhibited PDGF-induced keratocyte spreading and migration. Relatively low levels of keratocyte-induced matrix contraction were also maintained in PDGF, and the amount of PDGF-induced collagen degradation was similar to that observed in S- controls. The collagen degradation pattern was consistent with membrane-associated MMP activity, and keratocytes showed positive staining for MT1-MMP, albeit weak. Both matrix contraction and collagen degradation were reduced by MMP inhibition. For most outcome measures, the inhibitory effect of BB-94 was significantly greater than that of GM6001. Overall, the data demonstrate for the first time that even under conditions in which low levels of contractility and extracellular matrix proteolysis are maintained, MMPs still play an important role in mediating cell spreading and migration within 3-D collagen matrices. This appears to be mediated at least in part by membrane-tethered MMPs, such as MT1-MMP.

Techniques for assessing 3-D cell-matrix mechanical interactions in vitro and in vivo.

Cellular interactions with extracellular matrices (ECM) through the application of mechanical forces mediate numerous biological processes including developmental morphogenesis, wound healing and cancer metastasis. They also play a key role in the cellular repopulation and/or remodeling of engineered tissues and organs. While 2-D studies can provide important insights into many aspects of cellular mechanobiology, cells reside within 3-D ECMs in vivo, and matrix structure and dimensionality have been shown to impact cell morphology, protein organization and mechanical behavior. Global measurements of cell-induced compaction of 3-D collagen matrices can provide important insights into the regulation of overall cell contractility by various cytokines and signaling pathways. However, to understand how the mechanics of cell spreading, migration, contraction and matrix remodeling are regulated at the molecular level, these processes must also be studied in individual cells. Here we review the evolution and application of techniques for imaging and assessing local cell-matrix mechanical interactions in 3-D culture models, tissue explants and living animals.

Investigating transcriptional differences in mechanotransductive and ECM related genes in cultured primary corneal keratocytes, fibroblasts and myofibroblasts.

After a stromal injury in the cornea, the release of growth factors and pro-inflammatory cytokines typically results in the activation of quiescent keratocytes toward migratory fibroblast and/or fibrotic myofibroblast phenotypes. The persistence of the myofibroblast phenotype can lead to corneal fibrosis and scarring, which are leading causes of blindness worldwide. The primary goal of this study was to establish comprehensive transcriptional profiles for cultured corneal keratocytes, fibroblasts, and myofibroblasts to gain insights into the mechanisms through which changes in phenotype may occur. Here, we cultured primary rabbit corneal keratocytes on collagen-coated glass coverslips in serum free media (SF), serum containing media (FBS), or in the presence of TGF-ß1 to induce keratocyte, fibroblast, and myofibroblast phenotypes, respectively. Total RNA was collected and sent to Novogene for bulk RNA sequencing. Subsequent bioinformatic analysis included gene expression quantification, differential expression, and functional analysis. When comparing FBS and TGF-ß1 conditions to SF, genes characteristic of a quiescent keratocyte phenotype were downregulated (e.g. KERA, LUM, ALDH1A1), while genes commonly associated with fibroblasts or myofibroblasts were upregulated (e.g. VIM, TNC, FN1, ITGA5, ACTA2). Functional analysis of genes differentially expressed between fibroblasts and keratocytes highlighted pathways related to proliferation (e.g. DNA replication, PI3K-Akt signaling) and cell migration (e.g. Rap1 signaling, ECM-receptor interactions). Enriched pathways for the comparison of myofibroblasts to keratocytes included focal adhesion, regulation of actin cytoskeleton, hippo signaling, and ECM-receptor interaction pathways. Together, these pathways support changes in cytoskeletal organization, cell contractility, mechanotransduction, and cell-ECM interactions in myofibroblasts compared to keratocytes. Overall, these data demonstrate that there are distinct transcriptional differences between cultured corneal keratocytes, fibroblasts, and myofibroblasts. In our initial analysis, we have identified genes and signaling pathways that may play important roles in keratocyte differentiation, including many related to proliferation, cell mechanical activity, and ECM interactions. Furthermore, our findings reveal novel markers for each cell type as well as possible targets for modulating cell behavior and differentiation to promote physiological corneal wound healing.

Treatment with both TGF-ß1 and PDGF-BB disrupts the stiffness-dependent myofibroblast differentiation of corneal keratocytes.

During corneal wound healing, stromal keratocytes transform into a repair phenotype that is driven by the release of cytokines, like transforming growth factor-beta 1 (TGF-ß1) and platelet-derived growth factor-BB (PDGF-BB). Previous work has shown that TGF-ß1 promotes the myofibroblast differentiation of corneal keratocytes in a manner that depends on PDGF signaling. In addition, changes in mechanical properties are known to regulate the TGF-ß1-mediated differentiation of cultured keratocytes. While PDGF signaling acts synergistically with TGF-ß1 during myofibroblast differentiation, how treatment with multiple growth factors affects stiffness-dependent differences in keratocyte behavior is unknown. Here, we treated primary corneal keratocytes with PDGF-BB and TGF-ß1 and cultured them on polyacrylamide (PA) substrata of different stiffnesses. In the presence of TGF-ß1 alone, the cells underwent stiffness-dependent myofibroblast differentiation. On stiff substrata, the cells developed robust stress fibers, exhibited high levels of ⍺-SMA staining, formed large focal adhesions (FAs), and exerted elevated contractile forces, whereas cells in a compliant microenvironment showed low levels of ⍺-SMA immunofluorescence, formed smaller focal adhesions, and exerted decreased contractile forces. When the cultured keratocytes were treated simultaneously with PDGF-BB however, increased levels of ⍺-SMA staining and stress fiber formation were observed on compliant substrata, even though the cells did not exhibit elevated contractility or focal adhesion size. Pharmacological inhibition of PDGF signaling disrupted the myofibroblast differentiation of cells cultured on substrata of all stiffnesses. These results indicate that treatment with PDGF-BB can decouple molecular markers of myofibroblast differentiation from the elevated contractile phenotype otherwise associated with these cells, suggesting that crosstalk in the mechanotransductive signaling pathways downstream of TGF-ß1 and PDGF-BB can regulate the stiffness-dependent differentiation of cultured keratocytes. Statement of Significance: In vitro experiments have shown that changes in ECM stiffness can regulate the differentiation of myofibroblasts. Typically, these assays involve the use of individual growth factors, but it is unclear how stiffness-dependent differences in cell behavior are affected by multiple cytokines. Here, we used primary corneal keratocytes to show that treatment with both TGF-ß1 and PDGF-BB disrupts the dependency of myofibroblast differentiation on substratum stiffness. In the presence of both growth factors, keratocytes on soft substrates exhibited elevated ⍺-SMA immunofluorescence without a corresponding increase in contractility or focal adhesion formation. This result suggests that molecular markers of myofibroblast differentiation can be dissociated from the elevated contractile behavior associated with the myofibroblast phenotype, suggesting potential crosstalk in mechanotransductive signaling pathways downstream of TGF-ß1 and PDGF-BB.

Corneal stromal cells use both high- and low-contractility migration mechanisms in 3-D collagen matrices.

Corneal keratocyte migration can impact both corneal clarity and refractive outcome following injury or refractive surgery. In this study, we investigated how culture conditions, ECM properties, and Rho kinase activity regulate the mechanics of keratocyte migration, using a nested collagen matrix model. Time-lapse imaging demonstrated that both serum and PDGF stimulate keratocyte migration into the outer matrix. Although the velocity of cell migration was similar, cells in serum were bipolar and induced significant matrix deformation during migration, whereas PDGF induced extension of branching dendritic processes with smaller, more localized force generation. These differences in cell-induced matrix reorganization were verified with a global matrix contraction assay and confocal reflection imaging, using both bovine and rat tail collagen. When constructs were detached from the substrate to lower the effective stiffness, migration was significantly reduced in serum; but was unchanged in PDGF. These differences in migration mechanics were mediated, in part, by Rho kinase. Overall, corneal keratocytes can effectively migrate through collagen matrices using varying degrees of cellular force generation. Low-contractility migration may facilitate keratocyte repopulation of the stroma following surgery or injury, without altering the structural and mechanical properties that are critical to maintaining corneal transparency.

Sutureless Conjunctiva-Sparing Posterior Ptosis Repair Surgery: A Novel Technique.

PURPOSE: Müller muscle-conjunctival resection (MMCR) is a popular posterior/internal surgical approach to cases of mild to moderate blepharoptosis with good levator function. MMCR necessitates the removal of healthy conjunctiva and exposes the cornea to suture material. The goal of this study is to describe a novel sutureless conjunctiva-sparing Müllerectomy (CSM) surgery and demonstrate its long-term efficacy, efficiency, and safety. DESIGN: IRB approved retrospective study of patients undergoing sutureless conjunctiva-sparing posterior ptosis repair surgery. METHODS: The medical records of 100 patients (171 eyes) who underwent sutureless CSM with a minimum follow-up interval of 6 months were retrospectively reviewed. Photographs were analyzed using ImageJ software. Outcome measures were derived from margin reflex distance 1 (MRD1) and palpebral fissure height (PFH) at various postoperative timepoints. RESULTS: Mean ΔMRD1 and ΔPFH at 6 months were 2.85 ± 0.98 mm and 2.60 ± 1.38 mm, respectively. Symmetry within 1 mm was observed 91% of cases. Sutureless CSM took 4.42 minutes on average compared to 8.45 minutes for traditional MMCR. There were no corneal abrasions or ocular complications. The reoperation rate was 2.3% (1 case of overcorrection and 3 cases of undercorrection) per eye. CONCLUSIONS: Sutureless CSM is a promising alternative to traditional MMCR and sutured CSM based on long-term outcomes, symmetry, shorter operative time, and low complication rate. NOTE: Publication of this article is sponsored by the American Ophthalmological Society.

Loss of Corneal Nerves and Corneal Haze in Patients with Fuchs' Endothelial Corneal Dystrophy with the Transcription Factor 4 Gene Trinucleotide Repeat Expansion.

Objective: Seventy percent of Fuchs' endothelial corneal dystrophy (FECD) cases are caused by an intronic trinucleotide repeat expansion in the transcription factor 4 gene (TCF4). The objective of this study was to characterize the corneal subbasal nerve plexus and corneal haze in patients with FECD with (RE+) and without the trinucleotide repeat expansion (RE-) and to assess the correlation of these parameters with disease severity. Design: Cross-sectional, single-center study. Participants: Fifty-two eyes of 29 subjects with a modified Krachmer grade of FECD severity from 1 to 6 were included in the study. Fifteen of the 29 subjects carried an expanded TCF4 allele length of ≥ 40 cytosine-thymine-guanine repeats (RE+). Main Outcomes Measures: In vivo confocal microscopy assessments of corneal nerve fiber length (CNFL), corneal nerve branch density, corneal nerve fiber density (CNFD), and anterior corneal stromal backscatter (haze); Scheimpflug tomography densitometry measurements of haze in anterior, central, and posterior corneal layers. Results: Using confocal microscopy, we detected a negative correlation between FECD severity and both CNFL and CNFD in the eyes of RE+ subjects (Spearman ρ = -0.45, P = 0.029 and ρ = -0.62, P = 0.0015, respectively) but not in the eyes of RE- subjects. Additionally, CNFD negatively correlated with the repeat length of the expanded allele in the RE+ subjects (Spearman ρ = -0.42, P = 0.038). We found a positive correlation between anterior stromal backscatter and severity in both the RE+ and RE- groups (ρ = 0.60, P = 0.0023 and ρ = 0.44, P = 0.024, respectively). The anterior, central, and posterior Scheimpflug densitometry measurements also positively correlated with severity in both the RE+ and RE- groups (P = 5.5 × 10-5, 2.5 × 10-4, and 2.9 × 10-4, respectively, after adjusting for the expansion status in a pooled analysis. However, for patients with severe FECD (Krachmer grades 5 and 6), the posterior densitometry measurements were higher in the RE+ group than in the RE- group (P < 0.05). Conclusions: Loss of corneal nerves in FECD supports the classification of the TCF4 trinucleotide repeat expansion disorder as a neurodegenerative disease. Haze in the anterior, central, and posterior cornea correlate with severity, irrespective of the genotype. Quantitative assessments of corneal nerves and corneal haze may be useful to gauge and monitor FECD disease severity in RE+ patients.