Impact of implementation of polymerase chain reaction on diagnosis, treatment, and clinical course of Acanthamoeba keratitis.

PURPOSE: Acanthamoeba keratitis (AK) is a painful and possibly sight-threatening ocular infection. While the correct diagnosis and specific treatment in the early stages significantly improve the prognosis, the disease is often misdiagnosed and in clinical examination confused with other forms of keratitis. Polymerase chain reaction (PCR) for the detection of AK was first introduced in our institution in December 2013 to improve the timely diagnosis of AK. The aim of this study was to assess the impact of implementation of Acanthamoeba PCR on the diagnosis and treatment of the disease in a German tertiary referral center. PATIENTS AND METHODS: Patients treated for Acanthamoeba keratitis between 1st of January 1993 and 31st of December 2021 in the Department of Ophthalmology of the University Hospital Duesseldorf were identified retrospectively via in-house registries. Evaluated parameters include age, sex, initial diagnosis, method of correct diagnosis, duration of symptoms until correct diagnosis, contact lens use, visual acuity, and clinical findings as well as medical and surgical therapy by keratoplasty (pKP). In order to assess the impact of implementation of Acanthamoeba PCR, the cases were divided into two groups (before (pre-PCR group) and after PCR implementation (PCR group). RESULTS: Seventy-five patients with Acanthamoeba keratitis were included (69.3% female, median age 37 years). Eighty-four percent (63/75) of all patients were contact lens wearers. Until PCR was available, 58 patients with Acanthamoeba keratitis were diagnosed either clinically (n = 28), by histology (n = 21), culture (n = 6), or confocal microscopy (n = 2) with a median duration until diagnosis of 68 (18; 109) days. After PCR implementation, in 17 patients, the diagnosis was established with PCR in 94% (n = 16) and median duration until diagnosis was significantly shorter with 15 (10; 30.5) days. A longer duration until correct diagnosis correlated with a worse initial visual acuity (p = 0.0019, r = 0.363). The number of pKP performed was significantly lower in the PCR group (5/17; 29.4%) than in the pre-PCR group (35/58; 60.3%) (p = 0.025). CONCLUSIONS: The choice of diagnostic method and especially the application of PCR have a significant impact on the time to diagnosis and on the clinical findings at the time of confirmation of diagnosis and the need for penetrating keratoplasty. In contact lens-associated keratitis, the first crucial step is to take AK into consideration and perform a PCR test as timely confirmation of diagnosis of AK is imperative to prevent long-term ocular morbidity.

PREVALENCE OF SARS-CoV-2 RNA AND ANTIBODY DETECTION, AND VACCINATION STATUS IN PATIENTS WITH OCULAR VASCULAR OCCLUSION.

PURPOSE: To analyze the annual prevalence of ocular vascular occlusion in relation to COVID-19 infection and vaccination status in a prospective study. METHODS: All patients were examined for an active severe acute respiratory syndrome coronavirus 2 infection by RNA detection and for a previous infection by virus-specific antibody detection, and their vaccination status was documented. Data from pandemic year 2020 and previous years, before COVID-19 (2019, 2018, 2017), were retrospectively analyzed. RESULTS: In 2021, a total of 103 patients with the first diagnosis of ocular vascular occlusion were treated. Most frequent subdiagnoses were central retinal vein occlusion (20.4%), nonarteritic anterior ischemic optic neuropathy (18.4%), central retinal artery occlusion (13.6%), and branch retinal artery occlusion (12.6%). Thereof, only three patients (2.9%) presented with virus-specific severe acute respiratory syndrome coronavirus 2 antibodies, and none was PCR positive. Patients with preceded severe acute respiratory syndrome coronavirus 2 vaccination (59.2%) presented with comparable characteristics as unvaccinated patients with vascular occlusion regarding age, gender distribution, systemic risk factors, duration of symptoms, visual acuity, and the present subdiagnoses ( P > 0.05). The total number of cases in 2021 (103 cases) was comparable with the pandemic year 2020, at which no vaccination was available (114 cases), and to earlier years 2017, 2018, and 2019 without COVID-19 pandemic (100, 120, and 119 cases). Furthermore, we did not reveal any differences between pandemic and reference years regarding patients' characteristics ( P > 0.05). CONCLUSION: Our study did not reveal an increased annual prevalence of ocular vascular occlusions during COVID-19 pandemic years 2020 and 2021. Patients with previous COVID-19 vaccination did not present differences regarding the risk profile nor symptoms, compared with unvaccinated individuals.

Electrospun PCL Scaffolds as Drug Carrier for Corneal Wound Dressing Using Layer-by-Layer Coating of Hyaluronic Acid and Heparin.

Due to its ability to reduce scarring and inflammation, human amniotic membrane is a widely used graft for wound dressings after corneal surgery. To overcome donor dependency and biological variances in the donor tissue, artificial nanofibrous grafts acting as drug carrier systems are promising substitutes. Electrospun nanofibrous scaffolds seem to be an appropriate approach as they offer the properties of permeable scaffolds with a high specific surface, the latter one depending on the fiber diameter. Electrospun scaffolds with fiber diameter of 35 nm, 113 nm, 167 nm and 549 nm were manufactured and coated by the layer-by-layer (LbL) technology with either hyaluronic acid or heparin for enhanced regeneration of corneal tissue after surgery. Studies on drug loading capacity and release kinetics defined a lower limit for nanofibrous scaffolds for effective drug loading. Additionally, scaffold characteristics and resulting mechanical properties from the application-oriented characterization of suture pullout from suture retention tests were examined. Finally, scaffolds consisting of nanofibers with a mean fiber diameter of 113 nm were identified as the best-performing scaffolds, concerning drug loading efficiency and resistance against suture pullout.

[Corneal wound healing-Pharmacological treatment]. / Wundheilung der Kornea ­ Pharmakologische Therapie.

Physiological wound healing of the cornea is a complex process and involves numerous multifactorial tissue processes. A proper wound healing, especially without the formation of light-scattering scars, is essential to preserve the integrity and function of the cornea. Misdirected wound healing is of vast clinical relevance as it can lead to corneal fibrosis and the loss of optical transparency with subsequent reduction of visual acuity, up to blindness. In addition to the understanding of the pathophysiological mechanisms, the knowledge of therapeutic concepts and options for treating corneal wound healing disorders and fibrosis is essential to counteract a permanent damage of the cornea as early as possible. Nowadays, various pharmacological and surgical options are available for treatment. The decision, appropriate selection and indication for the optimal treatment depend primarily on the genesis and clinical appearance of the corneal wound, fibrosis or scar. The treatment of wound healing disorders ranges from the use of topical therapy and supportive measures up to tissue replacement procedures. As long as the mechanical stability of the cornea is intact and wound healing processes are still ongoing, a pharmacological modulation is reasonable, which is discussed in this article.

UV light-mediated corneal crosslinking as (lymph)angioregressive pretreatment to promote graft survival after subsequent high-risk corneal transplantation (CrossCornealVision): protocol for a multicenter, randomized controlled trial.

BACKGROUND: Good vision highly depends on the transparency of the cornea, which is the "windscreen" of the eye. In fact, corneal blindness due to transparency loss is the second most common cause of blindness worldwide, and corneal transplantation is the main cure. Importantly, the cornea is normally avascular but can secondarily be invaded by pathological (blood and lymphatic) vessels due to severe inflammation, and the survival prognosis of a corneal graft mainly depends on the preoperative vascular condition of the recipient's cornea. Whereas transplants placed into avascular recipient beds enjoy long-term survival rates of > 90%, survival rates significantly decrease in pathologically pre-vascularized, so-called high-risk recipients, which account for around 10% of all performed transplants in Germany and > 75% in lower and middle-income countries worldwide. METHODS: This parallel-grouped, open-randomized, multicenter, prospective controlled exploratory investigator-initiated trial (IIT) intends to improve graft survival by preconditioning pathologically vascularized recipient corneas by (lymph)angioregressive treatment before high-risk corneal transplantation. For this purpose, corneal crosslinking (CXL) will be used, which has been shown to potently regress corneal blood and lymphatic vessels. Prior to transplantation, patients will be randomized into 2 groups: (1) CXL (intervention) or (2) no pretreatment (control). CXL will be repeated once if insufficient reduction of corneal neovascularization should be observed. All patients (both groups) will then undergo corneal transplantation. In the intervention group, remaining blood vessels will be additionally regressed using fine needle diathermy (on the day of transplantation). Afterwards, the incidence of graft rejection episodes will be evaluated for 24 months (primary endpoint). Overall graft survival, as well as regression of corneal vessels and/or recurrence, among other factors, will be analyzed (secondary endpoints). DISCUSSION: Based on preclinical and early pilot clinical evidence, we want to test the novel concept of temporary (lymph)angioregressive pretreatment of high-risk eyes by CXL to promote subsequent corneal graft survival. So far, there is no evidence-based approach to reliably improve graft survival in the high-risk corneal transplantation setting available in clinical routine. If successful, this approach will be the first to promote graft survival in high-risk transplants. It will significantly improve vision and quality of life in patients suffering from corneal blindness. TRIAL REGISTRATION: ClinicalTrials.gov NCT05870566. Registered on 22 May 2023.

A novel pro-lymphangiogenic function for Th17/IL-17.

Th17 cells, in addition to their proinflammatory functions, have been recognized as potent inducers of angiogenesis in autoimmune diseases and malignancies. In the present study, we demonstrate distinct mechanisms by which IL-17 induces lymphangiogenesis. Using the mouse cornea micropocket and cell culture assays, our data demonstrate that IL-17 directly promotes growth of lymphatic vessels by inducing increased expression of prolymphangiogenic VEGF-D and proliferation of lymphatic endothelial cells. However, IL-17-induced growth of blood vessels is primarily mediated through IL-1ß secretion by IL-17-responsive cells. Furthermore, in vivo blockade of IL-17 in a preclinical model of Th17-dominant autoimmune ocular disease demonstrates a significant reduction in the corneal lymphangiogenesis and in the progression of clinical disease. Taken together, our findings demonstrate a novel prolymphangiogenic function for Th17/IL-17, indicating that IL-17 can promote the progression and amplification of immunity in part through its induction of lymphangiogenesis.

Anatomical and functional results after vitrectomy with conventional ILM peeling versus inverted ILM flap technique in large full-thickness macular holes.

BACKGROUND: Aim of the study was to compare success rate and functional outcome following pars plana vitrectomy (PPV) with conventional internal limiting membrane (ILM) peeling versus ILM flap technique for full-thickness idiopathic macular holes (FTMH). METHODS: Retrospective analysis of consecutive eyes with FTMH having undergone vitrectomy with sulfur hexafluoride (SF6) endotamponade 25% at the University Medical Center Rostock, Germany (2009-2020). Eyes were divided according to applied surgical technique (ILM peeling [group P] versus ILM flap [group F]). Inclusion criteria were macular hole base diameters (MH-BD) ≥ 400 µm plus axial length ≤ 26.0 mm. Each group was divided into two subgroups based on macular hole minimum linear diameter (MH-MLD): ≤ 400 µm and > 400 µm. Exclusion criteria were FTMH with MH-BD < 400 µm, trauma, myopia with axial length > 26.0 mm or macular schisis. Demographic, functional, and anatomical data were obtained pre- and postoperatively. Preoperative MH-BD and MH-MLD were measured using optical coherence tomography (OCT; Spectralis®, Heidelberg Engineering GmbH, Heidelberg, Germany). Main outcome parameter were: primary closure rate, best-corrected visual acuity (BCVA), and re-surgery rate. RESULTS: Overall 117 eyes of 117 patients with FTMH could be included, thereof 52 eyes underwent conventional ILM peeling (group P) and 65 additional ILM flap (group F) technique. Macular hole closure was achieved in 31 eyes (59.6%) in group P and in 59 eyes (90.8%) in group F (p < 0.001). Secondary PPV was required in 21 eyes (40.4%) in group P and in 6 eyes (9.2%) in group F. Postoperative BCVA at first follow-up in eyes with surgical closure showed no significant difference for both groups (MH-MLD ≤ 400 µm: p = 0.740); MH-MLD > 400 µm: p = 0.241). CONCLUSION: Anatomical results and surgical closure rate following ILM flap technique seems to be superior to conventional ILM peeling for treatment of FTMH.

Schwann Cells Promote Myogenic Differentiation of Myoblasts and Adipogenic Mesenchymal Stromal Cells on Poly-ɛ-Caprolactone-Collagen I-Nanofibers.

For the purpose of skeletal muscle tissue engineering, different cell types have been investigated regarding their myogenic differentiation potential, including co-cultured myoblasts and adipogenic mesenchymal stromal cells (Mb/ADSC). As neural cells enhance synaptic junction formation, the aim of this study was to co-culture Schwann cells (SCs) with Mb/ADSC on biocompatible electrospun aligned poly-ε-polycaprolacton (PCL)-collagen I-nanofibers. It was hypothesized that SCs, as part of the peripheral nervous system, promote the myogenic differentiation of Mb/ADSC co-cultures. Mb/ADSC were compared to Mb/ADSC/SC regarding their capacity for myogenic differentiation via immunofluorescent staining and gene expression of myogenic markers. Mb/ADSC/SC showed more myotubes after 28 days of differentiation (p ≤ 0.05). After 28 days of differentiation on electrospun aligned PCL-collagen I-nanofibers, gene expression of myosin heavy chains (MYH2) and myogenin (MYOG) was upregulated in Mb/ADSC/SC compared to Mb/ADSC (p ≤ 0.01 and p ≤ 0.05, respectively). Immunofluorescent staining for MHC showed highly aligned multinucleated cells as possible myotube formation in Mb/ADSC/SC. In conclusion, SCs promote myogenic differentiation of Mb/ADSC. The co-culture of primary Mb/ADSC/SC on PCL-collagen I-nanofibers serves as a physiological model for skeletal muscle tissue engineering, applicable to future clinical applications.

Examining the Transmission of Visible Light through Electrospun Nanofibrous PCL Scaffolds for Corneal Tissue Engineering.

The transparency of nanofibrous scaffolds is of highest interest for potential applications like corneal wound dressings in corneal tissue engineering. In this study, we provide a detailed analysis of light transmission through electrospun polycaprolactone (PCL) scaffolds. PCL scaffolds were produced via electrospinning, with fiber diameters in the range from (35 ± 13) nm to (167 ± 35) nm. Light transmission measurements were conducted using UV-vis spectroscopy in the range of visible light and analyzed with respect to the influence of scaffold thickness, fiber diameter, and surrounding medium. Contour plots were compiled for a straightforward access to light transmission values for arbitrary scaffold thicknesses. Depending on the fiber diameter, transmission values between 15% and 75% were observed for scaffold thicknesses of 10 µm. With a decreasing fiber diameter, light transmission could be improved, as well as with matching refractive indices of fiber material and medium. For corneal tissue engineering, scaffolds should be designed as thin as possible and fabricated from polymers with a matching refractive index to that of the human cornea. Concerning fiber diameter, smaller fiber diameters should be favored for maximizing graft transparency. Finally, a novel, semi-empirical formulation of light transmission through nanofibrous scaffolds is presented.

Optimization of polycaprolactone - based nanofiber matrices for the cultivation of corneal endothelial cells.

Posterior lamellar transplantation of the eye' s cornea (DSAEK, DMEK) currently is the gold standard for treating patients with corneal endothelial cell and back surface pathologies resulting in functional impairment. An artificial biomimetic graft carrying human corneal endothelium could minimize the dependency on human donor corneas giving access to this vision-restoring surgery to large numbers of patients, thus reducing current long waiting lists. In this study, four groups of electrospun nanofibrous scaffolds were compared: polycaprolactone (PCL), PCL/collagen, PCL/gelatin and PCL/chitosan. Each of the scaffolds were tissue-engineered with human corneal endothelial cells (HCEC-B4G12) and analyzed with regard to their potential application as artificial posterior lamellar grafts. Staining with ZO-1 and Na+/K+-ATPase antibodies revealed intact cell functionalities. It could be shown, that blending leads to decreasing contact angle, whereby a heterogeneous blend morphology could be revealed. Scaffold cytocompatibility could be confirmed for all groups via live/dead staining, whereby a significant higher cell viability could be observed for the collagen and gelatine blended matrices with 97 ± 3% and 98 ± 2% living cells respectively. TEM images show the superficial anchoring of the HCECs onto the scaffolds. This work emphasizes the benefit of blended PCL nanofibrous scaffolds for corneal endothelial keratoplasty.

Midterm results of cultivated autologous and allogeneic limbal epithelial transplantation in limbal stem cell deficiency.

BACKGROUND: Limbal stem cell deficiency (LSCD) leads to growth of abnormal fibro-vascular pannus tissue onto the corneal surface as well as chronic inflammation and impaired vision. Our aim was to investigate the clinical outcome of ocular surface reconstruction in LSCD using limbal epithelial cells expanded on amniotic membrane (AM). METHODS: Forty-four eyes of 38 patients (27 male, 11 female) with total (n = 32) or partial (n = 12) LSCD were treated by transplantation of autologous (n = 30) or allogeneic (n = 14) limbal epithelial cells expanded on intact AM. LSCD was caused by chemical and thermal burns (n = 22), pterygium (n = 9), congenital aniridia (n = 6), tumor excision (n = 2), perforating eye injury, mitomycin C, epidermolysis bullosa, bilateral graft-versus-host disease and chlamydial conjunctivitis (each n = 1). RESULTS: Mean follow-up time was 28.5 +/- 14.9 months. The corneal surface could be reconstructed to full stability in 30 (68%), and clear central cornea was achieved in 37 (84%) eyes. Grafting was significantly more successful in eyes treated by autologous than by allogeneic transplantation (76.7 vs. 50%, p < 0.05). The corneal surface could be successfully restored in 10 (83.3%) eyes with partial LSCD and in 20 (63.3%) eyes with total LSCD. Visual acuity (VA) increased significantly in 32 (73%) eyes, was stable in 10 (23%) eyes and decreased in 2 (4%) eyes. Mean VA increased significantly (p < 0.0001), from preoperative 1.7 +/- 0.9 log MAR (20/1,000) to 0.9 +/- 0.7 log-MAR (20/160). VA increased significantly after both autologous (p < 0.0001) and allogeneic transplantation (p < 0.005). CONCLUSIONS: In most patients with LSCD, transplantation of limbal epithelium cultivated on intact AM restores the corneal surface and results in significantly increased VA.

Biomechanical assessment of healthy and keratoconic corneas (with/without crosslinking) using dynamic ultrahigh-speed Scheimpflug technology and the relevance of the parameter (A1L-A2L).

AIMS: To examine corneal biomechanics in healthy and keratoconic eyes, with or without crosslinking obtained by ultrahigh-speed Scheimpflug measurements (Corvis ST). METHODS: One hundred and seventeen eyes were studied in three groups: group 1 (n=39) contained keratoconic eyes without crosslinking. Group 2 (CXL; n=28) comprised keratoconic eyes after crosslinking. These were compared with a control group (n=50 matched healthy eyes). In addition, 10 keratoconus patients, before and after CXL treatment, respectively, were examined. RESULTS: The novel parameter A1L-A2L demonstrated highly significant differences between crosslinked corneas and untreated keratoconic or healthy corneas. Velocity during second applanation (A2V) and deformation amplitude (DA) were significantly increased in crosslinked keratoconic eyes both compared with untreated keratoconic eyes and with healthy controls. Radius at highest curvature also was significant among all groups. Inward applanation length (A1L) was significantly increased in controls, whereas outward applanation length (A2L) was significantly reduced in crosslinked keratoconic eyes compared with both other groups. The follow-up analysis revealed statistically significant changes in pachymetry and intraocular pressure and showed tendencies towards significance in applanation times 1 and 2 and in DA. CONCLUSIONS: Both A2V and A2L are viable parameters to discriminate healthy from keratoconic but also crosslinked from non-crosslinked keratoconic corneas. The difference of A1L-A2L could reliably discriminate crosslinked from non-crosslinked and healthy corneas. Follow-up examination in a small cohort allows distinction between crosslinked and untreated keratoconus in follow-up examinations. The difference of A1L-A2L could reliably discriminate crosslinked from non-crosslinked and healthy corneas. Measurements of corneal deformation using dynamic ultrahigh-speed Scheimpflug technology are reproducible and provide useful information about keratoconus assessment and biomechanics. Therefore, the Corvis ST seems to provide useful technology to monitor therapeutic success of crosslinking treatment.

[Novel concepts for treatment of the corneal endothelium with nanoparticles]. / Neuartige Konzepte zur Behandlung des Hornhautendothels mittels Nanopartikel.

BACKGROUND: The number of corneal transplantations has increased in Germany in recent years. One reason is an earlier time point of surgery due to less invasive posterior lamellar grafting techniques. To date, penetrating and lamellar corneal transplantations are the only established therapeutic options to treat corneal diseases resulting from endothelial cell pathologies. AIM: This review article provides an insight into nanoparticle-related translational strategies to improve or to avoid corneal transplantation. RESULTS: Nanoparticle-based strategies for optimization of the corneal endothelium have different fields of application: 1. during donor cornea cultivation in culture medium, 2. for single cell injection therapies and 3. to treat the patients' own endothelium in an effort to avoid transplantation. CONCLUSION: Several translational concepts exist to improve or to avoid grafting of a donor cornea. The coming decade will provide established alternatives to conventional corneal transplantation.

Argon Cold Plasma-A Novel Tool to Treat Therapy-resistant Corneal Infections.

PURPOSE: To test whether therapy-resistant corneal infections can be successfully treated with argon cold plasma to reduce or eliminate pathogen microorganisms without affecting corneal cell viability. DESIGN: First-in-human case series and experimental study. METHODS: Cold plasma effects on viability of primary human corneal limbal epithelial cells were studied using exposure times from 0.5 to 10 minutes (metabolic activity, oxidative stress, apoptosis). Disinfective potential of cold plasma was tested against common pathogens (Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Pseudomonas aeruginosa, and Candida albicans) on culture medium and evaluated by counting colony-forming units and optical density measurements, as well as against S aureus in a human cornea infection model. Additionally, in a first-in-human trial 4 patients with therapy-resistant corneal ulcers were treated to evaluate the clinical potential of cold plasma. RESULTS: Cells treated for 0.5-5 minutes completely recovered within 24 hours without changes in morphology; only 10-minute treatment impaired the cells permanently. No evident oxidative stress, apoptosis, or damage to the corneal structure could be found. All pathogens were susceptible to cold plasma treatments, with different levels of sensitivity. The condition of all 4 patients significantly improved after cold plasma treatment combined with antibiotic therapy. CONCLUSIONS: Our results indicate that argon cold plasma treatment reduces or eliminates common pathogens without impairing corneal epithelial cells in vitro, ex vivo, and in direct application on patients' eyes. We conclude that argon cold plasma therapy offers a potential supplement or alternative therapy for therapy-resistant corneal infections. A larger, comparative study is necessary to further confirm these findings.

Gene Therapy for Modulation of T-Cell-Mediated Immune Response Provoked by Corneal Transplantation.

Corneal transplantation (keratoplasty) is the most common type of tissue replacement in the world. The increased rate of graft rejection after keratoplasty is a central problem for repeated transplantations and in inflamed host corneas. It has been shown that apoptosis of grafted epithelium has a role in corneal allograft rejection. This study focused on the T-cell response triggered in BALB/c mice after allogeneic corneal transplantation with and without anti-apoptotic p35-transduced epithelium. To restrict p35 expression to the epithelial cells, modified allogeneic composite grafts were created. As a result, it was found that the proportion of alloreactive CD4+ T cells in postoperatively removed cervical lymph nodes was reduced in the p35-transduced group compared to the allogeneic control group. Diminished priming of the CD4+ T cells was supported by significantly decreased proliferation and lower interferon gamma secretion when compared to allogeneic engraftments. The reduced priming of CD4+ lymphocytes is the first confirmation of the functionality of p35 in the epithelium of corneal grafts to alter the development of the recipient's immune response. Thus, modification of allosensibilization seems to be a promising tool for reducing graft-mediated immune response following corneal transplantation.

The influence of donor factors on corneal organ culture contamination.

PURPOSE: To evaluate the contamination rate and the corresponding spectrum of microbes and to identify donor risk factors for corneal organ culture contaminations. METHODS: A total of 3306 organ-cultured donor corneas were included in the study. We performed a retrospective database analysis to evaluate donor factors such as gender, age, death-to-explantation interval (DEI), procurement site and cause of death and to determine their influence on donor cornea contaminations. Odds ratios (ORs) were calculated for each factor. RESULTS: The overall contamination rate was 7.8% (n = 259). Younger donor age (OR: 2.2, p = 0.003, chi-squared test), a DEI of more than 24 hr (OR: 1.6, p < 0.001), hospitalization prior to death (OR: 2.2, p < 0.001) and death caused by sepsis (OR: 2.7, p < 0.001) were associated with an increased risk of contamination, whereas donor gender did not have an effect on donor cornea contaminations. The most frequently isolated microbes were Enterococci (19%), Staphylococci (10.8%) and Candida (37.4%). CONCLUSION: This study helps to estimate the contamination risk of a cultured cornea based on specific donor factors. However, donors with risk factors should not be generally excluded from cornea donation. Further studies including antibiograms might clarify whether a change in the antibiotic composition of the culture medium would be useful to deal with the increasing number of multi-resistant microbes.

Lamin Cleavage: A Reliable Marker for Studying Staurosporine-Induced Apoptosis in Corneal Tissue.

Purpose: The aims of this study were to identify a robust apoptosis marker suitable for both quantification and back-to-back analyses of programmed cell death and to define specific upstream targets for apoptosis in corneal cells. Methods: Apoptotic cleavage of initiator caspases and their downstream targets such as lamins and poly-ADP ribose polymerase was investigated in human corneal endothelial cells (HCEC-12), keratocytes (HCK), epithelial cells (HCEp), and full-thickness corneas using Western blotting and confocal microscopy following apoptosis induction with staurosporine. We specifically focused on nuclear lamins, which have important structural and regulatory functions in the cell nucleus. Results: The cleavage of lamin A in HCEC-12 was significantly increased following apoptotic induction compared with HCK. More importantly, lamin A cleavage was detected in a dose-dependent manner in full-thickness corneal tissue by both Western blot analysis and fluorescence microscopy. Our study also demonstrates that HCEp show approximately three-fold increase in caspase 6 cleavage compared with endothelial cells or keratocytes. The presence of cleaved caspase 9 was lower in endothelial cells compared with epithelial cells and keratocytes. Conclusions: We successfully established lamin A cleavage as a quantifiable marker of apoptosis in both corneal cells and tissue. Quantification of lamin A cleavage by Western blotting followed by a back-to-back analysis with fluorescence microscopy was studied for the first time in the experimental (donor) corneal tissue. Screening of downstream apoptosis proteins and establishing cell type-specific protocols allowed us to identify possible targets (caspases, Apaf-1, etc.) for protective therapeutic approaches.

Influence of operating parameters on the suture retention test for scaffolds in ophthalmology.

The strength of a suture used to attach a graft to a patient can be quantified using the suture retention test. In order to gain deeper insight on the influence of testing and application conditions testing parameters were varied. Different pull rates, suture and jaw positions, and suture materials were tested to show their influence on the result of the test. The results of the different states were analyzed using statistical tests. Based on the statistic analysis conditions for testing with a maximum of accuracy and as close as possible to in vivo conditions have been revealed.

Adhesion and metabolic activity of human corneal cells on PCL based nanofiber matrices.

In this work, polycaprolactone (PCL) was used as a basic polymer for electrospinning of random and aligned nanofiber matrices. Our aim was to develop a biocompatible substrate for ophthalmological application to improve wound closure in defects of the cornea as replacement for human amniotic membrane. We investigated whether blending the hydrophobic PCL with poly (glycerol sebacate) (PGS) or chitosan (CHI) improves the biocompatibility of the matrices for cell expansion. Human corneal epithelial cells (HCEp) and human corneal keratocytes (HCK) were used for in vitro biocompatibility studies. After optimization of the electrospinning parameters for all blends, scanning electron microscopy (SEM), attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), and water contact angle were used to characterize the different matrices. Fluorescence staining of the F-actin cytoskeleton of the cells was performed to analyze the adherence of the cells to the different matrices. Metabolic activity of the cells was measured by cell counting kit-8 (CCK-8) for 20days to compare the biocompatibility of the materials. Our results show the feasibility of producing uniform nanofiber matrices with and without orientation for the used blends. All materials support adherence and proliferation of human corneal cell lines with oriented growth on aligned matrices. Although hydrophobicity of the materials was lowered by blending PCL, no increase in biocompatibility or proliferation, as was expected, could be measured. All tested matrices supported the expansion of human corneal cells, confirming their potential as substrates for biomedical applications.

Poly (glycerol sebacate)-poly (ε-caprolactone) blend nanofibrous scaffold as intrinsic bio- and immunocompatible system for corneal repair.

A major challenge in corneal tissue engineering and lamellar corneal transplantation is to develop synthetic scaffolds able to simulate the optical and mechanical properties of the native cornea. As a carrier, the graft scaffolds should provide the basis for anchorage, repair and regeneration. Although quite a number of scaffolds have been engineered to date, they have not been able to simultaneously recapitulate chemical, mechanical, and structural properties of the corneal extracellular matrix (ECM). Here, we examined different compositions of elastomeric biodegradable poly (glycerol sebacate) (PGS)-poly (ε-caprolactone) (PCL) nanofibrous scaffolds with respect to their cyto- and immunocompatibility. These scaffolds were semi-transparent with well-defined mechanical properties and direct positive effects on viability of human corneal endothelial cells (HCEC) and human conjunctival epithelial cells (HCjEC). Moreover, within 3days HCEC established monolayers with the hexagonal morphology typical for this cell type. All PGS-PCL mixtures analyzed did not trigger effects in granulocytes, naïve and activated peripheral blood mononuclear cells (PBMCs). However, scaffolds with a higher content of PGS-PCL ratio showed the best cell organization, cyto- and immunocompatibility. Subsequently, this PGS-PCL composition could be used for further development of clinical constructs to support corneal tissue repair. STATEMENT OF SIGNIFICANCE: In corneal tissue engineering a major challenge is the development of synthetic scaffolds with similar properties to native cornea. In our recent works, we introduced the biodegradable, polymeric nanofibrous scaffolds with similar optical and mechanical properties for corneal regeneration and here we examined the cyto- and immunocompatibility of biodegradable nanofibrous scaffolds in contact to white blood cells. Directing the alignment of human corneal cells by nanofibrous scaffolds and high viability of cells was detected by forming of endothelium monolayer with hexagonal morphology on the nanofibrous scaffold. In addition, our results for the first time show that these nanofibrous scaffolds did not trigger effects in white blood cells. These results highlight the considerable translational potential of the nanofibrous scaffolds to clinical applications.