Impact of crosslinking/riboflavin-UVA-photodynamic inactivation on viability,apoptosis and activation of human keratocytes in vitro

Riboflavin-UVA photodynamic inactivation is a potential treatment altemative in therapy resistant infectious keratitis. The purpose of our study was to determine the impact of riboflavin-UVA photodynamic inactivation on viability, apop- tosis and activation of human keratocytes in vitro. Primary human keratocytes were isolated from human corneal buttons and cultured in DMEM/Ham＇s F12 medium supplemented with 10% fetal calf serum. Keratocytes underwent UVA light illumination （375 nm） for 4.10 minutes （2 J/cm2） during exposure to different concentrations of riboflavin. Twenty-four hours after treatment, cell viability was evaluated photometrically, whereas apoptosis, CD34 and alpha-smooth muscle actin （α-SMA） expression were assessed using flow cytometry. We did not detect significant changes in cell viability, apoptosis, CD34 and α-SMA expression in groups only treated with riboflavin or UVA light. In the group treated with riboflavin-UVA-photodynamic inactivation, viability of keratocytes decreased significantly at 0.1% riboflavin （P〈0.01） while the percentage of CD34 （P〈0.01 for both 0.05% and 0.1% riboflavin） and alpha-SMA positive keratocytes （P〈0.01 and P〈0.05 for 0.05% and 0.1% riboflavin, respectively） increased significantly compared to the controls. There was no significant change in the percentage of apoptotic keratocytes compared to controls at any of the used ribo- flavin concentrations （P=0.09 and P=0.13）. We concluded that riboflavin-UVA-photodynamic-inactivation decreases viability of myofibroblastic transformation and multipotent haematopoietic stem cell transformation; however, it does not have an impact on apoptosis of human keratocytes in vitro.

Using bovine pituitary extract to increase proliferation of keratocytes and maintain their phenotype in vitro

AIM:To investigate the effects of bovine pituitary extract on the proliferation of keratocytes and maintaining the keratocyte phenotype/n v/tro.METHODS:Single keratocytes were isolated by enzyme digestion for in vitro culture.Three groups were designed according to the different culture media:a bovine pituitary extract(BPE)group,a fetal bovine serum(FBS)group and the control group.The phenotypes and proliferation of cultured cells were evaluated by morphology,immunofluorescent staining and mRNA expression of CD34,Lumican,VSX1,α-SMA and proliferating cell nuclear antigen(PCNA).in the BPE group,cells underwent serial subcultivation,and their phenotypes were identified by immunofluorescent staining.To analyze the proliferation of keratocytes in different concentrations of BPE,six different concentrations were designed to ascertain the most appropriate amount.RESULTS:In the BPE group,the cells spread out and presented dendritic morphology,and their dendrites connected to one another to form networks.On the third passage,most cells maintained their phenotype.In the FBS group,the cells exhibited a dendritic appearance in early cultured stages,but their morphology subsequently changed into a fibroblast-like shape.The number ofdendritic cells in BPE group was more than FBS and control groups.Immunofluorescent staining and realtime polymerase chain reaction(PCR)confirmed that few keratocytes underwent fibroblastic transformation in the BPE and control groups,and that proliferation was higher in the BPE group than in the control group.Although the proliferation was higher in the FBS group,many keratocytes underwent fibroblastic transformation.The analysis of cell morphology and mRNA expressions of CD34,PCNA and VSX1 in six group showed that different concentrations of BPE affected the proliferation obviously but didn’t affect the keratocyte phenotype,and the concentration of 40μg/mL was the most appropriate one.CONCLUSION:BPE can improve the proliferation of keratocytes and maintain their phenotype in vitro.Many keratocytes can be harvested rapidly and provide seeds for the construction of corneal stroma.

A cellular and proteomic approach to assess proteins extracted from cryopreserved human amnion in the cultivation of corneal stromal keratocytes for stromal cell therapy

Background:Human corneal stromal keratocytes propagated in culture media supplemented with human amnion extract(AME)can correct early corneal haze in an animal model.Clinical application of cultivated keratocytes is limited by infectious disease screening before amnion products can be used in humans.It remains unclear if AME from cryopreserved versus fresh human amnion can support human keratocyte propagation,and which components of the extract promote keratocyte growth.Methods:Three placentas were collected for the preparation of fresh and cryopreserved amnion tissues followed by homogenization and protein extraction.AME protein profiles were studied using isobaric tagging for relative and absolute quantitation(iTRAQ)proteomics.Enriched gene ontology(GO)terms and functional classes were identified.Primary human keratocytes from 4 donor corneas were cultured in media supplemented with fresh AME(F-AME)or cryopreserved AME(C-AME).Cell viability,proliferation and keratocyte marker expression were examined by confocal immunofluorescence and flow cytometry.Results:AME proteomics revealed 1385 proteins with similar expression levels(between 0.5-and 2-fold)between Fand C-AME,while 286 proteins were reduced(less than 0.5-fold)in C-AME.Enriched GO term and biological pathway analysis showed that those proteins with comparable expression between F-AME and C-AME were involved in cell metabolism,epithelial-mesenchymal transition,focal adhesion,cell-extracellular matrix interaction,cell stress regulation and complement cascades.Human corneal stromal keratocytes cultured with F-AME or C-AME showed similar morphology and viability,while cell proliferation was mildly suppressed with C-AME(P>0.05).Expression of aldehyde dehydrogenase 3A1(ALDH3A1)and CD34 was similar in both cultures.Conclusion:AME from cryopreserved amnion had limited influence on keratocyte culture.It is feasible to use protein extract from cryopreserved amnion to propagate human keratocytes for potential translational applications.

Assessment of the effects of intrastromal injection of adipose-derived stem cells in keratoconus patients

·AIM: To evaluate the efficacy and safety of intrastromal transplantation of adipose-derived stem cells(ASCs) in keratoconus patients.·METHODS: This study was conducted on 8 eyes of 8 patients with moderate to severe keratoconus. In the patients, ophthalmic assessments including visual acuity, refraction, slit lamp examination, fundoscopy, corneal topography, and confocal microscopy were performed. Autologous stem cells were used. The isolated stem cells were injected into the corneal stroma by using femtosecond laser. Surgical procedure was similar to intracorneal ring implantation. All patients were re-assessed 1, 3, and 6mo after surgery.·RESULTS: The baseline mean visual acuity was 0.48±0.18 and improved to 0.66±0.17 after surger y and final acuity increased by 1.85±0.80 lines(P=0.001).The mean spherical refraction of patients improved 0.34 ± 0.35 D(P=0.039), and the mean cylindrical refraction of patients improved 0.84±0.23 D(P=0.016). The mean flat keratometry decreased 0.78±0.71 D(P=0.017), and the mean steep keratometry decreased 0.59±0.68 D(P=0.023). The mean central corneal thickness of patients improved of 6.29±4.47 μm(P=0.03). The mean keratocyte density at the anterior and middle stroma of cornea increased(P<0.05) but remained stable at the posterior stroma after 6mo. All patients had no complications and their corneas remained transparent. ·CONCLUSION: Intrastromal transplantation of ASCs has positive effects on vision and refractive parameters in most patients with keratoconus. After six months, visual acuity improved moderately, corneal parameters reduced slightly, and stromal keratocytes density increased. This modality is safe, and patients do not have any complications.

Acellular porcine corneal matrix as a carrier scaffold for cultivating human corneal epithelial cells and fibroblasts in vitro

AIM：To investigate the feasibility of corneal anterior lamellar reconstruction with human corneal epithelial cells and fibroblasts,and an acellular porcine cornea matrix（APCM） in vitro.·METHODS：The scaffold was prepared from fresh porcine corneas which were treated with 0.5%sodium dodecyl sulfate（SDS）solution and the complete removal of corneal cells was confirmed by hematoxylin-eosin（HE）staining and 4’,6-diamidino-2-phenylindole（DAPI）staining.Human corneal fibroblasts and epithelial cells were cultured with leaching liquid extracted from APCM,and then cell proliferative ability was evaluated by MTT assay.To construct a human corneal anterior lamellar replacement,corneal fibroblasts were injected into the APCM and cultured for 3d,followed by culturing corneal epithelial cells on the stroma construction surface for another 10d.The corneal replacement was analyzed by HE staining,and immunofluorescence staining.·R ESULTS：Histological examination indicated that there were no cells in the APCM by HE staining,and DAPI staining did not detect any residual DNA.The leaching liquid from APCM had little influence on the proliferation ability of human corneal fibroblasts and epithelial cells.At 10d,a continuous 3 to 5 layers of human corneal epithelial cells covering the surface of the APCM was observed,and the injected corneal fibroblasts distributed within the scaffold.The phenotype of the construction was similar to normal human corneas,with high expression of cytokeratin 12 in the epithelial cell layer and high expression of Vimentin in the stroma.·CONCLUSION：Corneal anterior lamellar replacement can be reconstructed in vitro by cultivating human corneal epithelial cells and fibroblasts with an acellular porcine cornea matrix.This laid the foundation for the further transplantation in vitro.

Expression of visual system homeobox 1 in human keratoconus

AIM: To investigate the expression of visual system homeobox 1(VSX1) and myofibroblast marker alpha smooth muscle actin(α-SMA) in keratoconus(KC). METHODS: Thirty corneal tissue were collected from KC patients after corneal transplantation and 15 normal donor corneas were obtained. All corneal tissues divided into 4 parts for different detections. Scanning electron microscopy was used to observe the ultrastructure of the specimens. VSX1 and α-SMA localization in cornea tissues was detected using immunofluorescence histochemistry. Reverse transcription-quantitative polymerase chain reaction(RT-qPCR) and Western blot were performed to analyze the expression level of VSX1 and α-SMA. RESULTS: Compared to normal cornea tissue, the collagen fibers in KC stroma were distortional and attenuated and keratocytes were abnormally changed. VSX1 and α-SMA located in the corneal stroma. The mRNA and protein expression level of VSX1 in KC were about 3 times as high as that of normal tissue(P<0.001). α-SMA was hardly expressed in the normal corneas, however, its expression in the KC was about 1.5 times higher than that of the normal corneas(P<0.0001). CONCLUSION: Compared with normal corneal the expression of VSX1 and α-SMA in KC both increased. VSX1 is related to the activation of keratocytes and involved in the pathogenesis of keratoconus.

An Association of Vitamins A and E with Hyaluronic and Lactobionic Acids may Prevent Molecular Changes Associated with Keratocyte to Myofibroblast Transition

Inflammatory events in the corneal stroma may activate keratocytes and trigger their transition towards myofibroblasts,which now produce different extracellular matrix(ECM)proteins thus causing corneal opacification.Corneal haze is a frequent side effect after photorefractive keratectomy(PRK)to correct high myopia.Currently,a preventive treatment with mitomycin-c can be used to limit the occurrence of this phenomenon.However,mitomycin-c is a toxic drug,not devoid of side effects,which may occasionally involve the corneal endothelium.Therefore,we have searched for a less risky,natural way,to prevent keratocytes transition.To this purpose,we have used as markers of the phenotype switch the proteins lumican(highly expressed by keratocytes and much less by myofibroblasts)and smooth muscle actin(αSMA)(highly expressed by myofibroblasts and poorly found in keratocytes),beside Fibronectin(Fn),the expression of which is also increased by transforming growth factor-beta(TGFβ)treatment.Treatment of human keratocytes with TGFβwas used to induce the protein shift.Among different possible candidates,we have found that vitamins A and E,hyaluronic and lactobionic acids may prevent,either alone,or much better in association,the shift in the ratio between lumican andαSMA and the increased Fn expression.In conclusion,it could be speculated that topic treatment of the ocular surface with an association of these four compounds could be able to prevent or at least limit the occurrence of post-PRK corneal haze,with the additional advantage of lubrication,hydration and antioxidant defense exerted by these molecules.

Clinical and microstructural changes with different iontophoresis-assisted corneal cross-linking methods for keratoconus

AIM: To compare the clinical and microstructural changes induced by different transepithelial iontophoresis-assisted corneal cross-linking(I-CXL) methods for keratoconus. METHODS: A total of 42 eyes of 42 patients with progressive keratoconus were divided into two groups. Group A received I-CXL for 5 min, while group B received I-CXL for 10 min. Visual acuity, optical coherence tomography(OCT), specular microscopy and confocal microscopy were evaluated preoperatively and at 1, 3, 6, and 12 mo postoperatively. RESULTS: Twelve months after the operation, uncorrected visual acuity(UCVA) and corrected distance visual acuity(CDVA) were improved in both groups, with a better outcome in the I-CXL 10 min group(P=0.025, 0.021, respectively). Kmax values decreased by 0.94±3.00 D in the I-CXL 10 min group(P=0.033) but increased by 1.87±3.29 D in the I-CXL 5 min group(P=0.012). OCT scans showed that the demarcation line was most visible and substantially deeper in the I-CXL 10 min group. Confocal microscopy showed greater anterior stromal keratocyte decreases in the I-CXL 10 min group than in the I-CXL 5 min group at 3 and 6 mo postoperatively(P<0.001); however, anterior stromal keratocytes and subbasal nerve density were not significantly different between the two groups at 12 mo postoperatively. CONCLUSION: I-CXL for 10 min more effectively halts the progression of keratoconus than I-CXL for 5 min after 12 mo of follow-up. However, long-term studies are needed to evaluate the efficacy and safety of I-CXL.