[Good practice procedures for acquisition and preparation of cryopreserved human amniotic membranes from donor placentas]. / Gute fachliche Praxis zur Gewinnung und Herstellung von kryokonservierter humaner Amnionmembran aus Spenderplazenta.

Within the framework of obtaining a valid authorization for tissue preparation of cryopreserved human amniotic membranes at the Paul Ehrlich Institute, pursuant to § 21a paragraph 1 of the German Medicines Act (AMG), parts of the existing good practice procedures for acquisition of cryopreserved human amniotic membranes from donor placentas were reviewed and supplemented by new knowledge. The present good practice procedures were formulated in cooperation with members of the section for tissue transplantation and biotechnology of the German Ophthalmological Society. The current revised version is presented in this article.

[Ocular graft versus host disease : Corneal complications]. / Okuläre Graft-versus-Host-Disease : Korneale Komplikationen.

BACKGROUND: Ocular graft-versus-host disease (GvHD) following allogeneic blood stem cell transplantation leads to immunologically induced alterations in many ocular tissues, particularly at the ocular surface. Within the framework of the main topic, this article focuses primarily on corneal complications in chronic ocular GvHD. OBJECTIVE: This article aims to promote understanding of the influencing factors, diagnostics, and therapeutic options pertaining to corneal complications in ocular GvHD. Furthermore, the possibilities for prevention are discussed. MATERIALS AND METHODS: This analysis is based on a literature review as well as on data from the Ophthalmology Clinic at the University Hospital Essen. RESULTS: Corneal complications often occur secondarily in ocular GvHD, as a consequence of severe inflammatory alterations of the conjunctiva or eyelid. Spontaneous corneal perforations associated with only mild symptoms are less common during the course of disease. From the ophthalmologist's perspective, it is important that the inflammatory activity of all the different ocular tissues is considered. Treatment may follow a stepwise scheme that includes substitution, immunosuppression, and surgical rehabilitation. CONCLUSION: Systematic diagnosis of ocular GvHD helps to prevent corneal complications or support early therapeutic intervention. An interdisciplinary approach to diagnosis and treatment planning is recommended, in order to optimize local and systemic immunosuppressive therapy.

[Limbal epithelial stem cell transplantation : Current state and perspectives]. / Limbusstammzelltransplantation : Aktueller Stand und Perspektiven.

Homeostasis of the corneal surface is maintained by epithelial stem cells localized in the limbus. Multiple intrinsic factors or external injuries can destroy the delicate microenvironment of limbal epithelial stem cells causing a state which is termed limbal stem cell deficiency (LSCD). In such cases, re-epithelialization of the cornea is drastically impeded and conjunctival epithelium starts to extend beyond the limbus and to invade the corneal surface. In partial LSCD, a superficial keratectomy combined with an amniotic membrane is advised and helpful to restore an intact, healthy ocular surface. In complete LSCD, stem cell transplantation is the only curative option. Before any reconstruction, causative factors and comorbidities should be eliminated or at least optimized. In cases of unilateral LSCD, stem cells can be obtained from the contralateral eye. Advanced surgical and cultivation techniques pursue a gentle, tissue-saving procedure of harvesting a limbal biopsy from the only healthy functioning eye. Patients with bilateral involvement can be treated with allogeneic tissue, but will require long-term systemic immunosuppressive therapy. Another newer option is the use of autologous, but noncorneal epithelial cells as a tissue source, e.g., buccal mucosa. Future studies will focus on the further development of cellular expansion and/or the establishment of new alternative sources for replacing limbal epithelial stem cells.

[Clinical, morphological and molecular biological characteristics of the aging eye]. / Klinische, morphologische und molekularbiologische Charakteristika des alternden Auges.

BACKGROUND: The physiological aging of the eye is associated with loss of visual function. Age-related changes of the eye can result in ophthalmological diseases. The aim of this article is to display morphological, histological and molecular biological alterations of the aging eye. MATERIAL AND METHODS: A web-based search and review of the literature for aging of the visual system including cornea, lens, vitreous humor, retina, retinal pigment epithelium (RPE), choroidea and optic nerve were carried out. The most important results related to morphological, histological and molecular biological changes are summarized. RESULTS: Age-related, morphological alterations can be found in preretinal structures, e. g. cornea, lens and vitreous humor, as well as neuronal structures, such as the retina. In addition to negligible clinical signs of the aging eye, there are clinically relevant changes which can develop into pathological ophthalmological diseases. These transitions from age-related alterations to relevant ophthalmological diseases, e. g. age-related macular degeneration and glaucoma are continuous. CONCLUSION: An understanding of aging could provide predictive factors to detect the conversion of physiological aging into pathological conditions. The derivation of physiological markers or new approaches to detection and treatment of disease-related entities associated with the risk factor aging are desirable. Translational approaches in clinical and basic science are necessary to provide new therapeutic options for relevant ophthalmological diseases in the future.

[Long-term results of autologous transplantation of limbal epithelium cultivated ex vivo for limbal stem cell deficiency]. / Langzeitergebnisse zur autologen Transplantation von ex vivo kultiviertem Limbusepithel bei limbaler Stammzellinsuffizienz.

OBJECTIVE: This study reports the long-term clinical outcome of autologous limbal epithelial cells cultivated ex vivo on intact amniotic membranes (AM) for ocular surface reconstruction in limbal stem cell deficiency (LSCD). PATIENTS AND METHODS: A total of 61 eyes from 57 patients (46 males and 11 females) with LSCD were treated by transplantation of autologous limbal epithelial cells cultivated on intact AM. The etiology of the LSCD was chemical and thermal burns (n = 34), recurrent or primary large-sized pterygium (n = 12), mitomycin C and tumor excision-induced LSCD (n = 9), severe infectious keratitis (n = 3), perforating injury, epidermolysis bullosa and contact lens-associated keratopathy (each n = 1). Only eyes with a follow-up time of at least 12 months were included in the analysis. The main outcome end points were restoration of ocular surface integrity and improvement of visual acuity (VA). RESULTS: The mean follow-up time was 50.8 ± 32.7 months. An entirely stable corneal surface was reconstructed in 46 (75.4%) eyes. Visual acuity significantly increased in 40 (65.6 %) eyes, was stable in 12 (19.7%) eyes and decreased in 9 eyes (14.8%). The mean visual acuity significantly increased (p < 0.0001) from 1.4 ± 0.91 LogMAR preoperatively to 0.8± 0.67 LogMAR postoperatively. CONCLUSION: Transplantation of limbal epithelium cultivated ex vivo on intact AM leads to restoration of a stable corneal surface and resulted in a significant increase of visual acuity in most cases of LSCD. Autologous transplantation of cultivated limbal epithelium showed an excellent prognosis and outcome after long-term follow-up.

Evaluation of a new protocol for sterility controls of corneal culture medium.

Careful testing for microbial contamination is essential for corneal transplants. Sterility tests are performed on the antibiotics containing culture medium leaving the problem that antibiotics might compromise the test results. In this study a protocol for the application of the automated BacT/Alert system for sterility testing of corneal cell culture medium was examined. Corneal culture medium in combination with an antibiotics degrading enzyme were injected in resin containing test bottles of the BacT/Alert system named FA plus (intended for aerobic microorganisms) or FN plus (intended for anaerobic microorganisms) depending on their aerobic or anaerobic nature. Additionally i-FA plus(aerobic test bottle for industrial use) bottles were used. Microbial test strains on the basis of the European Pharmacopaea (Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans, Aspergillus brasiliensis and Clostridium sporogenes) with the addition of Propioniobacterium acnes were added to the test bottles. The bottles were incubated at two different temperatures for 14 days. The time to detection (TTD) was monitored for each bottle. Growth of the test strains except European Pharm was detected in the FA and FN Plus bottles. The TTD for the strains was 44 ± 1.5 h (P. aeruginosa), 57.7 ± 2.2 h (B. subtilis), 56 ± 1 h (S. aureus), 26.3 ± 1 h (C. sporogenes), 223 ± 4.6 h (P. acnes), 64.4 ± 10 (C. albicans). A. brasiliensis was detected in i-FA Plus bottles with a TTD of 94.9 ± 3.7 h. The application of BacT/Alert FA Plus and FN Plus resin bottles in combination with a penicillin degrading enzyme is able to detect small scale microbial contamination with different microorganisms in antibiotic containing corneal culture medium. For detection of Aspergillus brasiliensis in the medium the (i-) FA Plus bottles should be used.

[Validation of an automatic test system for sterile testing of amniotic membranes]. / Validierung eines automatischen Testsystems für die Steriltestung von Amnionmembran.

PURPOSE: The use of cryopreserved amniotic membranes for the treatment of diseases and injuries of the surface of the eye is an established procedure in ophthalmological surgery. Before clinical use of cryopreserved amniotic membranes (AM) a careful testing for microbial contamination is essential to ensure a safe application. In this study the use of the BacT/Alert® test system was evaluated for screening of microbial growth in AMs. MATERIALS AND METHODS: Minced fresh and cryopreserved AMs (approximately 5 × 5 cm in size) were injected with 10 ml of balanced salt solution in separate culture media test bottles and 10 ml of cryopreservation medium bacterial and fungal test strains according to European Union (EU) regulations were applied to test the performance of the system. Approximately 10-100 colony forming units were applied on the samples prior to injection in the corresponding test bottles. Bottles were incubated at 37 °C for 7 days. Positive controls contained only balanced salt solution and the test strains while negative controls contained the test material without microbial test strains. RESULTS: Growth of the test strains was detected in all inoculated samples from non-processed and cryopreserved AM within the 7-day incubation period. In samples of the cryopreservation medium only growth of the fungus Candida albicans could be detected. CONCLUSIONS: The automated BacT/Alert test system is suitable for testing of microbial safety of amniotic membranes but not for testing the cryopreservation medium in clinical practice according to EU regulations.

[Short-term and long-term complications after transplantation of cultivated limbal epithelium]. / Kurz- und Langzeitkomplikationen nach Transplantation von kultiviertem Limbusepithel.

Recent advances in tissue engineering have facilitated the development of new strategies in ocular surface reconstruction. Limitations and possibilities of ex vivo cultivation and limbal epithelium cell culture techniques as well as the short and long-term complications after transplantation of ex vivo expansion of cultivated limbal epithelium for the treatment of limbal stem cell deficiency are summarized in this review.

[The biological basis of limbal stem cell deficiency]. / Biologische Grundlagen der Limbusstammzellinsuffizienz.

Limbal stem cell deficiency results from the loss of tissue regenerating stem and progenitor cells. Corneal epithelial regeneration is maintained by stem and progenitor cells which reside in the schlerocorneal limbus. They possess stem cell characteristics and can be stimulated to proliferate by external signals. The limbus is the stem cell niche for corneal epithelial stem cells and forms a unique microenvironment in which stem cell characteristics are conserved. Regulation of limbal epithelial stem cells is produced by a network of signals within the niche which governs cell fate decisions with regards to proliferation, differentiation or maintenance of a quiescent status.

[Ocular surface reconstruction in limbal stem cell deficiency : Transplantation of cultivated limbal epithelium]. / Oberflächenrekonstruktion bei Limbusstammzellinsuffizienz : Transplantation von Limbusgewebe - Kultivierungsverfahren.

Various ocular surface diseases are caused by loss of corneal epithelial stem cells or dysfunction of the limbal stem cell niche. Besides conventional transplantation of autologous or allogenic limbal tissue, recent advances in tissue engineering have led to the development of new culture and expansion techniques of human limbal stem and progenitor cells (LSPC) as a new strategy to successfully treat limbal stem cell deficiency (LSCD). From a small autologous limbal biopsy with a limited amount of LSPC an epithelium ready for transplantation is achieved. Autologous grafting of cultured limbal epithelium led in most of the treated cases to a successful reconstruction of the corneal surface. Alternative methods which have recently been introduced to treat LSCD use other stem cell sources including the transplantation of oral mucosal epithelium. In this article the challenges and controversies associated with these stem cell culture techniques for ocular surface reconstruction are reviewed.

The effect of long-term storage on the biological and histological properties of cryopreserved amniotic membrane.

PURPOSE: Cryopreserved amniotic membrane (AM) is widely used in ophthalmology because of its anti-angiogenic, anti-inflammatory, and wound healing promoting capabilities. A common method to conserve the tissue is the storage in cryo-medium containing 50% glycerol at -80°C. The aim of this study was to examine the influence of storage time on the sterility as well as the histological and biological properties of cryopreserved AM. METHODS: Amniotic membrane from different donors was stored in cell culture media containing 50% glycerol for different time periods, on average 4 months (group 1), 15 months (group 2), and 24 months (group 3), at -80°C. Samples of the tissue and cryo-medium were examined for bacterial and fungal contamination. Tissue samples were incubated in 0.5 ml/cm(2) serum-free medium at 37°C. The medium was changed after 1, 2, and 3 days. The proteins released by AM were TCA-precipitated and the presence of the proteins TIMP-1 and IL-1ra was analyzed using Western blotting and semi quantified by means of image analysis. Integrity of the amniotic epithelium and the basement membrane components collagen IV, collagen VII, laminin, laminin 5, and fibronectin were examined by haematoxylin eosin stain and immunohistochemistry in cryosections of AM. RESULTS: None of the examined samples showed bacterial or fungal contamination. The soluble proteins TIMP-1 and IL-1ra were found in all samples of medium incubated for all time periods. The examined proteins were detectable after one-day incubation but the staining signal diminished significantly in the second and third wash after 48 hr and 72 hr. Differences in the intensity of the Western blot signal between the three particular groups were statistically not significant. The epithelia of all samples were intact. The basement membranes of all samples showed a similar distribution of collagen IV, collagen VII, laminin, laminin 5, and fibronectin. CONCLUSIONS: Long-term storage of amniotic membrane in cell culture media with 50% glycerol does not significantly impair sterility, histology, or biological properties of AM.

[Amniotic membrane transplantation in herpetic corneal infections]. / Amnionmembrantransplantation bei herpetischen Hornhautinfektionen.

Severe infectious corneal ulceration such as herpetic stromal keratitis commonly causes loss of vision and may lead to blindness. Treatment depending on the underlying disease includes antimicrobial medication and the development of surgical strategies to restore the integrity of the corneal ocular surface. Ulcerative herpetic stromal keratitis and/or neurotrophic keratopathy with the risk of corneal perforation are still clinically challenging conditions in ophthalmic surgery of the ocular surface. Since the introduction of newly developed preservation methods, amniotic membrane (AM) functioning as a basement membrane substitute has gained widespread popularity in ocular surface reconstruction. Various ways of clinical application such as the use of AM as a graft, patch or culture substrate and carrier system to expand ocular surface epithelia have been recently reported. In this article, the basis and clinical application of amniotic membrane transplantation for the management of corneal infections with Herpes simplex and Herpes zoster virus are reviewed.