M. Bischoff

Analysis of the changes in keratoplasty indications and preferred techniques.

Background Recently, novel techniques introduced to the field of corneal surgery, e.g. Descemet membrane endothelial keratoplasty (DMEK) and corneal crosslinking, extended the therapeutic options. Additionally contact lens fitting has developed new alternatives. We herein investigated, whether these techniques have affected volume and spectrum of indications of keratoplasties in both a center more specialized in treating Fuchs’ dystrophy (center 1) and a second center that is more specialized in treating keratoconus (center 2). Methods We retrospectively reviewed the waiting lists for indication, transplantation technique and the patients’ travel distances to the hospital at both centers. Results We reviewed a total of 3778 procedures. Fuchs’ dystrophy increased at center 1 from 17% (42) to 44% (150) and from 13% (27) to 23% (62) at center 2. In center 1, DMEK increased from zero percent in 2010 to 51% in 2013. In center 2, DMEK was not performed until 2013. The percentage of patients with keratoconus slightly decreased from 15% (36) in 2009 vs. 12% (40) in 2013 in center 1. The respective percentages in center 2 were 28% (57) and 19% (51). In both centers, the patients’ travel distances increased. Conclusions The results from center 1 suggest that DMEK might increase the total number of keratoplasties. The increase in travel distance suggests that this cannot be fully attributed to recruiting the less advanced patients from the hospital proximity. The increase is rather due to more referrals from other regions. The decrease of keratoconus patients in both centers is surprising and may be attributed to optimized contact lens fitting or even to the effect corneal crosslinking procedure.

Wachstumsfaktoren und Interleukine in Amniongewebehomogenat

PURPOSE Application of amniotic membrane homogenate eye drops may be a potential treatment alternative for therapy resistant corneal epithelial defects. The purpose of this study was to determine the concentrations of epidermal growth factor (EGF), fibroblast growth factor basic (bFGF), hepatocyte growth factor (HGF), keratinocyte growth factor (KGF), interleukin-6 (IL-6) and interleukin-8 (IL-8) in amniotic membrane homogenates. METHODS Amniotic membranes of 8 placentas were prepared and thereafter stored at - 80 °C using the standard methods of the LIONS Cornea Bank Saar-Lor-Lux, Trier/Westpfalz. Following defreezing, amniotic membranes were cut in two pieces and homogenized in liquid nitrogen. One part of the homogenate was prepared in cell-lysis buffer, the other part was prepared in PBS. The tissue homogenates were stored at - 20 °C until enzyme-linked immunosorbent assay (ELISA) analysis for EGF, bFGF, HGF, KGF, IL-6 and IL-8 concentrations. RESULTS Concentrations of KGF, IL-6 and IL-8 were below the detection limit using both preparation techniques. The EGF concentration in tissue homogenates treated with cell-lysis buffer (2412 pg/g tissue) was not significantly different compared to that of tissue homogenates treated with PBS (1586 pg/g tissue, p = 0.72). bFGF release was also not significantly different using cell-lysis buffer (3606 pg/g tissue) or PBS treated tissue homogenates (4649 pg/g tissue, p = 0.35). HGF release was significantly lower using cell-lysis buffer (23,555 pg/g tissue), compared to PBS treated tissue (47,766 pg/g tissue, p = 0.007). CONCLUSION Containing EGF, bFGF and HGF, and lacking IL-6 and IL-8, the application of amniotic membrane homogenate eye drops may be a potential treatment alternative for therapy-resistant corneal epithelial defects.

Growth Factor and Interleukin Concentrations in Amniotic Membrane-Conditioned Medium

ABSTRACT Purpose: Application of amniotic membrane-conditioned medium (AMM) eye drops is a potential treatment alternative for therapy-resistant corneal epithelial defects. Our purpose was to determine the concentration of growth factors epidermal growth factor (EGF), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), transforming growth factor β1 (TGFβ1), fibroblast growth factor basic (FGFb), and interleukins, IL-1β, IL-6, IL-8, in AMM following different preparation methods. Methods: Amniotic membranes of 10 placentas were prepared and thereafter stored at −80°C using the standard method of our LIONS Cornea Bank. Following defreezing, amniotic membrane pieces with a standard size were inserted in a 12-well plate either complete or cut in small pieces, and 2000 µl DMEM culture medium was added. EGF, NGF, VEGF, TGFβ1, FGFb, IL-1β, IL-6, and IL-8 concentrations in the culture medium were determined following 8, 48, and 96 hours, and 1, 2, 3, and 4 weeks of incubation using enzyme-linked immunosorbent assay (ELISA). Results: Concentrations of NGF, VEGF, TGFβ1, and IL-1ß were beyond the detection limit at all time points. EGF concentrations were between 0.14 and 0.80 ng/g tissue, FGFb between 0.48 and 2.89 ng/g tissue, IL-6 between 0.11 and 1.41 ng/g tissue, and IL-8 between 0.32 and 6.18 ng/g tissue. A significant difference between both preparation methods was shown for the IL-6 concentration after 8 and 48 hours (p < 0.001; p = 0.01) and in IL-8 concentration after 8 and 96 hours and after 3 weeks (p = 0.02; p = 0.002; p = 0.027). Conclusion: AMM containing EGF and FGFb, and IL-6 and IL-8 AMM is a potential nonsurgical treatment alternative of therapy-resistant corneal epithelial defects. However, the most effective preparation method and the optimal harvesting time point are yet to be determined.

Wachstumsfaktorenkonzentration im Kulturüberstand von Keratozyten mit humanem Serum in vitro

PURPOSE Application of serum eye drops is an alternative treatment option for therapy-resistant corneal epithelial defects. In case of persisting epithelial defects, cytokines, which are secreted from stromal keratocytes, may play a role in epithelial wound healing. Our aim was to analyze fibroblast growth factor basic (FGFb), hepatocyte growth factor (HGF), keratinocyte growth factor (KGF) and transforming growth factor β1 (TGF-β1) concentration in the supernatant of keratocytes, after incubation with human serum (HS). MATERIALS AND METHODS Serum eye drops of 10 patients were prepared using the standards of the LIONS Eye Bank Saar-Lor-Lux, Trier/Westpfalz, and were stored at - 80 °C. Primary human keratocytes were isolated from human corneoscleral rings using collagenase A (1 mg/ml) (n = 1) and were cultured in DMEM/Hams culture medium with 10% fetal bovine serum (FBS). Thereafter, keratocyte cultures were incubated in 15 or 30% HS (in DMEM/F14 without FBS) and FGFb, HGF, KGF and TGF-β1 concentration was determined with an enzyme-linked immunoabsorbent assay (ELISA) from the supernatant of the culture after 24 hours. We used 15 or 30% HS without keratocytes (under the same storage conditions) as controls. RESULTS HGF concentration was, for both HS concentrations, significantly higher in the supernatant of keratocytes, than in HS controls (without keratocytes) following 24 hours (p < 0.01). FGFb concentration was significantly increased in 30% HS with keratocytes compared to 30% HS without keratocytes after 24 hours (p < 0.01). TGF-β1 and KGF concentrations remained unchanged through keratocytes. CONCLUSION HGF and FGFb concentrations increase in the supernatant of keratocytes, 24 hours after incubation with human serum. These concentration changes may play a role in wound healing of epithelial defects.

Amnionmembransuspension und autologes Serum – spielt der Inhalt eine Rolle für die Wundheilung?

Autologous serum (AS) and amniotic membrane transplantation (AMT) are used in the treatment of several ocular surface diseases. AMT is associated with a surgical intervention. Surgery could be avoided by using eye drops prepared from an amniotic membrane homogenate (AMH) or amniotic membrane suspension (AMS). EGF, bFGF, IL-6 and IL-8 were detected in AMS. However, EGF and bFGF concentrations in AMS were about 1.7-17× lower than in AMH, and IL-6 and IL-8 could not be detected in AMH. 100 % AMS, 15 and 30 % AMH significantly decreased proliferation of human corneal epithelial cells (HCECs) compared to controls (p = <0.002 for all), but 15 and 30 % AMS did not affect proliferation. Migration increased significantly compared to controls with 15 and 30 % AMS (p < 0.001), but did not change significantly with 15 or 30 % AMH (p = 0.153 and p = 0.083). Proliferation of HCECs was significantly greater with 15 % AS than with 30 % AS (p < 0.001). HCEC migration was significantly greater with 30 % AS than with 5 % AS (p < 0.01). In summary, 15 and 30 % AMS and 15 and 30 % AS exhibit the best supportive effect on human corneal epithelial cells. Nevertheless, we always have to keep in mind that individual growth factor concentrations exhibit high inter-individual fluctuations in AS or AMS eye drops.