RESUMO
The complex anatomy of the cornea and the subsequent keratocyte-fibroblast transition have always made corneal stromal regeneration difficult. Recently, 3D printing has received considerable attention in terms of fabrication of scaffolds with precise dimension and pattern. In the current work, 3D printable polymer hydrogels made of GelMA/agarose are formulated and its rheological properties are evaluated. Despite the variation in agarose content, both the hydrogels exhibited G'>G'' modulus. A prototype for 3D stromal model is created using Solid Works software, mimicking the anatomy of an adult cornea. The fabrication of 3D-printed hydrogels is performed using pneumatic extrusion. The FTIR analysis speculated that the hydrogel is well crosslinked and established strong hydrogen bonding with each other, thus contributing to improved thermal and structural stability. The MTT analysis revealed a higher rate of cell proliferation on the hydrogels. The optical analysis carried out on the 14th day of incubation revealed that the hydrogels exhibit transparency matching with natural corneal stromal tissue. Specific protein marker expression confirmed the keratocyte phenotype and showed that the cells do not undergo terminal differentiation into stromal fibroblasts. The findings of this work point to the potential of GelMA/A hydrogels as a novel biomaterial for corneal stromal tissue engineering.
RESUMO
AIM: To evaluate the clinical efficacy of small-diameter acellular porcine corneal stroma (SAPS) for the treatment of peripheral corneal ulceration (PCU). METHODS: This retrospective clinical study included 18 patients (18 eyes) with PCU between April 2018 and December 2020. All patients had PCU and underwent lamellar keratoplasty with SAPS. Observation indicators included preoperative and postoperative best-corrected visual acuity (BCVA) and transparency of SAPS. The infection control rate in the surgical eye-lesion area was also calculated. RESULTS: Eighteen patients underwent lamellar keratoplasty with SAPS to treat PCU. None of the patients experienced rejection after 6mo (18/18) and 12mo (16/16) of follow-up. The BCVA (0.47±0.30) at the 6mo follow-up after operation was significantly improved compared with the baseline (0.99±0.80), and the difference was statistically significant (Z=-3.415, P<0.05). The BCVA at the 12mo follow-up after operation was not statistically significant compared to the 6mo (Z=0, P=1). With time, the SAPS graft gradually became transparent. At the 6mo (18/18) and 12mo (16/16) follow-up, none of the patients had recurrent corneal infection. CONCLUSION: SAPS is clinically effective in the treatment of PCU, improving the patient's BCVA and reducing the incidence of rejection after keratoplasty.
RESUMO
A corneal stroma circular ring has captured by smartphone adaptor slit lamp camera after smile for 1 week.
RESUMO
PURPOSE: To verify corneal biomechanical changes, poststromal augmentation using myopic small-incision lenticule extraction's (SMILEs) lenticules in advanced keratoconus (KCN) through Corvis ST (Oculus, Wetzlar, Germany). MATERIALS AND METHODS: A clinical trial enrolled 22 advanced KCN patients. We implanted lenticules exceeding 100 µ according to a nomogram and evaluated biomechanical factors through Corvis ST at 3-, 6-, and 24-month postimplantation. We examined parameters during the first applanation (A1), second applanation (A2), highest concavity (HC)/max concavity events, and Vinciguerra screening parameters, as recently established criteria derived from the ideal blend of diverse biomechanical and ocular factors and formulated through the application of logistic regression. Regression analyses explored relationships with age, mean keratometry value, thickness, sphere, cylinder, and best-corrected visual acuity. RESULTS: Patients were well matched for age, intraocular pressure, and central corneal thickness (CCT). The mean spherical equivalent decreased from -13.48 ± 2.86 Diopters (D) to -8.59 ± 2.17 D (P < 0.007), and mean keratometry decreased from 54.68 ± 2.77 D to 51.95 ± 2.21 D (P < 0.006). Significant increases were observed in HC time (HCT), Radius-central curvature radius at the HC state-, peak distance (PD) during HC state, CCT, first applanation time, and stiffness parameter (A1T and SP-A1), whereas HC deformation amplitude, maximum deformation amplitude ratio at 2 mm, Corvis Biomechanical Index (CBI), integrated radius (IR), second applanation deformation amplitude (A2DA), first applanation velocity and deflection amplitude (A1V and A1DeflA) significantly decreased postlenticule implantation. Multivariable regression revealed age positively correlated with SP-A1 (P = 0.003) and negatively with HC delta Arc length (P = 0.007). Mean K positively correlated with CCT (P = 0.05) and negatively with CBI (P = 0.032). Best-corrected visual acuity positively correlated with HCT (P = 0.044), and the cylinder positively correlated with PD (P = 0.05) and CCT (P = 0.05) whereas negatively with IR (P = 0.025). CONCLUSIONS: Stromal augmentation using myopic SMILE lenticules induces significant corneal biomechanical changes in KCN.
RESUMO
PURPOSE: The human cornea is thicker in the periphery than the center and it has been suggested that this must be due to greater numbers of lamellae in the peripheral corneal stroma. The purpose of this study was to use high-resolution ultrastructural imaging to determine if the greater thickness of the peripheral cornea is due to the presence of more lamellae or if there is some other anatomical explanation. METHODS: In this study, full thickness corneas from three human donors were processed for light microscopy (LM) and transmission electron microscopy (TEM). Images were taken in three distinct stromal regions (anterior, middle, and posterior) from the central and peripheral cornea. Stromal thickness was evaluated by LM while TEM was used to evaluate numbers and thicknesses of lamellae, mean collagen fibril diameter, and mean collagen fibril density. RESULTS: Mean stromal thickness was significantly thinner in the central (415 ± 34 µm) compared to the peripheral (536 ± 29 µm) cornea (P = 0.009). Numbers of lamellae were not significantly different between central (246 ± 14) and peripheral (251 ± 14) cornea. Average lamellar thickness was not different across all regions of the cornea, except for the peripheral posterior where the lamellae were approximately 50 % thicker (P < 0.05). Collagen fibril diameters were larger in the peripheral cornea by approximately 30 % when compared to the central cornea, in all regions (P < 0.01). CONCLUSIONS: This study shows that it is an increase peripheral posterior lamellar thickness, rather than an increase in the number of lamellae, that accounts for the increase in corneal stromal thickness in the periphery of the human cornea. While collagen fibril diameters are greater throughout the peripheral stroma, the lamellae in the mid and anterior peripheral stroma are not thicker than centrally.
Assuntos
Córnea , Substância Própria , Humanos , Substância Própria/ultraestrutura , Substância Própria/diagnóstico por imagem , Córnea/ultraestrutura , Doadores de Tecidos , Pessoa de Meia-Idade , Masculino , Paquimetria Corneana , Microscopia Eletrônica de Transmissão , Feminino , Adulto , Idoso , Colágeno/metabolismo , Colágeno/ultraestruturaRESUMO
PURPOSE: This study aimed to measure the Raman spectrum of the human corneal stroma lens obtained from small incision lenticule extraction surgery (SMILE) in Asian myopic eyes using a confocal Raman micro-spectrometer built in the laboratory. METHODS: Forty-three myopic patients who underwent SMILE with equivalent diopters between - 4.00 and - 6.00 D were selected, and the right eye data were collected. Corneal stroma lenses were obtained during surgery, and the Raman spectra were measured after air drying. The complete Raman spectrum of human myopic corneal stroma lens tissue was obtained within the range of 700-4000 cm-1. RESULTS: Thirteen characteristic peaks were found, with the stronger peaks appearing at 937 cm-1, corresponding to proline, valine, and the protein skeleton of the human myopic corneal stroma lens; 1243 cm-1, corresponding to collagen protein; 1448 cm-1, corresponding to the collagen protein and phospholipids; and 2940 cm-1, corresponding to the amino acid and lipids, which was the strongest Raman peak. CONCLUSION: These results demonstrated that Raman spectroscopy has much potential as a fast, cost-effective, and reliable diagnostic tool in the diagnosis and treatment of eye diseases, including myopia, keratoconus, and corneal infection.
Assuntos
Cirurgia da Córnea a Laser , Ceratomileuse Assistida por Excimer Laser In Situ , Miopia , Humanos , Substância Própria/cirurgia , Acuidade Visual , Miopia/diagnóstico , Miopia/cirurgia , Ceratomileuse Assistida por Excimer Laser In Situ/métodos , Colágeno , Lasers de Excimer , Refração OcularRESUMO
PURPOSE: During life up to 70% of aniridia subjects develop aniridia-associated keratopathy (AAK). AAK is characterized by limbal stem cell insufficiency, impaired corneal epithelial cell differentiation and abnormal cell adhesion, which leads to centripetal spreading vascularization, conjunctivalization, and thickening of the cornea. Our aim was to examine the subbasal nerve plexus and central corneal stromal microstructure in subjects with congenital aniridia, using in vivo confocal laser scanning microscopy CLSM. METHODS: 31 eyes of 18 patients (55.6% males, mean age: 25.22 ± 16.35 years) with congenital aniridia and 46 eyes of 29 healthy subjects (41.4% males, mean age 30 ± 14.82 years) were examined using the Rostock Cornea Module of Heidelberg Retina Tomograph-III. At the subbasal nerve plexus, corneal nerve fiber density (CNFD), corneal nerve fiber length (CNFL), corneal total branch density (CTBD), and corneal nerve fiber width (CNFW) were analyzed using ACCMetrics software. Keratocyte density in the anterior, middle and posterior stroma was assessed manually. RESULTS: The CNFD (2.02 ± 4.08 vs 13.99 ± 6.34/mm2), CNFL (5.78 ± 2.68 vs 10.56 ± 2.82 mm/mm2) and CTBD (15.08 ± 15.62 vs 27.44 ± 15.05/mm2) were significantly lower in congenital aniridia subjects than in controls (p < 0.001 for all). CNFW was significantly higher in aniridia subjects than in controls (0.03 ± 0.004 vs 0.02 ± 0.003 mm/mm2) (p = 0.003). Keratocyte density was significantly lower in all stromal layers of aniridia subjects than in controls (p < 0.001 for all). Stromal alterations included confluent keratocytes, keratocytes with long extensions and hyperreflective dots between keratocytes in aniridia. CONCLUSIONS: Decrease in CNFD, CNFL, and CTBD, as well as increase in CNFW well refer to the congenital aniridia-associated neuropathy. The decreased keratocyte density and the stromal alterations may be related to an increased cell death in congenital aniridia, nevertheless, stromal changes in different stages of AAK have to be further analyzed in detail.
Assuntos
Aniridia , Substância Própria , Microscopia Confocal , Fibras Nervosas , Humanos , Aniridia/diagnóstico , Feminino , Masculino , Adulto , Substância Própria/patologia , Substância Própria/inervação , Fibras Nervosas/patologia , Adulto Jovem , Adolescente , Pessoa de Meia-Idade , Nervo Oftálmico/patologia , CriançaRESUMO
PURPOSE: To report a case of ulcerative keratopathy following implantation of acellular porcine corneal stroma (APCS) in a patient with keratoconus (KC). METHODS: A 58 year-old patient initially presented with an ulcerative keratopathy in the left eye. Previously, several corneal procedures (including radial keratotomy, laser-in-situ keratomileusis, crosslinking) were performed for KC. Eight months ago, an APCS lenticule (Xenia corneal implant, Gebauer Medizintechnik GmbH, Neuhausen, Germany) was implanted into a stromal pocket because of progressive keratectasia. Visual acuity was hand movement. Anterior segment optical coherence tomography showed a space between the APCS lenticule and the host stroma. Excimer laser-assisted penetrating keratoplasty (PKP, 8.0/8.1 mm) was performed in the left eye. The corneal explant was investigated by light and transmission electron microscopy. RESULTS: Best-corrected visual acuity was 20/40 six weeks after PKP. Light microscopy demonstrated a stromal ulceration down to the APCS lenticule. No stromal cells could be found within the APCS lenticule eight months after implantation. The APCS lenticule did not show a green stain of the collagens with Masson-Goldner staining and exhibited a strong Periodic acid-Schiff positive reaction. Electron microscopy of the APCS lenticule revealed cross-linked collagen lamellae without cellular components. Close to the interface, corneal collagen lamellae of the host cornea were disorganized. Few vital keratocytes were present on the surface of the lenticule and appeared to cause mechanical disruption of the host stroma along the lenticule-stroma interface. CONCLUSION: APCS implantation may lead to severe complications such as ulcerative keratopathy in otherwise uncomplicated KC corneas. In such cases, excimer laser-assisted PKP or Deep Anterior Lamellar Keratoplasty are the methods of choice to restore visual acuity.
Assuntos
Substância Própria , Úlcera da Córnea , Ceratocone , Acuidade Visual , Ceratocone/cirurgia , Ceratocone/fisiopatologia , Humanos , Substância Própria/cirurgia , Substância Própria/patologia , Pessoa de Meia-Idade , Animais , Suínos , Acuidade Visual/fisiologia , Úlcera da Córnea/cirurgia , Úlcera da Córnea/etiologia , Úlcera da Córnea/diagnóstico , Masculino , Tomografia de Coerência Óptica , Ceratoplastia Penetrante , Topografia da CórneaRESUMO
Keratoconus (KC) is a degenerative disorder resulting from the degradation of the stromal collagen fibril network in the cornea, leading to its thinning and conical deformation. Various studies have established animal models of KC by using the collagenase type II enzyme to gain a better understanding of the pathogenesis, however, long-term monitoring or follow-up of the models have not been reported so far. This study evaluates the long-term stability of collagenase type II-induced KC in a rabbit model. Six New Zealand rabbits were divided into 4 study groups with 3 eyes per group. The groups were control (group 1), 0.5% proparacaine + 5 min collagenase treatment on day 0 and day 30 (group 2), 0.5% proparacaine + 10 min collagenase treatment on day 0 (group 3) and, mechanical debridement + 2 min collagenase treatment on day 0 (group 4). Inflammation was observed in group 4 till week 10. Significant decrease in the central corneal thickness was observed in group 3 by week 4 (p < 0.001) however, the thickness was regained in the subsequent follow-ups in all the groups. Keratography results showed no changes in Km values but an increased astigmatic power in all groups. Scanning electron microscopy images revealed thinner collagen fibrils arranged in a mesh-like pattern above the uniform layer of the collagen lamellae in the central part of the treated corneas. Similarly, histological staining revealed loosely packed stromal fibrils in the anterior portion of the cornea which corroborates with the immunofluorescent staining results. This study revealed the remodeling of the corneal structure by eight weeks of collagenase treatment. Consequently, the possibility of creating a rabbit keratoconus model induced by collagenase may warrant further consideration.
Assuntos
Ceratocone , Propoxicaína , Coelhos , Animais , Ceratocone/induzido quimicamente , Ceratocone/tratamento farmacológico , Ceratocone/metabolismo , Córnea/metabolismo , Colágeno/metabolismo , Colagenases , Progressão da DoençaRESUMO
Corneal injury-induced stromal scarring causes the most common subtype of corneal blindness, and there is an unmet need to promote scarless corneal wound healing. Herein, a biomimetic corneal stroma with immunomodulatory properties is bioengineered for scarless corneal defect repair. First, a fully defined serum-free system is established to derive stromal keratocytes (hAESC-SKs) from a current Good Manufacturing Practice (cGMP)-grade human amniotic epithelial stem cells (hAESCs), and RNA-seq is used to validate the phenotypic transition. Moreover, hAESC-SKs are shown to possess robust immunomodulatory properties in addition to the keratocyte phenotype. Inspired by the corneal stromal extracellular matrix (ECM), a photocurable gelatin-based hydrogel is fabricated to serve as a scaffold for hAESC-SKs for bioengineering of a biomimetic corneal stroma. The rabbit corneal defect model is used to confirm that this biomimetic corneal stroma rapidly restores the corneal structure, and effectively reshapes the tissue microenvironment via proteoglycan secretion to promote transparency and inhibition of the inflammatory cascade to alleviate fibrosis, which synergistically reduces scar formation by ≈75% in addition to promoting wound healing. Overall, the strategy proposed here provides a promising solution for scarless corneal defect repair.
Assuntos
Lesões da Córnea , Substância Própria , Animais , Humanos , Coelhos , Biomimética , Córnea , Lesões da Córnea/terapia , Lesões da Córnea/patologia , Cicatriz/patologiaRESUMO
BACKGROUND: Corneal tissues indirectly obtain nutritional needs and oxygen to maintain their homeostasis, and therefore, benzalkonium chloride (BAC) containing ocular instillations for medical therapy may, in turn, induce toxic effects more than expected in corneal tissues, especially the inside stroma layer. METHODS: To evaluate the effects of very low concentrations (10-8%, 10-6%, or 10-4%) of BAC on human corneal stroma, we used two-dimensional (2D) cultures of human corneal stromal fibroblast (HCSF) cells and carried out the following analyses: (1) cell viability measurements, (2) Seahorse cellular bio-metabolism analysis, and (3) the expression of ECM molecules and endoplasmic reticulum (ER) stress-related molecules. RESULTS: In the absence and presence of 10-8%, 10-6%, or 10-4% concentrations of BAC, cell viability deteriorated and this deterioration was dose-dependent. The results showed that maximal mitochondrial respiration was decreased, the mRNA expression of most of ECM proteins was decreased, and ER stress-related molecules were substantially and dose-dependently down-regulated in HCSFs by the BAC treatment. CONCLUSIONS: The findings reported herein indicate that the presence of BAC, even at such low concentrations, is capable of causing the deterioration of cellular metabolic functions and negatively affecting the response to ER stress in HCSF cells resulting in a substantially decreased cellular viability.
Assuntos
Compostos de Benzalcônio , Sobrevivência Celular , Substância Própria , Conservantes Farmacêuticos , Humanos , Compostos de Benzalcônio/toxicidade , Substância Própria/efeitos dos fármacos , Substância Própria/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Conservantes Farmacêuticos/toxicidade , Relação Dose-Resposta a Droga , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Ceratócitos da Córnea/efeitos dos fármacos , Ceratócitos da Córnea/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
ABSTRACT Purpose: This study aimed to report an experiment designed to determine anatomical changes in porcine corneas following placement of a novel polymer implant into the cornea. Methods: An ex vivo porcine eye model was used. A novel type I collagen-based vitrigel implant (6 mm in diameter) was shaped with an excimer laser on the posterior surface to create three planoconcave shapes. Implants were inserted into a manually dissected stromal pocket at a depth of approximately 200 μm. Three treatment groups were defined: group A (n=3), maximal ablation depth 70 μm; Group B (n=3), maximal ablation depth 64 μm; and group C (n=3), maximal ablation depth 104 μm, with a central hole. A control group (D, n=3) was included, in which a stromal pocket was created but biomaterial was not inserted. Eyes were evaluated by optical coherence tomography (OCT) and corneal tomography. Results: Corneal tomography showed a trend for a decreased mean keratometry in all four groups. Optical coherence tomography showed corneas with implants placed within the anterior stroma and visible flattening, whereas the corneas in the control group did not qualitatively change shape. Conclusions: The novel planoconcave biomaterial implant described herein could reshape the cornea in an ex vivo model, resulting in the flattening of the cornea. Further studies are needed using in vivo animal models to confirm such findings.
RESUMO Objetivo: Relatar um experimento projetado para determinar alterações anatômicas em córneas porcinas após a colocação de um novo implante de polímero na córnea. Métodos: Foi utilizado olho de porco ex vivo. Um novo agente modelador biocompatível, de colágeno tipo 1, com 6mm de diâmetro foi moldado com excimer laser em sua face posterior, para criar três formatos planocôncavos. Os implantes foram inseridos dentro de um bolsão, dissecado manualmente, a 200 micrômetros (μm). Foram definidos três grupos de tratamento: grupo A (n=3), teve a profundidade máxima de ablação de 70 μm; o grupo B (n=3), profundidade máxima de ablação de 64 μm; e o grupo C (n=3), profundidade máxima de ablação de 104 μm, com buraco central. O grupo controle, D (n=3), foi incluído, com a criação do bolsão estromal, porém sem inserir o material. A avaliação desses olhos foi realizada por tomografia de coerência óptica (OCT) e por tomografia corneana. Resultados: A tomografia corneana mostrou uma tendência para diminuição da ceratometria média em todos os 4 grupos. A tomografia de coerência óptica mostrou córneas com implantes localizados no estroma anterior e aplanamento visível, enquanto as córneas não mudaram qualitativamente o formato no grupo controle. Conclusões: O novo implante de biomaterial planocôncavo descrito aqui foi capaz de remodelar a córnea em modelo de animal ex vivo, resultando no aplanamento corneano. Novos estudos são necessários usando modelos animais in vivo para confirmar tais achados.
RESUMO
PURPOSE: We investigated healing pattern of an incisional wound in corneal stroma of lumican-null (KO) mice. METHODS: C57BL/6 mice (wild-type, WT) and lumican-null (knockout, KO) mice were used. A linear full-thickness incision was produced in one cornea of each mouse. After intervals of healing, the corneas were processed for the following analyses. Histology was employed to measure the distance between each edge of the disrupted Descemet's membrane at the center of the cornea. Immunohistochemistry and real-time RT-PCR were employed to evaluate the expression of wound healing-related components in the tissue. Cultured ocular fibroblasts were obtained from cornea and sclera of WT and KO postnatal day 1 pups. The cells were subjected to examination for cell proliferation and expression of wound healing-related gene products. In vitro gel contraction assay was used to asses cell contractile activity of WT and KO cells. RESULTS: At day 5 of incision, the distance between the disrupted Descemet's membrane was larger in a KO mouse as compared with a WT mouse. Myofibroblast appearance in the wound was suppressed by the loss of lumican. The loss of lumican downregulated TGFß1's effects on mRNA expression of α-smooth muscle actin and collagen Ia1 in cultured ocular fibroblasts. Cell proliferation rate increased in injured stroma, which was further supported by in vitro datum of cell proliferation augmentation by the loss of lumican. Loss of lumican suppressed cell-mediated gel contraction. CONCLUSION: Loss of lumican perturbs the healing of penetrating incision in mouse corneal stroma in association with suppression of myofibroblast generation.
Assuntos
Substância Própria , Cicatrização , Animais , Camundongos , Substância Própria/patologia , Lumicana/metabolismo , Camundongos Endogâmicos C57BL , Cicatrização/fisiologia , Córnea/patologiaRESUMO
Cornea transplantation is one of the most commonly performed allotransplantations worldwide. Prolonged storage of donor corneas leads to decreased endothelial cell viability, severe stromal edema, and opacification, significantly compromising the success rate of corneal transplantation. Corneal stroma, which constitutes the majority of the cornea, plays a crucial role in maintaining its shape and transparency. In this study, we conducted proteomic analysis of corneal stroma preserved in Optisol-GS medium at 4 °C for 7 or 14 days to investigate molecular changes during storage. Among 1923 identified proteins, 1634 were quantifiable and 387 were significantly regulated with longer preservation. Compared to stroma preserved for 7 days, proteins involved in ocular surface immunomodulation were largely downregulated while proteins associated with extracellular matrix reorganization and fibrosis were upregulated in those preserved for 14 days. The increase in extracellular matrix structural proteins together with upregulation of growth factor signaling implies the occurrence of stromal fibrosis, which may compromise tissue clarity and cause vision impairments. This study is the first to provide insights into how storage duration affects corneal stroma from a proteomic perspective. Our findings may contribute to future research efforts aimed at developing long-term preservation techniques and improving the quality of preserved corneas, thus maximizing their clinical application.
Assuntos
Criopreservação , Proteômica , Humanos , Criopreservação/métodos , Córnea , Substância Própria/metabolismo , Matriz Extracelular , Gentamicinas/metabolismo , Misturas Complexas/metabolismoRESUMO
Lacking specimens is the biggest limitation of studying the mechanical behaviors of human corneal. Extracting stress-strain curves is the crucial step in investigating hyperelastic and anisotropic properties of human cornea. 15 human corneal specimens extracted from the small incision lenticule extraction (SMILE) surgery were applied in this study. To accurately measure the personalized true stress-strain curve using corneal lenticules, the digital image correlation (DIC) method and finite element method were used to calibrate the stress and the strain of the biaxial extension test. The hyperelastic load-displacement curves obtained from the biaxial extension test were performed in preferential fibril orientations, which are arranged along the nasal-temporal (NT) and the superior-inferior (SI) directions within the anterior central stroma. The displacement and strain fields were accurately calibrated and calculated using the digital image correlation (DIC) method. A conversion equation was given to convert the effective engineering strain to the true strain. The stress field distribution, which was simulated using the finite element method, was verified. Based on this, the effective nominal stress with personalized characteristics was calibrated. The personalized stress-strain curves containing individual characteristic, like diopter and anterior surface curvature, was accurately measured in this study. These results provide an experimental method using biaxial tensile test with corneal lenticules. It is the foundation for investigating the hyperelasticity and anisotropy of the central anterior stroma of human cornea.
Assuntos
Córnea , Substância Própria , Humanos , Anisotropia , Calibragem , Análise de Elementos FinitosRESUMO
To examine the epithelial-mesenchymal transition (EMT) that is induced on the human corneal stroma, two- and three-dimensional (2D and 3D) cultures of human corneal stroma fibroblasts (HCSFs) were used. In this study, HCSF 2D monolayers and 3D spheroids were characterized by (1) scanning electron microscopy (SEM), (2) trans-endothelial electrical resistance (TEER) measurements and fluorescein isothiocyanate (FITC)-dextran permeability, (3) cellular metabolic measurements, (4) the physical properties of 3D HCSF spheroids, and (5) the extracellular matrix (ECM) molecule gene expressions, including collagen (COL) 1, 4 and 6, and fibronectin (FN), a tissue inhibitor of metalloproteinase (TIMP) 1-4, matrix metalloproteinase (MMP) 2, 3, 9 and 14, and several endoplasmic reticulum (ER) stress-related factors. In the 2D HCSFs, TGF-ß2 concentration-dependently generated (1) a considerable increase in ECM deposits revealed by SEM, (2) an increase in TEER values and a decrease in FITC-dextran permeability, (3) increases in both mitochondrial and glycolytic functions, and a substantial upregulation of COL1, COL4, FN, αSMA, TIMP1, TIMP, and most ER stress-related genes and the downregulation of COL6 and MMP3. In the case of 3D spheroids, TGF-ß2 induced the downsizing and stiffening of 3D spheroids and the upregulation of COL6, MMP14, and most ER stress-related genes. These findings suggest that TGF-ß2 significantly induced a number of EMT-associated biological events including planar proliferation, cellular metabolic functions, and the production of ECM molecules in the 2D cultured HCSF cells, but these effects were significantly less pronounced in the case of 3D HCSF spheroids.
RESUMO
This study investigated the interplay between transforming growth factor beta (TGF-ß1/T1 and TGF-ß3/T3), and sex hormone receptors using our 3D in vitro cornea stroma model. Primary human corneal fibroblasts (HCFs) from healthy donors were plated in transwells at 106 cells/well and cultured for four weeks. HCFs were supplemented with stable vitamin C (VitC) and stimulated with T1 or T3. 3D construct proteins were analyzed for the androgen receptor (AR), progesterone receptor (PR), estrogen receptor alpha (ERα) and beta (ERß), luteinizing hormone receptor (LHR), follicle-stimulating hormone receptor (FSHR), gonadotropin-releasing hormone receptor (GnRHR), KiSS1-derived peptide receptor (KiSS1R/GPR54), and follicle-stimulating hormone subunit beta (FSH-B). In female constructs, T1 significantly upregulated AR, PR, ERα, FSHR, GnRHR, and KiSS1R. In male constructs, T1 significantly downregulated FSHR and FSH-B and significantly upregulated ERα, ERß, and GnRHR. T3 caused significant upregulation in expressions PR, ERα, ERß, LHR, FSHR, and GNRHR in female constructs, and significant downregulation of AR, ERα, and FSHR in male constructs. Semi-quantitative Western blot findings present the interplay between sex hormone receptors and TGF-ß isoforms in the corneal stroma, which is influenced by sex as a biological variable (SABV). Additional studies are warranted to fully delineate their interactions and signaling mechanisms.
Assuntos
Substância Própria , Fator de Crescimento Transformador beta3 , Humanos , Feminino , Masculino , Receptor alfa de Estrogênio , Receptores de Kisspeptina-1 , Receptor beta de Estrogênio/genética , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta , Hormônio FoliculoestimulanteRESUMO
Corneal activated keratocytes (CAKs) -representing the injured phenotype of corneal stromal cells- are associated with several corneal diseases. Inflammatory cytokines are the key drivers of CAK formation subsequently leading to fibrogenesis. This study aimed to investigate the effect of adlay seed extract on the expression of genes involved in inflammation (IL-6, IL-1b, LIF) and fibrogenesis (TGF-ß) in CAK cells. CAKs (106 cells/10 cm2) were exposed to methanolic (MeOH) and residual (Res) extract of adlay seed (1 mg/ml, 24 h). The control group received the vehicle solution without extract at the same time and condition. Then, RNA extraction, cDNA synthesis, and real-time PCR were performed to quantify the relative expression of IL-6, IL-1b, LIF, and TGF-ß in the treated vs. control cells. This study showed that the MeOH extract of adlay seed could significantly downregulate the expression of IL-6 and IL-1b in the CAKs, while the Res extract led to a significant decrease in TGF-ß gene expression. We showed that CAK treatment with adlay seed extract could decrease the expression of genes related to inflammation and fibrogenesis. However, the genes to be targeted depended on the method of extraction. This proof-of-concept study could provide groundwork for the treatment of corneal stromal diseases and ocular regenerative medicine in the future.
Assuntos
Doenças da Córnea , Interleucina-6 , Humanos , Interleucina-6/genética , Ceratócitos da Córnea , Inflamação , Córnea , Metanol , Extratos Vegetais/farmacologiaRESUMO
Purpose: Compare the safety and efficacy of wavefront-guided photorefractive keratotomy (PRK) 6 months after cross-linking (CXL) to wavefront-guided PRK alone for refractive correction in patients with bilateral asymmetric corneal topography. Methods: Prospective randomized clinical trial with 16 patients (32 eyes). CXL with subsequent PRK after 6 months in one eye, and PRK alone was performed in contralateral eyes. The follow-up was 10 years. We analyzed visual outcomes, Scheimpflug topography, and corneal haze evaluation. Results: Eyes in the PRK group showed better results than in the CXL + PRK group. Mean postoperative CDVA was 0.044 logmar (SD, 0.073) in the PRK group and 0.1 logmar (SD, 0.21) in the CXL + PRK group, the mean sphere was + 0.21 (SD, 0.6) D in the PRK group and 0.87 (SD, 2.3) D in the CXL + PRK group, and mean SE was -0.35 (SD, 0.65) D in the PRK group and 0.62 (SD, 2.32) D in the CXL + PRK group. In one patient, a steepening of 2.5 D and a thinning of 17 µm occurred in PRK alone group. Two patients in the CXL + PRK group presented corneal haze. The overall complication rate was 18,75% (haze and ectasia). Conclusion: Non-simultaneous CXL and PRK procedures yielded good refractive results, but worse than those obtained with PRK alone. Although one patient in the PRK group developed corneal ectasia, the CXL + PRK group had a higher loss of vision lines, indicating less safety.
Assuntos
Crosslinking Corneano , Opacidade da Córnea , Humanos , Topografia da Córnea , Dilatação Patológica , Seguimentos , Estudos ProspectivosRESUMO
BACKGROUND: To evaluate the ability of anterior segment optical coherence tomography (AS-OCT) to visualize the anatomic features of the pterygium and its invasion of the corneal layers. METHODS: Seventy-five eyes of 54 patients diagnosed with pterygium were included. All subjects underwent complete ophthalmologic examinations, including AS-OCT. The limbus-apex distance, vertical height at the limbus, invasion of the Bowman's and stromal layers, and other morphologic structures of the pterygium tissue were analyzed. RESULTS: The mean age of the patients was 49.67 ± 16.49 (20-85) years. The mean apex-limbus distance was 2548.37 ± 1026.32 (933-4597) µm, and the mean vertical height at the limbus was 4843.89 ± 1374.10 (1740-7784) µm. A space was observed beneath the pterygium tissue in 44 (58.67%) eyes. The mean width and height of this space were 1756.33 ± 560.22 (1009-3095) µm and 231.70 ± 85.88 (109-465) µm, respectively. Invasion of the Bowman's layer was apparent in 74 (98.67%) eyes, and invasion of the stromal layer was detected in 33 (44%) eyes. A hyperreflective layer was observed beneath the epithelial layer at the edge of the pterygium apex in 31 (41.33%) eyes. In 24 (92.31%) of the 26 advanced pterygium cases and 20 (40.82%) of the 49 early pterygium cases, a subpterygium space was found beneath the lesion (p = 0.0001). CONCLUSION: AS-OCT enables measurement of the actual size and thickness of pterygia, assessment of invasion of the Bowman's and stromal layers of the cornea, and evaluation of the pterygium structure. Over half of the eyes exhibited space beneath the pterygium.