Keratocyte Proliferation Research Articles

Mesenchymal stem cells derived-exosomes enhanced amniotic membrane extract promotes corneal keratocyte proliferation.

Amniotic membrane extract (AME) and Wharton's jelly mesenchymal stem cells derived-exosomes (WJ-MSC-Exos) are promising therapeutic solutions explored for their potential in tissue engineering and regenerative medicine, particularly in skin and corneal wound healing applications. AME is an extract form of human amniotic membrane and known to contain a plethora of cytokines and growth factors, making it a highly attractive option for topical applications. Similarly, WJ-MSC-Exos have garnered significant interest for their wound healing properties. Although WJ-MSC-Exos and AME have been used separately for wound healing research, their combined synergistic effects have not been studied extensively. In this study, we evaluated the effects of both AME and WJ-MSC-Exos, individually and together, on the proliferation of corneal keratocytes as well as their ability to promote in vitro cell migration, wound healing, and their impact on cellular morphology. Our findings indicated that the presence of both exosomes (3 × 105 Exo/mL) and AME (50 μg/mL) synergistically enhance the proliferation of corneal keratocytes. Combined use of these solutions (3 × 105 Exo/mL + 50 μg/mL) increased cell proliferation compared to only 50 μg/mL AME treatment on day 3 (**** p < 0.0001). This mixture treatment (3 × 105 Exo/mL + 50 μg/mL) increased wound closure rate compared to isolated WJ-MSC-Exo treatment (3 × 105 Exo/mL) (*p < 0.05). Overall, corneal keratocytes treated with AME and WJ-MSC-Exo (3 × 105 Exo/mL + 50 μg/mL) mixture resulted in enhanced proliferation and wound healing tendency. Utilization of combined use of AME and WJ-MSC-Exo can pave the way for a promising foundation for corneal repair research.

Climate change with increasing seawater temperature will challenge the health of farmed Atlantic Cod (Gadus morhua L.)

Aquaculture is one of the fastest growing food production sectors in the world and further expansion is expected throughout the 21st century. However, climate change is threatening the development of the sector and action is needed to prepare the industry for the coming challenges. Using downscaled temperature projections based on the Intergovernmental Panel on Climate Change (IPCC) climate projection (Shared Socioeconomic Pathway, SSP2-4.5), we analysed potential future temperatures at a selected Atlantic cod (Gadus morhua L.) farm site in Northern Norway. Results showed that the farming area may experience increased temperatures the next 10–15 years, including more days with temperatures above 17°C. Based on the predicted future conditions, we designed a study with Atlantic cod (Gadus morhua L.) to evaluate effects from high temperature alone and in combination with Fransicella noatunensis infection. Fish were kept at 12°C and 17°C for eight weeks and samples of skin and spleen collected at different timepoints were analysed with transcriptomics, histology, scanning electron microscopy and immunohistochemistry. Results showed that high temperature had a stronger effect on the barrier functions of skin than the infection. Increased temperature induced gene expression changes in skin and spleen, heat shock protein 47 and cold inducible RNA binding protein were identified as potential gene markers for thermal stress. The effect of bacterial challenge was small at 12°C. At high temperature, the development of severe pathology in spleen coincided with a significant decrease of immunoglobulins transcripts, which contrasted with the activation of multiple immune genes. In addition, we used an in vitro model of skin biopsies and scale explants exposed to hydrogen peroxide (H2O2) to assess the effects of thermal and oxidative stress. High temperature and H2O2 reduced proliferation and migration of keratocytes, and increased expression of stress markers, and compounding effects were observed with combined stressors. Results suggest that the projected increased seawater temperature will pose a significant threat to Norwegian cod farming, affecting various biological processes and making fish more vulnerable to stressors and pathogens. Cod farming needs high attention to temperature changes, and special precautions should be taken if the temperature increases beyond cods’ thermal optimum.

Comparative analysis of the regenerative potential of blood derivatives on a cell model of corneal stromal injury

BACKGROUND: Agents based on blood derivatives contain substances that accelerate the healing of wound defects. Nowadays data have been accumulated that various blood derivatives have different effects on regeneration processes. Therefore, a comparative study of the effect of several types of such preparations on the corneal stromas cell culture is relevant.&#x0D; AIM: Impact assessment of 3 different blood derivatives on the processes of proliferation, migration and cell death of keratocytes in vitro.&#x0D; MATERIAL AND METHODS: This study was conducted on a primary cell culture of human corneal keratocytes. Blood samples were obtained from the cubital vein of healthy volunteers after signing informed consent to participate in the study. Monolayer wound healing test, formazan test to assess proliferation, staining for Annexin V to assess the processes of apoptosis and necrosis were perfomed. Moreover, effect evaluation of platelet-rich plasma, intact serum and processed serum were performed.&#x0D; RESULTS: It was shown that all 3 stimulants have a positive effect on the regeneration of the experimental stromal defect, but it is implemented in different ways. Processed whey showed the greatest stimulating effect on the process of cell migration. This serum reduced the level of cell death, but had almost no effect on proliferation processes. It is advisable to further study different types of stimulants to obtain processed serum. Intact serum increased the percentage of dividing cells more than others, but also proportionally increased the percentage of death in culture. Platelet-rich plasma showed the weakest effect on the migratory ability of keratocytes, but it still differed significantly from the control. This stimulator had little effect on the process of cell division, and had no effect on the overall level of cell death.&#x0D; CONCLUSION: Experimental data can be taken into account in choosing of the management in clinical practice and personalized usage of blood derivatives, specifically in prescribing some blood derivatives to accelerate regeneration, and other derivatives for reducing cell deaths processes.

Intra-stromal injection of honey-treated keratocytes as a cell-based therapy for experimental corneal laceration.

This study aimed to investigate the potential of honey-supplemented medium (HSM) for expanding corneal keratocytes and its transplantation in a model of corneal laceration. Keratocytes were cultured in 1 % HSM- or 10 % fetal bovine serum (FBS)-supplemented medium for 24 h. The effect of HSM on keratocyte proliferation was evaluated using the MTT assay. The relative expression of Lum, Kera, and ALDH3A1, known markers of native keratocytes, was quantified by real-time PCR. The safety and efficacy of HSM-treated keratocyte intrastromal injection in a rabbit model of corneal laceration were also evaluated. The MTT assay showed that HSM treatment didnot significantly affect cell viability compared to FBS-supplemented medium (84.71±2.38 vs. 100.08±10.92, respectively; p=0.076). Moreover, HSM-treated keratocytes had significantly increased expression of Lum, Kera,andALDH3A1 compared to cells treated with FBS, while theexpression of the proliferation biomarker Thy-1 did notsignificantly differ between the two treatments. Intrastromal injection of HSM-treated keratocytes in the laceration animal model was safe and uneventful, resulting in less stromal inflammation and neovascularization, and consequently, better final architecture with less residual haze compared to the group injected with FBS-treated keratocytes. These findings suggest that honey is a suitable supplement for keratocyte treatment and corneal cell therapy. The use of HSM may have potential applications in the treatment of corneal injuries and diseases.

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-β1) 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-β1-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.

Effect of Thyroxine on Transforming Growth Factor β1, Collagen I, and V Expression in Keratoconus Corneal Fibroblasts and Keratocytes, in Vitro

ABSTRACT Background Keratoconus (KC) is a corneal disorder, associated with oxidative stress, hypoxia and as several times discussed, potentially with thyroid gland dysfunction. We aimed to investigate the effect of thyroxine on transforming growth factor β1 (TGF-β1), collagen I and V (Col I and V) expression in human corneal fibroblasts (HCFs) and human keratocytes of KC corneas, in vitro. Methods Primary human KC-keratocytes and normal keratocytes were isolated and cultured as corneal fibroblasts or keratocytes. The effect of 0.1 µg/ml and 1.0 µg/ml thyroxine on TGF-β1, Col I and Col V expression was investigated by qPCR, Western blot, and ELISA. Proliferation assay was performed using BrdU ELISA to observe the 24h effect of 1.0 µg/ml thyroxine on keratocytes, in vitro. Results TGFB1 mRNA expression of normal keratocytes increased following 1.0 µg/ml thyroxine stimulation for 24 h (p = .036), without changes in protein expression. Col I protein expression of KC-HCFs increased following 1.0 µg/ml thyroxine stimulation for 24 h (p = .0003). Proliferation of normal and KC keratocytes increased following a 7-day growth period and 24 hours thyroxine administration (p = .018; p = .024). Conclusions Thyroxine may affect the Col I protein expression in KC-HCFs, but not in KC keratocytes, in vitro. Thyroxine administration has no effect on TGF-β1, collagen I and V expression of keratoconus keratocytes. Therefore, an increased thyroxine concentration alone seems not to be causally related to the development of keratoconus.

L-carnitine suppresses transient receptor potential vanilloid type 1 activity and myofibroblast transdifferentiation in human corneal keratocytes

Corneal stromal wound healing is a well-balanced process promoted by overlapping phases including keratocyte proliferation, inflammatory-related events, and tissue remodeling. L-carnitine as a natural antioxidant has shown potential to reduce stromal fibrosis, yet the underlying pathway is still unknown. Since transient receptor potential vanilloid 1 (TRPV1) is a potential drug target for improving the outcome of inflammatory/fibrogenic wound healing, we investigated if L-carnitine can mediate inhibition of the fibrotic response through suppression of TRPV1 activation in human corneal keratocytes (HCK). We determined TRPV1-induced intracellular calcium transients using fluorescence calcium imaging, channel currents by planar patch-clamping, and cell migration by scratch assay for wound healing. The potential L-carnitine effect on TRPV1-induced myofibroblast transdifferentiation was evaluated by immunocytochemical detection of alpha smooth muscle actin. RT-PCR analysis confirmed TRPV1 mRNA expression in HCK. L-carnitine (1 mmol/l) inhibited either capsaicin (CAP) (10 µmol/l), hypertonic stress (450 mOsmol/l), or thermal increase (>43 °C) induced Ca2+ transients and corresponding increases in TRPV1-induced inward and outward whole-cell currents. This was accompanied by suppression of injury-induced increases in myofibroblast transdifferentiation and cell migration. In conclusion, L-carnitine contributes to inhibit stromal scarring through suppressing an injury-induced intrinsic TRPV1 activity that is linked with induction of myofibroblast transdifferentiation in HCK cells.

Effect of spirulina on corneal epithelial wound healing in zebrafish

This study investigated the regenerative and healing effect of spirulina on corneal epithelial injury by acid burn in zebrafish. Corneal epithelium of adult zebrafish was evaluated at 0, 12, 24, 48 h after acid burn injury with or without immersion with 0.02 and 0.04 mg/mL of spirulina water extract. The pathological changes of corneal epithelium were assessed by light microscopy. Gene and protein expressions of proliferating cell nuclear antigen (PCNA), matrix metalloproteinase (MMP) 9 and MMP13 were determined by immunohistochemistry or real-time PCR. Corneal epithelium was denuded totally after acid burn and gradually regenerated. PCNA-positive cells significantly increased in spirulina treated group. MMP9 and MMP13 mRNA transcripts were significantly decreased in spirulina treated groups. In conclusion, spirulina has regenerative and healing properties by increased keratocyte proliferation, and inhibited extracellular matrix degradation during the regeneration process of corneal epithelial injury by acid burn in zebrafish.

Short- and Long-Term Stability of Plasma Rich in Growth Factors Eye Drops.

To analyze whether plasma rich in growth factors (PRGFs) eye drops preserve their activity and biological properties after storage for 9 and 12 months at -20°C, and at 4°C, and at room temperature (RT) for 3 and 7 days in comparison to fresh samples (t0). PRGF eye drops were obtained from 6 healthy donors. Then, they were stored for 9 and 12 months at -20°C. At each time, different PRGF eye drops samples were thawed and maintained at RT or at 4°C for 3 and 7 days. Platelet-derived growth factor-AB, epidermal growth factor, transforming growth factor-β1, vascular endothelial growth factor, angiopoietin-1, and thrombospondin-1 were analyzed at each time and temperature of storage. In addition, the pH level, the microbial contamination, and the proliferative potential on primary human corneal stromal fibroblasts human keratocytes of each obtained PRGF eye drops were also evaluated. All growth factor levels were preserved at each time and storage condition. No differences were observed on the human keratocytes proliferation after treatment with PRGF eye drops at any studied time or temperature. No microbial contamination was observed in any of the PRGF eye drops. Finally, the pH levels increased significantly after 9 and 12 months of storage at -20°C compared with t0. PRGF eye drops can be stored for up to 12 months without reduction of the main growth factors and proteins and without any microbial contamination. Furthermore, the biological activity of the PRGF eye drops is maintained after storing for 3 and 7 days at 4°C or at RT.

Regulation of Keratocyte Phenotype and Cell Behavior by Substrate Stiffness.

Corneal tissue engineering is an alternative way to solve the problem of lack of corneal donor tissue in corneal transplantation. Keratocytes with a normal phenotype and function in tissue-engineered cornea would be critical for corneal regeneration. Although the role of extracellular/substrate material stiffness is well-known for the regulation of the cell phenotype and cell behavior in many different cell types, its effects in keratocyte culture have not yet been thoroughly studied. This project studied the effect of substrate stiffness on the keratocyte phenotype marker expression and typical cell behavior (cell adhesion, proliferation, and migration), and the possible mechanisms involved. Human primary keratocytes were cultured on tissue culture plastic (TCP, ∼106 kPa) or on plates with the stiffness equivalent of physiological human corneal stroma (25 kPa) or vitreous body (1 kPa). The expression of keratocyte phenotype markers, cell adhesion, proliferation, and migration were compared. The results showed that the stiffness of the substrate material regulates the phenotype marker expression and cell behavior of cultured keratocytes. Physiological corneal stiffness (25 kPa) superiorly preserved the cell phenotype when compared to the TCP and 1 kPa group. Keratocytes had a larger cell area when cultured on 25 kPa plates as compared to on TCP. Treatment of cells with NSC 23766 (Rac1 inhibitor) mimicked the response in the cell phenotype and behavior seen in the transition from soft materials to stiff materials, including the cytoskeletal structure, expression of keratocyte phenotype markers, and cell behavior. In conclusion, this study shows that substrate stiffness regulates the cell phenotype marker expression and cell behavior of keratocytes by Rac1-mediated cytoskeletal reorganization. This knowledge contributes to the development of corneal tissue engineering.

Vitamin C Loaded Poly(urethane-urea)/ZnAl-LDH Aligned Scaffolds Increase Proliferation of Corneal Keratocytes and Up-Regulate Vimentin Secretion.

A novel poly(urethane-urea) (PUU) based on poly(glycolide-co-ε-caprolactone) macro-diol with tunable mechanical properties and biodegradation behavior is reported for corneal stromal tissue regeneration. Zn-Al layered double hydroxide (LDH) nanoparticles were synthesized and loaded with vitamin C (VC, VC-LDH) and dispersed in the PUU to control VC release in the cell culturing medium. To mimic the corneal stromal EC, scaffolds of the PUU and its nanocomposites with VC-LDH (PUU-LDH and PUU-VC-LDH) were fabricated via electrospinning. Average diameters of the aligned nanofibers were recorded as 325 ± 168, 343 ± 171, and 414 ± 275 nm for the PUU, PUU-LDH, and PUU-VC-LDH scaffolds, respectively. Results of hydrophilicity and mechanical properties measurements showed increased hydrophobicity and reduced tensile strength and elongation at break upon addition of nanoparticles to the PUU scaffold. VC release studies represented that intercalation of the drug in Zn-Al-LDH controlled the burst release and extended the release period from a few hours to 5 days. Viability and proliferation of stromal keratocyte cells on the scaffolds were investigated via AlamarBlue assay. After 24 h, the cells showed similar viability on the scaffolds and the control. After 1 week, the cells showed some degree of proliferation on the scaffolds, with the highest value recorded for PUU-VC-LDH. SEM images of the scaffolds after 24 h and 1 week confirmed good penetration and attachment of keratocytes on all the scaffolds and the cells oriented with the direction of nanofibers. After 1 week, the PUU-VC-LDH scaffold was fully covered by the cells. Immunocytochemistry assay (ICC) was performed to investigate secretion of vimentin protein, ALDH3A1, and α-SMA by the cells. After 24h and 1 week, remarkably higher levels of vimentin and ALDH3A1 and lower level of α-SMA were secreted by keratocytes on PUU-VC-LDH compared to those on the PUU and PUU-LDH scaffolds and the control. Our results suggest that the aligned PUU-VC-LDH is a promising candidate for corneal stromal tissue engineering due to the presence of zinc and vitamin C.

Diamond Burr Polishing of Bowman’s Layer in the Treatment of Subepithelial Fibrosis after Radial Keratotomy (Clinical Case)

Radial keratotomy (RK) was widely used at the end of the last century for refractive purposes. However, ophthalmologists are increasingly faced with negative long-term consequences after RK now. A relatively rare complication after RK is subepithelial fibrosis (according to some data, it occurs in 2.7 % of cases), which may be the result of abnormal activation or proliferation of stromal keratocytes (stromal fibroblasts) after surgical or accidental injury of the Bowman membrane. Phototherapeutic keratectomy and manual mechanical removal by the “scraper” are used for the removal of fibrous tissue, as well as a combination of these methods with mitomycin. Since laser energy can provoke a recurrence of fibrosis in the future due to the activation of stromal keratocytes and using “scraper” is not always possible to remove fibrotic tissue and the use of mitomycin is associated with a number of complications, the question of finding new methods for its removal with minimal risk of complications arises. The method of diamond burr polishing of the cornea, which developed by us for treating the syndrome of recurrent cornea erosion, was, in our opinion, the most optimal for this purpose. The method of diamond burr polishing of the cornea in this clinical case made it possible to remove the epithelial fibrosis zone without damaging the Bowman’s layer, thereby avoiding the recurrence of fibrosis in the postoperative period (the follow-up period was 2 years). The depth of the location pathological tissue location was assessed using the OCT of the cornea. The absence of recurrence is also confirmed biomicroscopically and with the OCT of the cornea. So, the method of diamond burr polishing of the Bowman’s layer can be a method of choice for the treatment of subepithelial fibrosis for patients after keratorefractive operations, being highly effective, accessible, easy to perform.

Cell growth and orientation of corneal stroma keratocytes on electrospun polycaprolactone-collagen scaffolds

Background & Aim Corneal cells, namely keratocytes, are a hallmark cell type for topical ocular drugs, eye drops, and drug toxicity studies. However, there has yet to exist in-vitro models of keratocytes mimicking natural corneal cells, which align in the direction of parallel collagen fibers in corneal stroma. By culturing cells on tissue-engineered scaffolds that closely mimic the structure of the stroma, cell growth and behavior should be improved. In this study, we investigated the effect of scaffold fiber orientation on the survivability and proliferation of corneal stroma keratocytes. Methods, Results & Conclusion Three different types of materials, polycaprolactone, polycaprolactone-jellyfish collagen blend, and polycaprolactone-bovine dermis atelocollagen blend, were electrospun to obtain nanofibrous scaffolds with both random and aligned fiber orientations. Keratocytes were cultured on the scaffolds over a duration of 4 days, and their survivability and cellular alignment was determined using a live and dead cell assay on day 1 and day 4. In addition, cell proliferation was measured using 4′,6-diamidino-2-phenylindole (DAPI) staining on day 1 and day 4. The results show that cells survive and proliferate better on the aligned fibers by 2-fold, with the orientation of cell growth matching that of the fibers, as shown in Fig. 1. Moreover, polycaprolactone-atelocollagen blended scaffolds favor cell survivability and proliferation as compared to pure polycaprolactone and polycaprolactone-jellyfish collagen scaffolds. Our polycaprolactone-atelocollagen scaffolds have potential applications in scaffold-based drug screening, drug toxicity testing and tissue-engineering for corneal regeneration. Corneal cells, namely keratocytes, are a hallmark cell type for topical ocular drugs, eye drops, and drug toxicity studies. However, there has yet to exist in-vitro models of keratocytes mimicking natural corneal cells, which align in the direction of parallel collagen fibers in corneal stroma. By culturing cells on tissue-engineered scaffolds that closely mimic the structure of the stroma, cell growth and behavior should be improved. In this study, we investigated the effect of scaffold fiber orientation on the survivability and proliferation of corneal stroma keratocytes. Three different types of materials, polycaprolactone, polycaprolactone-jellyfish collagen blend, and polycaprolactone-bovine dermis atelocollagen blend, were electrospun to obtain nanofibrous scaffolds with both random and aligned fiber orientations. Keratocytes were cultured on the scaffolds over a duration of 4 days, and their survivability and cellular alignment was determined using a live and dead cell assay on day 1 and day 4. In addition, cell proliferation was measured using 4′,6-diamidino-2-phenylindole (DAPI) staining on day 1 and day 4. The results show that cells survive and proliferate better on the aligned fibers by 2-fold, with the orientation of cell growth matching that of the fibers, as shown in Fig. 1. Moreover, polycaprolactone-atelocollagen blended scaffolds favor cell survivability and proliferation as compared to pure polycaprolactone and polycaprolactone-jellyfish collagen scaffolds. Our polycaprolactone-atelocollagen scaffolds have potential applications in scaffold-based drug screening, drug toxicity testing and tissue-engineering for corneal regeneration.

Experimental study on the biocompatibility of keratoprosthesis with improved titanium implant.

To investigate whether hydroxyapatite (HAp) coating can improve keratoprosthesis (KPro) implant biointegration, ultimately to decrease the risk of implant-associated complications. The modified titanium implant was designed and prepared for artificial cornea. The titanium implant was treated with sandblasting and hydroxyapatite coating by acid-base two-step method. Surface was analyzed by scanning electron microscopy (SEM), KPro implants coated with HAp and KPro implant sandblasted were implanted in rabbits. Tissue adhesion to the implant was assessed and compared to an unmodified implant by histopathology (HE), transmission electron microscopy (TEM) and SEM. SEM demonstrated successful deposition of HAp on titanium implant sandblasted (HA/SB-Ti). The hydroxyapatite coatings caused enhancement of keratocyte proliferation compared with unmodified implant surfaces. HAp coating significantly increased adhesion forces. HAp coating of implants reduced the inflammatory response around the KPro implants in vivo. HAp-coated surfaces for use in titanium KPro implant greatly enhanced adherence of the titanium KPro implant in the rabbit cornea.

Wound Healing, Inflammation, and Corneal Ultrastructure After SMILE and Femtosecond Laser–Assisted LASIK: A Human Ex Vivo Study

To assess the wound healing, inflammation, and tissue ultrastructure in the human corneal stroma after small incision lenticule extraction (SMILE) and femtosecond laser-assisted LASIK (FS-LASIK). Sixteen corneoscleral discs of 16 human donors unsuitable for corneal transplantation were obtained from an eye bank. Eight eyes underwent SMILE with -5.00 diopters (D) of myopic correction; in 3 of them the lenticule was not extracted. Further 5 donor corneas were subjected to FS-LASIK with -5.00 D ablation, and 3 eyes served as the control group without surgical intervention. Postoperatively, specimens were incubated in organ culture medium for 72 hours before being subjected to immunofluorescence staining for CD11b, Ki67, fibronectin, terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick-end labelling assay, and high-magnification scanning electron microscopy. Keratocyte apoptosis, keratocyte proliferation, and infiltration of immune cells were generally mild and comparable between FS-LASIK and SMILE (irrespective of surgical lenticule extraction). By staining for fibronectin, we observed a trend toward milder fibrotic response in the corneal stroma after SMILE than after FS-LASIK. On the contrary, scanning electron microscopy analysis revealed a smoother, more regular ultrastructural appearance of the residual corneal bed after FS-LASIK. Corneal stromal wound healing after SMILE and FS-LASIK was virtually identical with respect to keratocyte proliferation and apoptosis in the human donor eye model. Although reactive fibrosis adjacent to the laser application site appeared less marked after SMILE, the stromal bed after LASIK exhibited a smoother surface texture. [J Refract Surg. 2018;34(6):393-399.].

The Effect of Antiamoebic Agents on Viability, Proliferation and Migration of Human Epithelial Cells, Keratocytes and Endothelial Cells, In Vitro

ABSTRACTPurpose: To analyze the effect of diamidines (hexamidine-diisethionat (HD), propamidin-isethionate (PD), dibromopropamidine-diisethionat (DD)), and biguanides (polyhexamethylen biguanid (PHMB), chlorhexidine (CH)) on human corneal epithelial cell, keratocyte and endothelial cell viability, proliferation, and migration, in vitro.Methods: For epithelial and endothelial cells a human cell line and for keratocytes primary cultures were used (n = 6 each). We used 3.9x10−4–0.1% HD, PD or DD, 3.9x10−4–0.0125% PD, 7.8x10−5–0.02% PHMB or CH concentration for 24 h to determine viability (Cell Proliferation Kit XTT), proliferation (Cell Proliferation ELISA BrdU kit), and migration using wound healing assay. Viability/proliferation/migration values of each drug were summarized as “area under curve” (AUC) together with a Mann–Whitney test.Results: HCEC, keratocyte, and HCEC-12 viability AUC, comparing PD and PHMB (p ≤ 0.014 for all; PD better) or PD and HD (p ≤ 0.011 for all; PD better) differed significantly. Keratocyte and HCEC-12 viability AUC comparing CH and HD (p ≤ 0.027; CH better), HCEC-12 viability AUC comparing PD and HD (p = 0.005; PD better) and HCEC viability AUC comparing CH and PHMB (p = 0.014; CH better) differed significantly. HCEC proliferation AUC, comparing PD with PHMB, CH, DD, HD (p ≤ 0.016; PD worse for all) and keratocyte proliferation AUC, comparing PHMB with HD, PD (p = 0.004; p = 0.002; PHMB better for both), CH with HD, PD (p ≤ 0.001; CH better for both) and DD with PD (p = 0.043; DD better) differed significantly. Keratocyte migration AUC comparing PD with control, PHMB, CH, DD and HD differed significantly (p ≤ 0.012; PD worse for all).Conclusions: Propamidin-isethionate as diamidine and chlorhexidin as biguanide may be used clinically to reduce cytotoxicity of antiamoebic treatment on human corneal cells. Diamidines reduce proliferation of human epithelial cells and keratocytes more than biguanides and propamidin-isethionate reduces migration of keratocytes. Therefore, in spite of lower cytotoxicity, the inhibitory effect on proliferation and migration indicates that extended use of propamidin-isethionate should be avoided in patients.

Paracrine effects of mesenchymal stem cells on the activation of keratocytes

AimsThe purpose of this study is to investigate the impact of mesenchymal stem cell (MSC)-derived soluble factors on the function of keratocytes, with a particular focus on the processes involved...

Corticosteroids effects on LPS-induced rat inflammatory keratocyte cell model.

PurposeCorticosteroids are efficient anti-inflammation treatments. However, there are still arguments on whether it should be used in keratitis. This study was to observe the effect of corticosteroids on keratocytes both in normal condition and inflammation status in vitro.MethodsRat keratocytes were cultured and used for examination. 10 μg/ml lipopolysaccharide (LPS) was used to establish the inflammatory keratocyte cell model, and prednisolone acetate (PA), dexamethasone (Dex) and fluorometholone (Flu) were used as corticosteroids treatments. 5 d-growth curve and cell viabilities were assayed by CCK8, and cell morphologies and migration rate were studied. TNF-α, IL-6 and IL-1β levels were examined by ELISA. Western blotting was used to quantified type VI collagen (Col VI) and matrix metalloproteinase 9 (MMP9) expressions, and immunofluorescence staining assays of Col I and Col VI were carried out.ResultsIn normal condition, proliferation and migration of keratocytes were slightly influenced in PA, Dex and Flu groups. The secretion of Col I and Col VI was suppressed and MMP9 expression increased in corticosteroids groups. But no significant difference was seen in TNF-α, IL-6 and IL-1β expression levels. In inflammatory status, TNF-α, IL-6 and MMP9 levels increased in LPS group, while they significantly decreased in corticosteroids groups. Although keratocytes viabilities and migration were slightly affected in 24 h, no significant differences were seen between LPS group and corticosteroids groups in 5-d proliferation. Col I and Col VI secretion in LPS-keratocytes was maintained with corticosteroids treatments.ConclusionsCorticosteroids showed lightly effects on keratocytes proliferation and migration, but it successfully decreased TNF-α, IL-6 level and maintained the secretion of and Col I and Col VI, while suppressed the expression of MMP9 in LPS-induced keratocytes. PA was suggested to use in early stage of keratitis clinical treatment.

Arginase activity, urea, and hydroxyproline concentration are reduced in keratoconus keratocytes.

Keratoconus (KC) is a disease characterized by thinning and deformation of the cornea, but its etiology remains unknown. Seventy percent of the corneal stroma consists of collagen, which is composed of three intertwined polypeptide chains with glycine-hydroxyproline-proline repeats along their sequence. Arginase is a cytoplasmatic enzyme and catalyzes the conversion of arginine to urea and ornithine, which serves as a precursor for the endogenous synthesis of proline and hydroxyproline. The purpose of this study was to analyze arginase activity, as well as collagen and urea formation in normal and KC-keratocytes and to determine the impact of urea on keratocyte viability and proliferation in vitro. Primary human keratocytes were isolated by digestion in collagenase (1.0mg/mL) from surgically removed corneas of eight keratoconus patients and eight normal human corneal buttons and cultured in DMEM/Ham's F12 medium supplemented with 5% fetal calf serum. Arginase activity and urea concentration were measured in cell-lysates, hydroxyproline concentration in supernatant of cultured keratocytes using colorimetric assay. Cell viability and cell proliferation of cultured keratocytes were assessed after treatment with urea at concentrations up to10mM for 24h using assays for metabolic activity and DNA replication. Arginase activity and urea concentration in KC-keratocytes decreased by about 50% compared to normal keratocytes (p = 0.003 and p = 0.008). Hydroxyproline synthesized by cultured KC-keratocytes was also approximately 50% less compared to normal keratocytes (p = 0.02) and this difference decreased following treatment with 5.0 or 10.0mM urea (p = 0.02; 0.03), without any change in cell viability (p > 0.09). However, the urea treatment increased modestly (by 20%) the proliferation rate of KC-keratocytes (p = 0.04; 0.04; 0.04), without any effect on normal cultured keratocytes (p > 0.09). We identified suppressed arginase activity in the metabolic program of cultured keratoconus keratocytes. The level of urea, as one product of the enzyme arginase was also decreased. This results in impaired collagen synthesis, evidenced in the culture by reduced hydroxyproline concentration. In addition, our data showed that the other product of the arginase reaction, urea supports the proliferation of KC-keratocytes, without changes in their viability. The metabolic reprogramming of keratoconus keratocytes and its impact on development of a clinically detectable keratoconus disease has to be further analyzed.

In vitro characterization of electrochemically compacted collagen matrices for corneal applications

Loss of vision due to corneal disease is a significant problem worldwide. Transplantation of donor corneas is a viable treatment option but limitations such as short supply and immune-related complications call for alternative options for the treatment of corneal disease. A tissue engineering-based approach using a collagen scaffold is a promising alternative to develop a bioengineered cornea that mimics the functionality of native cornea. In this study, an electrochemical compaction method was employed to synthesize highly dense and transparent collagen matrices. We hypothesized that chemical crosslinking of electrochemically compacted collagen (ECC) matrices will maintain transparency, improve stability, and enhance the mechanical properties of the matrices to the level of native cornea. Further, we hypothesized that keratocyte cell viability and proliferation will be maintained on crosslinked ECC matrices. The results indicated that uncrosslinked and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide–N-hydroxysuccinimide (EDC–NHS) crosslinked ECC matrices were highly transparent with light transmission measurements comparable to native cornea. Stability tests showed that while the uncrosslinked ECC matrices degraded within 6 h when treated with collagenase, EDC–NHS or genipin crosslinking significantly improved the stability of ECC matrices (192 h for EDC–NHS and 256 h for genipin). Results from the mechanical tests showed that both EDC–NHS and genipin crosslinking significantly improved the strength and modulus of ECC matrices. Cell culture studies showed that keratocyte cell viability and proliferation are maintained on EDC–NHS crosslinked ECC matrices. Overall, results from this study suggest that ECC matrices have the potential to be developed as a functional biomaterial for corneal repair and regeneration.