Dataset Information

An Assay System to Evaluate Riboflavin/UV-A Corneal Phototherapy Efficacy in a Porcine Corneal Organ Culture Model.

ABSTRACT: The purpose of this study was to investigate the response of porcine corneal organ cultures to riboflavin/UV-A phototherapy in the injury healing of induced lesions. A porcine corneal organ culture model was established. Corneal alterations in the stroma were evaluated using an assay system, based on an automated image analysis method able to (i) localize the holes and gaps within the stroma and (ii) measure the brightness values in these patches. The analysis has been performed by dividing the corneal section in 24 regions of interest (ROIs) and integrating the data analysis with a "multi-aspect approach." Three group of corneas were analyzed: healthy, injured, and injured-and-treated. Our study revealed a significant effect of the riboflavin/UV-A phototherapy in the injury healing of porcine corneas after induced lesions. The injured corneas had significant differences of brightness values in comparison to treated (p < 0.00) and healthy (p < 0.001) corneas, whereas the treated and healthy corneas showed no significant difference (p = 0.995). Riboflavin/UV-A phototherapy shows a significant effect in restoring the brightness values of damaged corneas to the values of healthy corneas, suggesting treatment restores the injury healing of corneas after lesions. Our assay system may be compared to clinical diagnostic methods, such as optical coherence tomography (OCT) imaging, for in vivo damaged ocular structure investigations.

SUBMITTER: Perazzi A

PROVIDER: S-EPMC7652214 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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An Assay System to Evaluate Riboflavin/UV-A Corneal Phototherapy Efficacy in a Porcine Corneal Organ Culture Model.

Perazzi Anna A Gomiero Chiara C Corain Livio L Iacopetti Ilaria I Grisan Enrico E Lombardo Marco M Lombardo Giuseppe G Salvalaio Gianni G Contin Roberta R Patruno Marco M Martinello Tiziana T Peruffo Antonella A

Animals : an open access journal from MDPI 20200423 4

The purpose of this study was to investigate the response of porcine corneal organ cultures to riboflavin/UV-A phototherapy in the injury healing of induced lesions. A porcine corneal organ culture model was established. Corneal alterations in the stroma were evaluated using an assay system, based on an automated image analysis method able to (i) localize the holes and gaps within the stroma and (ii) measure the brightness values in these patches. The analysis has been performed by dividing the ...[more]

PMID: 32340101

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Project description:PurposePreviously, we found norepinephrine (NE) could affect the corneal epithelial integrity, herein we investigated the feasibility and safety of NE serving as a chemical enhancer to promote corneal penetration of riboflavin during transepithelial corneal crosslinking (CXL).MethodsThe dosage of NE that could promote riboflavin diffusion through the healthy epithelial barrier without inducing epithelial damage in C57BL/6 mice was determined. The safety of NE treatment was confirmed by morphological and histological examinations of the whole cornea. The efficacy of NE in promoting riboflavin penetration was verified by slit lamp and scanning electron microscope (SEM), and corneal biomechanical measurement after CXL. To better fit the clinical scenario, increased NE dosage and shortened riboflavin infiltration time were further evaluated.ResultsThe lowest dosage of NE (1 mg/mL) that facilitated transepithelial riboflavin permeation was 2 µL. No visible corneal structure alteration was observed after NE treatment. SEM indicated dissociation of intercellular junctions among corneal epithelial cells. Homogenous distribution of riboflavin throughout corneal stroma was observed. NE-treated corneas reached comparable biomechanical properties after CXL, including stress-relaxation curve and elastic modulus, with corneas treated with the commercially available transepithelial drug Peschke TE. To better fit the clinical scenario, increasing NE up to 5.5 µL helped riboflavin infiltrate the corneal stroma within 30 minutes. After CXL with 9 mW/cm2 ultraviolet-A (UVA) for 2.5 minutes, the cornea showed significantly enhanced corneal biomechanical properties with undisturbed corneal endothelium.ConclusionsNE serves as an effective enhancer in increasing riboflavin diffusion with limited impairment on corneal epithelium and has great potential for clinical application.Translation relevanceNE serves as an effective enhancer for riboflavin penetration and clinical transepithelial CXL.

Project description:Corneal collagen cross-linking (CXL) halts human corneal ectasias progression by increasing stromal mechanical stiffness. Although some reports describe that this procedure is effective in dealing with some infectious and immunologic corneal thinning diseases, there is a need for more animal models whose corneal thickness more closely resemble those occurring in these patients. To meet this need, we describe here high-intensity protocols that are safe and effective for obtaining CXL in rat corneas. Initially, a range of potentially effective UVA doses were evaluated based on their effectiveness in increasing tissue enzymatic resistance to dissolution. At UVA doses higher than a threshold level of 0.54 J/cm2, resistance to enzymatic digestion increased relative to that in non-irradiated corneas. Based on the theoretical threshold CXL dose, a CXL regimen was established in which the UVA tissue irradiance was 9 mW/cm2, which was delivered at doses of either 2.16, 2.7 or 3.24 J/cm2. Their dose dependent effects were evaluated on ocular surface morphological integrity, keratocyte apoptotic frequency, tissue thickness and endothelial cell layer density. Doses of 2.16 and 2.7 J/cm2 transiently decreased normal corneal transparency and increased thickness. These effects were fully reversed after 14 days. In contrast, 3.24 J/cm2 had more irreversible side effects. Three days after treatment, apoptotic frequency in the CXL-2.16 group was lower than that at higher doses. Endothelial cell losses remained evident only in the CXL-3.24 group at 42 days posttreatment. Stromal fiber thickening was evident in all the CXL-treated groups. We determined both the threshold UVA dose using the high-intensity CXL procedure and identified an effective dose range that provides optimal CXL with minimal transient side effects in the rat cornea. These results may help to provide insight into how to improve the CXL outcome in patients afflicted with a severe corneal thinning disease.

Project description:Acellular cornea derived hydrogels provide significant advantages in preserving native corneal stromal keratocyte cells and endothelial cells. However, for clinical application, hydrogel physical properties need to be improved, and their role in corneal epithelial wound healing requires further investigation. In this study, an acellular porcine corneal stroma (APCS) hydrogel (APCS-gel) was successfully prepared from 20 mg/ml APCS, demonstrated optimal light transmittance and gelation kinetic properties and retained critical corneal ECM of collagens and growth factors. Compared with fibrin gel, the APCS-gel had a higher porosity ratio and faster nutrition diffusion with an accompanying improvement in the proliferation of primary rabbit corneal epithelial cells (RCECs) and stromal cells (RCSCs). These corneal cell types also displayed improved viability and cellular infiltration. Furthermore, the APCS-gel provides significant advantages in the preservation of RCECs stemness and enhancement of corneal wound healing in vitro and in vivo. After 7 days of culture, 3-4 layers of RCECs were formed on the APCS-gel in vitro, while only 1-2 layers were found on the fibrin gel. More corneal stem/progenitor cell phenotypes (K12-, p63+, ABCG2+) and proliferation phenotypes (Ki67+) were detected on the APCS-gel than fibrin gel. Using a corneal epithelial wound healing model, we also found faster reepithelization in corneas that received APCS-gel compared to fibrin gel. Additionally, our APCS-gel demonstrated better physical and biological properties when compared to Tisseel, a clinically used type of fibrin gel. In conclusion, our APCS-gel provided better corneal epithelial and stromal cell biocompatibility to fibrin gels and due to its transparency and faster gelation time could potentially be superior for clinical purposes. STATEMENT OF SIGNIFICANCE: Extracellular matrix (ECM) can be used to provide tissue specific physical microstructure and biochemical cues for tissue regeneration. Here, we produced an ECM hydrogel derived from acellular porcine cornea stroma (APCS-gel) that retained critical biological characteristics of the native tissue and provided significant transparency and fast gelation time. Our data demonstrated that the APCS-gel was superior to clinically used fibrin gel, as the APCS-gel showed high porosity and permeability, better corneal stromal keratocytes infiltration, increased cellular proliferation and retention of corneal epithelial cells stemness. The APCS-gel improved corneal wound healing in vitro and in vivo. This APCS-gel may have clinical utility for corneal diseases, and the more general approach used to make this hydrogel might be used in other tissues.

Dataset Information

An Assay System to Evaluate Riboflavin/UV-A Corneal Phototherapy Efficacy in a Porcine Corneal Organ Culture Model.

Publications

An Assay System to Evaluate Riboflavin/UV-A Corneal Phototherapy Efficacy in a Porcine Corneal Organ Culture Model.

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