Autologous platelet concentrate in epiretinal membrane surgery: A single-centre prospective comparative non-inferiority study.

PURPOSE: The purpose of the study was to compare the anatomical and functional results including reading ability after epiretinal membrane (ERM) surgery in patients with and without the use of autologous platelet concentrate (APC). METHODS: Design: Prospective, comparative non-inferiority series. SETTING: Institutional. PATIENTS: 51 eyes of 51 patients, who underwent pars-plana vitrectomy (PPV) for ERM surgery. 29 eyes additionally received intraoperative APC, 22 eyes underwent standard procedure without APC use. OBSERVATIONS: anatomical and functional outcome parameters (central retinal thickness (CRT), best corrected visual acuity (BCVA) and reading ability (RA)) were compared between the two groups at 6 weeks and 6 months postoperatively. Subjective assessment of visual acuity and reading ability was also analysed. MAIN OUTCOME MEASURES: BCVA, RA and CRT. RESULTS: Both groups showed significant CRT reduction and RA improvement, while BCVA improvement was significant only in eyes with intraoperative APC use during the follow-up time of 6 months. There was no statistically significant difference between CRT reduction, BCVA and RA improvement between the groups. CONCLUSION: Intraoperative APC use for ERM surgery results in similar anatomical and functional outcomes compared with standard ERM surgery without APC use.

Visual acuity prediction on real-life patient data using a machine learning based multistage system.

In ophthalmology, intravitreal operative medication therapy (IVOM) is a widespread treatment for diseases related to the age-related macular degeneration (AMD), the diabetic macular edema, as well as the retinal vein occlusion. However, in real-world settings, patients often suffer from loss of vision on time scales of years despite therapy, whereas the prediction of the visual acuity (VA) and the earliest possible detection of deterioration under real-life conditions is challenging due to heterogeneous and incomplete data. In this contribution, we present a workflow for the development of a research-compatible data corpus fusing different IT systems of the department of ophthalmology of a German maximum care hospital. The extensive data corpus allows predictive statements of the expected progression of a patient and his or her VA in each of the three diseases. For the disease AMD, we found out a significant deterioration of the visual acuity over time. Within our proposed multistage system, we subsequently classify the VA progression into the three groups of therapy "winners", "stabilizers", and "losers" (WSL classification scheme). Our OCT biomarker classification using an ensemble of deep neural networks results in a classification accuracy (F1-score) of over 98%, enabling us to complete incomplete OCT documentations while allowing us to exploit them for a more precise VA modelling process. Our VA prediction requires at least four VA examinations and optionally OCT biomarkers from the same time period to predict the VA progression within a forecasted time frame, whereas our prediction is currently restricted to IVOM/no therapy. We achieve a final prediction accuracy of 69% in macro average F1-score, while being in the same range as the ophthalmologists with 57.8 and 50 ± 10.7 % F1-score.

Treated Cases of Retinopathy of Prematurity in Germany: 10-Year Data from the Retina.net Retinopathy of Prematurity Registry.

PURPOSE: To analyze changes in demographic parameters and retreatment patterns over a 10-year period in a clinical routine setting of infants with retinopathy of prematurity (ROP) requiring treatment documented in the German Retina.net ROP registry. DESIGN: Multicenter, noninterventional, observational registry study recruiting patients treated for ROP. SUBJECTS: A total of 692 eyes of 353 infants treated for ROP were documented in the Retina.net ROP registry over a 10-year period between 2011 and 2020. These cases cover about 15% of all infants treated for ROP in Germany. METHODS: The Retina.net ROP registry was established in 2012 to jointly collect information on infants treated for ROP. The database collects information on demographic parameters (gestational age [GA], birth weight, neonatal comorbidities) as well as treatment parameters (type of treatment, weight and age at treatment, and stage of ROP). A total of 19 centers contributed to the analysis. This is the 10-year analysis of data from 2011 to 2020, in which we focus on changes over time regarding the respective parameters. MAIN OUTCOME MEASURES: Changes over time in demographic parameters and treatment patterns for ROP in Germany. RESULTS: The overall incidence of treatment requiring ROP was 3.5% of all infants screened for ROP at participating centers. Gestational age, weight at birth, and weight at treatment remained stable over the 10-year period, whereas postmenstrual and postnatal age at treatment increased moderately but statistically significantly over the years. The most prevalent ROP severity stage at treatment was stage 3+ in zone II (76.6% of all treated eyes). Treatment patterns changed considerably from predominantly laser treatments in 2011 (75% of all treated eyes) to predominantly ranibizumab treatments in 2020 (60.9% of all treated eyes). The overall retreatment rate was 15.6%. Retreatment rates differed between initial treatment modalities (14.1% after laser coagulation, 12% after bevacizumab and 24.5% after ranibizumab). Treatment-associated systemic or ophthalmic complications were rare. CONCLUSIONS: This data analysis represents one of the largest documented cohorts of infants treated for ROP. The data on demographic parameters and treatment patterns provide useful information for further improvement of ROP management. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

P04-A115†Serum containing organ culture of the human cornea - still state of the art?

PURPOSE: For decades, human corneas are prepared and stored in specialized tissue banks prior to transplantation. Especially in Europe, storage takes place in 'organ culture', the storage in cell culture medium at approximately physiological temperature. Traditionally, a serum-containing medium is used for this purpose. However, the use of fetal calf serum has considerable disadvantages: there is a risk of disease transmission, availability may not always be guaranteed in the necessary quality, there are considerable differences from batch to batch, which is associated with batch testing required in each case, and last but not least, the extraction of serum from unborn calves is an ethical issue. METHODS: In recent years, several studies have focused on the improvement of organ culture conditions for donor corneas, including different serum-free media and alternative deswelling substances. Meanwhile, media are on the market which seem to be equivalent to serum-supplemented MEM. Nevertheless, serum-free medium has not yet found its way into routine organ culture of corneas. RESULTS: Our own preliminary studies have shown that despite the promising approaches, no satisfactory overall result could be achieved. Since only maintenance metabolism is required for storage of corneas until transplantation, in principle cultivation in the conventionally used medium seems possible without addition of serum at all. Corneas stored in this way had comparably endothelial cell density (ECD) to their counterpart stored in serum-supplemented medium. However, during the final evaluation after deswelling, the ECD dropped drastically.Engelmann et al. started research on the use of serum-free culture medium (SFM) for a long time and comparable or even superior ECD and viability could be demonstrated. So far, however, it has not been possible to define a deswelling medium adapted to these conditions.Also, a serum-free storage medium developed by Eurobio (CorneaSyn) could not completely convince, because although ECD of the examined corneas remained constant, the morphology of the cells changed. CONCLUSION: Since it is essential to intensify efforts towards a serum-free system it is planned to test serum substitutes and, if possible, also to replace the de-swelling additive dextran with a less harmful alternative to guarantee the quality of cornea grafts in the future.

Induction of vascular endothelial growth factor-A165a in human retinal and endothelial cells in response to glyoxal.

Low-density lipoprotein (LDL) apheresis is effective and safe for patients with diabetes, proteinuria, and dyslipidemia. Diabetes mellitus is accompanied by ocular microvascular complications like retinal neovascularization or diabetic macular edema. These are leading causes of blindness and can be mediated by abnormal vessel growth and increased vascular permeability due to elevated levels of vascular endothelial growth factor (VEGF) in diabetic patients. In this study, we established methods to study the expression of different VEGF isoforms in human retinal and endothelial cells. The VEGF-A165a isoform is much higher expressed in retinal cells, compared to endothelial cells. Stimulation with glyoxal as a model of oxidative stress under diabetic conditions lead to a pronounced induction of VEGF-A165a in human retinal and endothelial cells. These data suggest that diabetes and oxidative stress induce VEGF-A isoforms which could be relevant in regulating the ingrowths of novel blood vessels into the retina in diabetic patients.

Opportunities of Digital Infrastructures for Disease Management-Exemplified on COVID-19-Related Change in Diagnosis Counts for Diabetes-Related Eye Diseases.

Background: Retrospective research on real-world data provides the ability to gain evidence on specific topics especially when running across different sites in research networks. Those research networks have become increasingly relevant in recent years; not least due to the special situation caused by the COVID-19 pandemic. An important requirement for those networks is the data harmonization by ensuring the semantic interoperability. Aims: In this paper we demonstrate (1) how to facilitate digital infrastructures to run a retrospective study in a research network spread across university and non-university hospital sites; and (2) to answer a medical question on COVID-19 related change in diagnostic counts for diabetes-related eye diseases. Materials and methods: The study is retrospective and non-interventional and runs on medical case data documented in routine care at the participating sites. The technical infrastructure consists of the OMOP CDM and other OHDSI tools that is provided in a transferable format. An ETL process to transfer and harmonize the data to the OMOP CDM has been utilized. Cohort definitions for each year in observation have been created centrally and applied locally against medical case data of all participating sites and analyzed with descriptive statistics. Results: The analyses showed an expectable drop of the total number of diagnoses and the diagnoses for diabetes in general; whereas the number of diagnoses for diabetes-related eye diseases surprisingly decreased stronger compared to non-eye diseases. Differences in relative changes of diagnoses counts between sites show an urgent need to process multi-centric studies rather than single-site studies to reduce bias in the data. Conclusions: This study has demonstrated the ability to utilize an existing portable and standardized infrastructure and ETL process from a university hospital setting and transfer it to non-university sites. From a medical perspective further activity is needed to evaluate data quality of the utilized real-world data documented in routine care and to investigate its eligibility of this data for research.

Unusual initial manifestation of choroidal melanoma in a 46-year-old adult with rapid growth over 9 months.

Purpose: To report a case with unusual initial manifestation of uveal melanoma in a 46-year-old adult. Observations: A 46-year-old man demonstrated a mid-peripheral temporal partially hyperpigmented lesion in his left eye. Initial clinical appearance as well as multimodal imaging approaches were similar to a chorioretinitic disease. Over the course of 9 months, a massive tumor growth and exudative retinal detachment could be observed. The diagnosis of choroidal melanoma was histopathologically confirmed after endoresection. Conclusions and importance: Classical choroidal melanoma manifest as pigmented, prominent tumors with >2 mm tumor thickness, exudative retinal detachment, and orange pigment. In our case, none of these classical clinical characteristic signs of melanoma were present at initial presentation, but the tumor underwent progressive development with significant tumor growth and exudative retinal detachment during the following 9 months.

Efficacy of combining intravitreal injections of ranibizumab with micropulse diode laser versus intravitreal injections of ranibizumab alone in diabetic macular edema (ReCaLL): a single center, randomised, controlled, non-inferiority clinical trial.

BACKGROUND: To evaluate if a combination therapy with micropulse diode laser (MPL) shows non-inferiority on visual acuity (BCVA) within 12 months in comparison to standard therapy, i.e. intravitreal injection of ranibizumab alone. SETTING: Institutional. Prospective randomized single-center trial. METHODS: Patients with diabetic macular edema (DME) received three intravitreal injections of 0.5 mg ranibizumab during the upload phase and were then randomised 1:1 to receive either the same dosage of ranibizumab (0.5 mg) injections pro re nata alone (IVOM-Group; n = 9), or with two additional treatments with micropulse diode laser (IVOM+Laser-Group; n = 10). The primary endpoint was change in BCVA after 12 months. Secondary endpoints were change in central macular thickness and overall number of ranibizumab injections. RESULTS: BCVA increased significantly in both groups (IVOM: + 5.86, p < 0.001; IVOM+Laser: + 9.30; p < 0.001) with corresponding decrease in central macular thickness (IVOM: - 105 µm, p < 0.01; IVOM+Laser: - 125 µm; p < 0.01). Patients with additional laser treatment had better visual improvement (group comparison p = 0.075) and needed fewer ranibizumab injections (cumulative proportion of injections 9.68 versus 7.46 in IVOM-Group and IVOM+Laser-Group, respectively). CONCLUSION: Non-inferiority of combination therapy in comparison to standard therapy alone could be demonstrated. Patients with additional laser therapy needed fewer ranibizumab injections. TRIAL REGISTRATION: Registered 10 February 2014 on ClinicalTrials.gov; NCT02059772 .

From Cord to Eye: Wharton Jelly-Derived Stem Cells Differentiate Into Corneal Endothelial-Like Cells.

PURPOSE: A malfunction of the corneal endothelium leading to corneal opacity is one of the main causes of impaired vision. Currently, keratoplasty is the one and only donor cornea-dependent treatment, and this calls for alternatives because of the worldwide lack of donor corneas. Recently, the topography of Descemet membrane (DM) has been discovered as a feasible stem cell differentiation tool. With this study, we further confirm this mechanotransductive system by using preinduced Wharton jelly-derived mesenchymal stem cells (WJ-EPCs). METHODS: To measure the mechanotransductive potential of Descemet-like topography (DLT), WJ-EPCs were cultivated on collagen imprints with DLT. Changes in the gene and protein expressions of corneal endothelial cells (CECs), typical markers such as zonula occludens (ZO-1), sodium/potassium (Na/K)-ATPase, paired-like homeodomain 2 (PITX2), and collagen 8 (COL-8) were measured. In addition, CEC functionality has been evaluated by measuring the relative potential differences in a 2-compartment system and by measuring corneal transparency in an ex vivo rabbit cornea model. To confirm the activity of WJ-EPCs, rabbit CECs were restless deleted by collagen digestion of a thin layer of rabbit Descemet membrane. RESULTS: The proper CEC-typical hexagonal morphology of WJ-EPCs in combination with a significant expression of ZO-1, Na/K-ATPase, PITX2, and COL-8 could be demonstrated. In addition, the WJ-EPCs were able to build up a relative potential difference of 40 mV and to keep corneas clear and transparent. CONCLUSIONS: These data indicate that a well-characterized, functional CEC monolayer was developed by using a DLT-mediated mechanotransductive differentiation of WJ-EPCs.

To Peel or Not to Peel: Pars Plana Vitrectomy with Macular Membrane Peel in Eyes with Abnormalities of Vitreomacular Interface and Coexisting Dry Age-Related Macular Degeneration.

PURPOSE: To evaluate the outcome of macular surgery with ILM- and epiretinal membrane peel associated with significant dry age-related macular degeneration (AMD) as defined by the Age-Related Eye Disease Study (AREDS). PATIENTS AND METHODS: Institutional. Retrospective case-control study. A total of 42 pseudophacic eyes of 39 patients (7 with full thickness macular hole and 35 with epiretinal membrane) with coexisting dry AMD underwent pars plana vitrectomy (PPV) with internal limiting membrane (ILM) and epiretinal membrane peel. Preoperative and postoperative data including best corrected visual acuity (BCVA), AMD grade according to AREDS, central retinal thickness (CRT), development of choroidal neovascularization (CNV), and central retinal atrophy have been evaluated. Twenty-eight fellow eyes with dry AMD of the included 39 patients served as a control group. RESULTS: A significant improvement in the visual acuity could be observed after surgery (initial BCVA 0.47±0.31 logarithm of the minimal angle of resolution (logMAR) vs 0.33±0.29logMAR 9 months postoperatively; p=0.006). CRT decreased significantly after surgery (p<0.001). In the surgery group, there were 4 eyes (9.5%) with CNV and 1 eye (2.5%) with new central retinal atrophy development after surgery. All these eyes had preoperative AREDS 3 (4 eyes) or AREDS 4 (1 eye) AMD category. In the control group, there was 1 eye (4%) with CNV and 4 eyes (14%) with new central retinal atrophy development during the follow-up of 9 months. These eyes had initially AREDS 2 (1 eye), AREDS 3 (3 eyes) or AREDS 4 (1 eye) AMD category. CONCLUSION: Eyes with dry AMD of AREDS 3 and AREDS 4 with coexisting VMI abnormalities improve significantly after PPV with membrane peel. While there is a higher risk of CNV development after surgery (9.5%) in these eyes, the vitrectomy does not seem to accelerate central retinal atrophy progression compared to the fellow eyes course.

Descemet's Membrane Biomimetic Microtopography Differentiates Human Mesenchymal Stem Cells Into Corneal Endothelial-Like Cells.

PURPOSE: Loss of corneal endothelial cells (CECs) bears disastrous consequences for the patient, including corneal clouding and blindness. Corneal transplantation is currently the only therapy for severe corneal disorders. However, the worldwide shortages of corneal donor material generate a strong demand for personalized stem cell-based alternative therapies. Because human mesenchymal stem cells are known to be sensitive to their mechanical environments, we investigated the mechanotransductive potential of Descemet membrane-like microtopography (DLT) to differentiate human mesenchymal stem cells into CEC-like cells. METHODS: Master molds with inverted DLT were produced by 2-photon lithography (2-PL). To measure the mechanotransductive potential of DLT, mesenchymal stem cells were cultivated on silicone or collagen imprints with DLT. Changes in morphology were imaged, and changes in gene expression of CEC typical genes such as zonula occludens (ZO-1), sodium/potassium (Na/K)-ATPase, paired-like homeodomain 2 (PITX2), and collagen 8 (COL-8) were measured with real-time polymerase chain reaction. At least immunofluorescence analysis has been conducted to confirm gene data on the protein level. RESULTS: Adhesion of MSCs to DLT molded in silicone and particularly in collagen initiates polygonal morphology and monolayer formation and enhances not only transcription of CEC typical genes such as ZO-1, Na/K-ATPase, PITX2, and COL-8 but also expression of the corresponding proteins. CONCLUSIONS: Artificial reproduction of Descemet membrane with respect to topography and similar stiffness offers a potential innovative way to bioengineer a functional CEC monolayer from autologous stem cells.

Enteric-coated mycophenolate sodium in the treatment of non-infectious intermediate uveitis: results of a prospective, controlled, randomised, open-label, early terminated multicentre trial.

BACKGROUND/AIMS: To evaluate the efficacy, safety and tolerability of enteric-coated mycophenolate sodium (EC-MPS) in combination with low-dose corticosteroids compared with a monotherapy with low-dose corticosteroids in subjects with non-infectious intermediate uveitis (IU). METHODS: Open-label, prospective, controlled, randomised multicentre trial. Patients were randomised in a 1:1 ratio to either the treatment group (prednisolone plus EC-MPS) or control group (prednisolone monotherapy). Patients in the control group who relapsed within 6 months changed to the crossover group (prednisolone plus EC-MPS). Maximum treatment duration was 15 months. The primary endpoint was the time to first relapse in the treatment group and control group. RESULTS: Forty-one patients at eight sites were analysed. Twenty-two patients were allocated to the treatment group, with 19 patients in the control group. A first relapse occurred in 9 patients (40.9%) in the treatment group and 15 patients (78.9%) in the control group (p=0.03). The median time to the first relapse was >15 months for the treatment group and 2.8 months for the control group (p=0.07). The probability of relapse-free survival at month 15 was estimated to be 52.9% in the treatment group and 19.7% in the control group (p=0.01). 15 patients changed to the crossover group. Of these, only four patients developed a second relapse. No safety concerns arose during the trial. Only one patient had to discontinue EC-MPS due to increased liver enzymes. CONCLUSION: EC-MPS can be considered an effective and well-tolerated immunosuppressive drug to prevent relapses in patients with chronic IU. TRIAL REGISTRATION NUMBER: EUDRACT number: 2009-009998-10, Results.

Intra-individual variability and circadian rhythm of vascular endothelial growth factors in subjects with normal glucose tolerance and type 2 diabetes.

Increased levels of systemic vascular endothelial growth factors (VEGFs) in patients with diabetes are associated with increased risk of microvessel disease. On the other hand, low VEGF levels after intravitreal antibody application may be associated with acute cardiovascular complications and treatment failure. Individual levels of systemic VEGF vary in a wide range depending on analytical methods and quality of diabetes control. So far only limited information exists on intraindividual fluctuations over longer periods and circadian rhythms. We analysed the intraindividual variance of VEGF-A, VEGF-C and placental growth factor (PLGF) in CTAD (citrate-theophylline-adenine-dipyridamol) plasma as well as VEGF-A in serum over a period of 6 months in patients with stable controlled type 2 diabetes (10 M, 10 F) and age and sex matched subjects with normal glucose tolerance (NGT). Furthermore, circadian levels of VEGFs were controlled hourly from 7:30 a.m. to 7:30 p.m. under standardized metabolic ward conditions. In addition, the relationship to metabolic, hormonal and inflammatory biomarkers was analyzed. VEGF-A, VEGF-C and PLGF remained stable in plasma and VEGF-A in serum over 6 months in both groups. No circadian change was observed in VEGF-A serum and plasma concentrations. A minor decrease of VEGF-C plasma levels was evident after 5 p.m. in both groups and a significant peak of PLGF concentrations occurred after lunch, which was more pronounced in T2DM. In multivariate analysis, only serum VEGF-A correlated to diabetes duration, whereas VEGF-C only correlated to HbA1c and fasting blood glucose. We did not observe significant intraindividual variances for VEGF-A in serum and VEGF-A, VEGF-C and PLGF in CTAD plasma over a period of 6 months. Taken together, a single morning measurement of systemic VEGF levels after 7:30 am appears to be a reliable parameter for the individual risk associated with abnormal VEGF concentrations in blood. TRIAL REGISTRATION: NCT02325271.

Effect of different culture media and deswelling agents on survival of human corneal endothelial and epithelial cells in vitro.

PURPOSE: To examine the effects of media and deswelling agents on human corneal endothelial and epithelial cell viability using a previously developed screening system. METHODS: The human corneal endothelial cell line HCEC-12 and the human corneal epithelial cell line HCE-T were cultured in four different corneal organ culture media (serum-supplemented: MEM +2 % FCS, CorneaMax®/CorneaJet®, serum-free: Human Endothelial-SFM, Stemalpha-2 and -3) with and without 6 % dextran T500 or 7 % HES 130/0.4. Standard growth media F99HCEC and DMEM/F12HCE-T served as controls. In additional controls, the stress inducers staurosporine or hydrogen peroxide were added. After 5 days in the test media, cell viability was assessed by flow cytometrically quantifying apoptotic and necrotic cells (sub-G1 DNA content, vital staining with YO-PRO-1® and propidium iodide) and intracellular reactive oxygen species (ROS). RESULTS: The MEM-based media were unable to support HCEC-12 and HCE-T survival under stress conditions, resulting in significantly increased numbers of apoptotic and necrotic cells. HCEC-12 survival was markedly improved in SFM-based media even under staurosporine or hydrogen peroxide. Likewise, HCE-T survival was improved in SFM with or without dextran. The media CorneaMax®, CorneaJet®, and CorneaMax® with HES supported HCEC-12 survival better than MEM-based media, but less well than SFM-based media. HCE-T viability was also supported by CorneaJet®, but not by CorneaMax® with or without HES. Stemalpha-based media were not suitable for maintaining viability of HCEC-12 or HCE-T in the applied cell culture system. CONCLUSIONS: The use of serum-supplemented MEM-based media for corneal organ culture should be discontinued in favour of serum-free media like SFM.

[Effect of autologous platelet concentrates on the anatomical and functional outcome of late stage macular hole surgery: A retrospective analysis]. / Wirkung von autologem Thrombozytenkonzentrat auf den anatomischen und funktionellen Erfolg bei der Chirurgie des Makulaforamens im Spätstadium : Eine retrospektive Analyse.

BACKGROUND: The macular hole (MH) is a disorder of the visual center of the retina in humans. An untreated MH leads to loss of central visual acuity and reading ability. Surgery for early-stage macular holes has been very successful for many years and leads to very good anatomical and functional results. Despite continuous improvement of surgical procedures, the outcome for the later stages of MH is still unsatisfactory. METHOD: In a retrospective analysis, we investigated the effect of autologous platelet concentrates in patients presenting later stages of MHs (stage III-IV) with respect to anatomic success (hole closure) and recovery of vision. The application of platelets was performed during retinal surgery (pars plana vitrectomy, ppV). In addition, selected platelet concentrates were qualitatively analysed for growth factors and platelet adhesion. RESULTS: In the first group, 74% of the patients showed a good anatomical macular hole closure. The analyses of the platelet concentrates indicated a possible wound-healing effect due to growth factors (e.g. the platelet-derived growth factor, PDGF) and lesser to the ability of the platelets to adhere after ristocetin administration. Further optimization of the production process of platelet concentrates and of the surgical procedure in the second group of patients showed an increase of the anatomical success (92%) and a very rapid increase of visual acuity within six weeks. DISCUSSION: In the past, the primary goal of MH surgery was to optimize the surgical procedures. Only few concepts focused on wound healing. Based on our data, we postulate the use of autologous platelet concentrates in MH surgery as a healing concept, which helps to increase the functional success of late-stage macular hole surgery.

Sutureless fixation of amniotic membrane for therapy of ocular surface disorders.

Amniotic membrane is applied to the diseased ocular surface to stimulate wound healing and tissue repair, because it releases supportive growth factors and cytokines. These effects fade within about a week after application, necessitating repeated application. Generally, amniotic membrane is fixed with sutures to the ocular surface, but surgical intervention at the inflamed or diseased site can be detrimental. Therefore, we have developed a system for the mounting of amniotic membrane between two rings for application to a diseased ocular surface without surgical intervention (sutureless amniotic membrane transplantation). With this system, AmnioClip, amniotic membrane can be applied like a large contact lens. First prototypes were tested in an experiment on oneself for wearing comfort. The final system was tested on 7 patients in a pilot study. A possible influence of the ring system on the biological effects of amniotic membrane was analyzed by histochemistry and by analyzing the expression of vascular endothelial growth factor-A (VEGF-A), hepatocyte growth factor (HGF), fibroblast growth factor 2 (FGF 2) and pigment epithelium-derived factor (PEDF) from amniotic membranes before and after therapeutic application. The final product, AmnioClip, showed good tolerance and did not impair the biological effects of amniotic membrane. VEGF-A and PEDF mRNA was expressed in amniotic membrane after storage and mounting before transplantation, but was undetectable after a 7-day application period. Consequently, transplantation of amniotic membranes with AmnioClip provides a sutureless and hence improved therapeutic strategy for corneal surface disorders.Trial Registration:ClinicalTrials.gov NCT02168790

Thermo-responsive cell culture carriers based on poly(vinyl methyl ether)-the effect of biomolecular ligands to balance cell adhesion and stimulated detachment.

Two established material systems for thermally stimulated detachment of adherent cells were combined in a cross-linked polymer blend to merge favorable properties. Through this approach poly(N-isopropylacrylamide) (PNiPAAm) with its superior switching characteristic was paired with a poly(vinyl methyl ether)-based composition that allows adjusting physico-chemical and biomolecular properties in a wide range. Beyond pure PNiPAAm, the proposed thermo-responsive coating provides thickness, stiffness and swelling behavior, as well as an apposite density of reactive sites for biomolecular functionalization, as effective tuning parameters to meet specific requirements of a particular cell type regarding initial adhesion and ease of detachment. To illustrate the strength of this approach, the novel cell culture carrier was applied to generate transplantable sheets of human corneal endothelial cells (HCEC). Sheets were grown, detached, and transferred onto planar targets. Cell morphology, viability and functionality were analyzed by immunocytochemistry and determination of transepithelial electrical resistance (TEER) before and after sheet detachment and transfer. HCEC layers showed regular morphology with appropriate TEER. Cells were positive for function-associated marker proteins ZO-1, Na+/K+-ATPase, and paxillin, and extracellular matrix proteins fibronectin, laminin and collagen type IV before and after transfer. Sheet detachment and transfer did not impair cell viability. Subsequently, a potential application in ophthalmology was demonstrated by transplantation onto de-endothelialized porcine corneas in vitro. The novel thermo-responsive cell culture carrier facilitates the generation and transfer of functional HCEC sheets. This paves the way to generate tissue engineered human corneal endothelium as an alternative transplant source for endothelial keratoplasty.

Serum-free medium and hydroxyethyl starch supports cell survival better than Minimal Essential Medium and dextran in organ-cultured mouse corneas.

BACKGROUND/AIMS: In a previous study, we observed a deleterious effect of serum-supplemented Minimal Essential Medium (MEM) on human corneal endothelial cell survival in a cell culture model. Consequently, here we studied the effects of conventional, serum-supplemented MEM and a serum-free medium in combination with two different deswelling substances on cell survival in whole corneas in a mouse model. METHODS: Murine corneas were cultured for 4 days in MEM+2% fetal calf serum (FCS) or serum-free Human Endothelial-SFM (SFM), both supplemented with either 6% dextran T500 or 7.5% hydroxyethyl starch (HES) 130/0.4. Cells were examined by differential interference contrast microscopy, H&E staining, immunocytochemistry for cleaved caspase-3, Bcl-2, haem oxygenase-1 and immunoblotting for cleaved caspase-3. RESULTS: In MEM, endothelial cells were almost completely lost after 4 days and the number of epithelial cells was markedly reduced. The remaining cells showed fragmented nuclei and were positive for cleaved caspase-3 and strongly positive for Bcl-2. Corneas cultured in SFM retained an almost closed layer of endothelial cells. Fewer cells were positive for Bcl-2, and only a few cells were positive for cleaved caspase-3 even under staurosporine administration. HES supplementation was well tolerated by corneal cells over 4 days, while a 4-day supplementation with dextran resulted in the loss of endothelial and epithelial cells. CONCLUSIONS: Serum-free medium, Human Endothelial-SFM, promoted cell survival during corneal organ culture better than MEM+2% FCS. HES 130/0.4 appeared to be tolerated better by the cells than dextran T500.

Semaphorin 3A alters endothelial cell immunogenicity by regulating Class II transactivator activity circuits.

BACKGROUND: Endothelial cells (ECs) play a pivotal role in the allogeneic immune response upon transplantation. Semaphorin 3A (Sema3A) was implicated in the modulation of EC growth, but its effects on immunogenicity were not previously investigated. STUDY DESIGN AND METHODS: ECs were transduced with a lentiviral vector encoding for the green fluorescence protein (GFP) sequence under the control of a Class II transactivator (CIITA)-dependent promoter. Upon stimulation of nonmodified ECs with recombinant Sema3A protein, mRNA and protein levels of CIITA, HLA-DR, and Sema3A receptors were evaluated. An enzyme-linked immunosorbent assay was developed to quantify Sema3A levels in the sera of kidney-transplanted patients. RESULTS: Sema3A stimulation of lentiviral vector encoding for the GFP sequence ECs caused a significant up regulation of the transgene expression, indicating an increase in CIITA levels. Stimulation of nonmodified ECs with Sema3A resulted in an up regulation of CIITA expression, which was associated with enhanced HLA-DR levels and an increase in alloreactive CD4+ T-cell proliferation. Sema3A receptor expression was enhanced by CIITA, establishing a positive feedback loop. Higher levels of Sema3A were observed in sera of patients presenting with organ rejection. CONCLUSION: This study links Sema3A signaling in ECs with increased CIITA levels and higher HLA-DR expression, resulting in CD4+ T-cell activation, which might have important implications for tissue and organ transplantation.