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Purpose: Choukroun's platelet-rich fibrin (PRF), a second-generation platelet concentrate, has unique morphological and chemical features and may be considered as a scaffold for scleral reinforcement and regeneration. The purpose of this study was to compare the use of xenogenic human-derived amniotic membrane (HAM), allogenic sclera, and autogenic PRF in rabbit lamellar scleral defect model with respect to both anatomical and immunohistochemical improvement. Methods: A total of 45 adult New Zealand rabbits were randomized into five groups: normal control; without surgical procedure, negative control; scleral defect model (SDM), xenogenic HAM; SDM+HAM graft, allogenic sclera; SDM+allogenic sclera graft, autogenic PRF; SDM+autogenic PRF graft. Clinical findings, Hematoxylin&Eozin (HE), Masson Trichrome, Verhoeff Acid Fuchsin, Transforming Growth Factor β Receptor 1, Fibroblast Growth Factor, Bone Morphogenetic Protein 2, collagen type 1, aggrecan, and Matrix Metalloproteinase 2 were evaluated. Results: Ocular surface inflammation was significantly lower in normal control and autogenic PRF groups (p <.001). Graft was avascular and not integrated to scleral wound area in 25% rabbits of allogenic sclera group (p =.02), was out of the scleral wound in 33.3% rabbits of xenogenic HAM group (p >.05), all the grafts were at the normal location and viable in autogenic PRF group. The inflammation and vascularization in autogenic PRF group was significantly lower than negative control and xenogenic HAM groups in HE (p <.001). The collagen score of negative control and xenogenic HAM groups were significantly lower than normal control (p <.001) and autogenic PRF (p <.001) groups. There were insignificant differences between allogenic sclera and autogenic PRF groups (p >.05). For immunohistochemistry, the closest values to normal control group were detected in autogenic PRF group for all immunomarkers. Conclusion: Autogenic PRF showed superior features via its excellent anatomical and chemical composition for scleral regeneration when compared to single-layered xenogenic HAM and allogenic sclera grafts. [ABSTRACT FROM AUTHOR]

Purpose: Adhesions between the extraocular muscles and surrounding tissues pose a main cause of failure of strabismus reoperations. Amniotic membrane (AM) transplantation during extraocular muscle surgery, to prevent the formation of adhesions, has been a subject of research during the past decade. This review aims to determine the value, indications, and tips on usage of AM transplantation during strabismus surgery. Materials and methods: All references cited in PubMed in English were searched using the key words: amniotic membrane strabismus or amniotic membrane extraocular muscles, and a brief summary of these was described. In addition, certain articles were chosen to provide introductory information on wound healing and fibrosis, AM properties and how it works after transplantation, and AM processing and preservation. Results: AM used for transplantation during extraocular muscle surgery may be cryopreserved, dried, or fresh. It may be oriented with its stroma or epithelium towards the muscle. It may or may not be fixed with sutures. What were the best choices? Various studies attempted to answer these questions. Many of the studies reviewed, however, were inconclusive or contradictory. Fresh AM seemed effective, but carried a risk of transmission of communicable diseases. Dried membrane was not of value in preventing adhesions. Histopathologically, cryopreserved membrane prevented the development of adhesions in the region of its presence, regardless of its orientation, and without the need for suture fixation. To accentuate this histopathological effect during clinical practice, it was recommended to utilize the largest segment possible of cryopreserved membrane and limit its usage to cases where adhesions are expected to be the main cause of failure of strabismus surgery. Conclusion: Cryopreserved AM transplantation was safe and histopathologically effective in preventing adhesions. This effect was, however, less pronounced clinically. Its use during strabismus reoperations is merited if previous recommendations and precautions are considered. [ABSTRACT FROM AUTHOR]

Purpose: The amniotic membrane (AM) is the transparent innermost layer of the placenta and it facilitates rapid wound healing in a diversity of ocular surface disorders. However, extended periods of cryopreservation before use induce significant impairment of cell viability due to oxidative stresses and inflammatory responses. We investigated the effect of supplementing ulinastatin (ULI), a known serine protease inhibitor, and relevant mechanisms of action in AM preservation solution through the hypothermic continuum on inflammatory and apoptotic signals and viability of AM tissue. Materials and methods: The expression of inflammatory signal factors, including high mobility group box 1 (HMGB1), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), receptor-interacting serine/threonine-protein kinase 3 (RIPK3) and anti-TNF-inducible gene 6 (TSG-6) which is a TNF-α-inducible anti-inflammatory protein, and the expression of apoptotic signal factors, including caspase (Cas)-9 and Cas-8, the initiators, and Cas-3, the executioner caspase and Bax were analyzed with or without ULI during hypothermic preservation of human AM. Subsequently, the actual viability of human AM tissue was verified with or without ULI supplementation throughout hypothermic continuum (both hypothermic- and cryopreservation). Results: Hypothermic AM preservation with ULI for 48 h resulted in downregulated expression of cold-inducible inflammatory factors, including HMGB1 and NF-κB, as well as RIPK3. In addition, ULI suppressed apoptotic signals related with Cas-9, Cas-8, and Cas-3 under hypothermic conditions. Furthermore, ULI supplementation during hypothermic- and cryopreservation of AM significantly enhanced viability of AM tissue and amniotic epithelial cells. Conclusions: Supplementation of ULI during human AM preservation through the hypothermic continuum may be a feasible dual anti-inflammatory and anti-apoptotic strategy that enhances the viability of AM tissue. [ABSTRACT FROM AUTHOR]

Purpose: To study the functional recovery of the superior rectus muscle (SRM) after its partial resection in a rabbit model with and without cryopreserved amniotic membrane (AM). Material and methods: Resection of the right and left SRMs of 30 rabbits was performed. On the left eyes, a single sheet of equine cryopreserved AM was placed covering the muscle edge sutured. Active and passive mechanical properties of muscles operated with and without AM were monitored over time at 30 (n = 10), 60 (n = 10), and 90 (n = 10) days after surgery. Muscle samples were extracted and electrically stimulated to register the force exerted by the samples, characterizing its active behavior. They were, then, subjected to stretching test to obtain its resistance to deformation, known as passive behavior. Moreover, right and left eyes of a control group (n = 5) were equally subjected to active and passive tests to characterize the physiological behavior of SRM muscles. Results: On active function examination, statistically significant differences were documented between the following: control vs AM and no AM at 30 days (p = 0.002 and p = 0.04, respectively). All other comparisons were insignificant (p > 0.05). On passive function analysis, significant differences were only found between control vs. no AM at 30 days (p = 0.004) and between AM vs. no AM at 30 days (p = 0.002). Indeed, muscle operated without AM did not recover a normal passive function until 60 days after surgery. Conclusion: Cryopreserved AM is effective in accelerating recovery of SRM passive function in rabbits. Nevertheless, AM produced no significant effect on recovery of SRM active function. [ABSTRACT FROM AUTHOR]

Purpose: To analyze the effects of different concentrations of amniotic membrane suspension (AMS) or amniotic membrane homogenate (AMH) on human corneal epithelial cell (HCEC) viability, migration and proliferation. Methods: Amniotic membranes (AMs) of 13 placentas were prepared and stored at −80°C. For AMS preparation, following de-freezing, AM pieces were inserted in six-well plates and 5 ml Dulbecco’s Modified Eagle’s Medium (DMEM)/F12 (with 5% fetal bovine serum, FBS) per gram tissue was added for 96 h. After removal of the AM, the remaining supernatant was collected for experiments. For AMH preparation, following de-freezing, AMs were homogenized in liquid nitrogen and 5 ml DMEM/F12 (with 5% FBS) per gram tissue was added. Following centrifugation, the supernatant was collected for experiments. HCECs were expanded and incubated in DMEM/F12, 5% FBS supplemented by 15%, 30% or 100% AMS or 15% or 30% AMH. Viability was analyzed using Cell Proliferation Kit XTT, migration using wound healing assay and proliferation by the cell proliferation ELISA BrdU kit. Results: HCEC viability remained unchanged using 15% or 30% AMS (p= 1.0 for both); however, it decreased significantly using 100% AMS (p< 0.001) or 15% (p= 0.041) or 30% AMH (p< 0.001), compared to controls. Using 15% or 30% AMS, HCEC migration increased significantly (p< 0.001 for both). Using 15% or 30% AMH (p= 0.153;p= 0.083), HCEC migration remained unchanged and 100% AMS inhibited HCEC migration (p< 0.001). Next, 15% and 30% AMS had no effect on HCEC proliferation (p= 0.454 andp= 0.119), but 100% AMS (p< 0.001) and 15% (p= 0.002) and 30% AMH (p= 0.001) inhibited HCEC proliferation significantly. Conclusion: With unchanged HCEC viability and proliferation and increased HCEC migration, 15% and 30% AMS application seems to be the most appropriate method to support epithelial healing. [ABSTRACT FROM AUTHOR]

Purpose: The aim of the present study was to compare the surgical results and recurrence rates of primary pterygium excision with conjunctival autografts versus amniotic membrane grafts fixated with fibrin glue. Materials and methods: In this prospective study, 73 eyes of 65 patients who had undergone conjunctival autograft group (CAG) (n:37) or amniotic membrane group (AMG) (n:36) after pterygium excision were evaluated. Fibrin glue was used for the fixation of grafts in both groups. The patients were followed up for 12 months. Postoperative complications were recorded. The rate of recurrence was defined as the primary outcome measure. Results: In the CAG, partial dehiscence of the graft was observed on the nasal edge on postoperative day 1 in two (5.4%) eyes that healed with secondary re-epithelialization at week 1. Twelve (32.4%) eyes showed a yellowish-orange or hemorrhagic edema of the graft on postoperative day 7 that spontaneously resolved in 1 to 3 weeks. In the AMG, partial dehiscence and folding of the amniotic membrane occurred in two (5.5%) eyes. Two eyes (5.4 %) in the CAG developed corneal recurrence and five eyes (13.8%) in the amniotic membrane graft group developed recurrence; one limbal (2.7%), four corneal recurrences (11.1%) (p = 0.25). Conclusion: Fibrin glue is a safe and effective method for attaching conjunctival or amniotic membrane grafts for wound closure following pterygium surgery. Although the results were not statistically significant, amniotic membrane grafting using fibrin glue seems to have a higher pterygium recurrence rate compared with conjunctival autografting. [ABSTRACT FROM PUBLISHER]

Purpose: Corneal epithelial wound healing is a major issue in ocular surface (OS) reconstruction. Aim of this study was to evaluate parameters of epithelial wound healing in vitro on transparent keratin films (KFs) derived from human hair in comparison with amniotic membrane (AM) and polystyrene. Materials and Methods: The human corneal epithelial cell line (HCE-T) was expanded on KF, AM and commercially available 24-well polystyrene cell culture plates in vitro to compare cell proliferation, migration and attachment by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, scratch-wound healing and adhesion assay. Cells cultured on KF and AM at an air-liquid interface for 14 d were stained with hematoxylin and eosin for histology. Results: The highest proliferation of HCE-T cells was observed on polystyrene at all time points ( p < 0.05). At a seeding density of 5 × 103 cells/well, no difference in proliferation was found between AM and KF after 24 h and 72 h ( p = 0.582 and p = 0.066), while higher proliferation was observed on AM compared to KF after 48 h ( p = 0.005). At a seeding density of 1 × 104 cells/well, no difference was found between AM and KF after 24 h ( p = 0.252), while higher proliferation was observed on AM compared to KF after 48 h and 72 h ( p = 0.001 and p = 0.003). The significantly fastest cell migration was observed on polystyrene at all time points ( p < 0.01). Cell migration was significantly higher on KF compared to AM at 48 h ( p < 0.05). After 30 min, there were significantly more cells attached to AM compared to polystyrene and KF ( p = 0.032 and p = 0.001). No significant difference in cell attachment was observed between KF and polystyrene ( p = 0.147). Histology demonstrated that HCE-T cells cultured on KF and AM at an air-liquid interface for 14 d form a multilayered epithelium similar to normal human corneal epithelium. Conclusion: Transparent KFs derived from human hair support proliferation, migration, adhesion and differentiation of HCE-T cells in vitro. Therefore, it could be a promising alternative to AM for OS reconstruction. [ABSTRACT FROM AUTHOR]

Severe ocular surface disorders can result in deficiency of limbal stem cells that is potentially associated with chronic inflammation, impaired vision and even blindness. Advanced stem cells deficiency requires reconstruction of the OS with autologous or allogeneic limbal stem cells. To address such deficiency, a limbal tissue biopsy is taken and limbal cells are expanded on a carrier, which then can be used for OS reconstruction. Human amniotic membrane - currently the most common carrier for transplantation of limbal epithelial stem cells - has the downsides of carrying the risk of disease transmission, limited transparency, variable and unstable quality and low mechanical strength. This article reviews the advantages and disadvantages of the established carrier materials for limbal stem cell transplantation, as well as discussing emerging alternatives, including carriers based on collagen, fibrin, siloxane hydrogel contact lenses, poly(&varepsilon;-caprolactone), gelatin-chitosan, silk fibroin, human anterior lens capsule, keratin, poly(lactide-co-glycolide), polymethacrylate, hydroxyethylmethacrylate and poly(ethylene glycol) for their potential use in the treatment of limbal stem cell deficiency. [ABSTRACT FROM AUTHOR]

Purpose: Corneal ulceration may be treated with human amniotic membrane (HAM) while neovascularization remains a common complication of corneal ulceration. As several factors contained within HAM may contribute to reduced corneal scarring, we investigated if HAM may be additionally loaded with bevacizumab and potentially serve as a carrier for anti-vascular endothelial growth factor (VEGF) drugs to provide constant VEGF blockade. Materials and Methods: Cryo-preserved HAM were incubated with different bevacizumab concentrations in organ culture medium for 2-5 d. Controls were incubated without bevacizumab. Then, all samples were placed into an organ culture medium without bevacizumab for 48 h, 72 h or for 5 d at 37 °C with the medium being changed at all time points. After that, VEGF165 was added to the supernatants for 24 h and free VEGF 165 was measured by ELISA. Results: Free VEGF was significantly blocked at 48 and 72 h ( p < 0.01). VEGF blockade was less pronounced after 1 week. However, as compared to control, VEGF was significantly blocked at all times ( p < 0.05). Conclusion: In this in-vitro setting, we could demonstrate effective VEGF-blockade for up to 1 week by incubating HAM with bevacizumab. HAMs might be potentially used as a carrier for drugs delivered to the cornea. [ABSTRACT FROM AUTHOR]

Purpose: Chemically defined bioadhesives (CDB) are 'two-component reactive adhesives' made from antibiotic food additives. They contain no animal-derived ingredients and are characterized by a high level of self-degradability, low toxicity, and strong bonding properties. Amniotic membrane (AM) transplantation is used to repair severe ocular surface disease in humans and horses. AM requires meticulous suturing to the injured cornea. The purpose of this study is to determine whether CDB can facilitate the suture-less placement and adherence of equine AM to porcine cadaver cornea. Design: Prospective study. Materials and Methods: To determine whether there are differences in ex vivo adhesion force between equine AM bonded with CDB to porcine cadaver cornea and equine AM sutured to porcine cadaver cornea. The following groups were tested ( n == 5 per group): (1) CDB group: trephined (8 mm in diameter) cornea was bonded with CDB to 8-mm-sized discs of AM. (2) Suture group: the 8-mm trephined cornea was sutured with 8-0 absorbable suture to the AM. (3) Control group 1: two sheets of nitrocellulose paper were bonded with CDB. (4) Control group 2: two sheets of nitrocellulose paper were attached with suture and tested. Each sample was anchored to a tensiometer materials testing machine, and the strength of adhesion measured and expressed in units of kilogram-force (kgf). Results: Mean ± SD strength of adhesion force found no statistically significant difference between CDB group (0.091 ± 0.054 kgf; 15 min of CDB adhesion time) and suture groups (0.095 ± 0.037 kgf), whereas there was significant difference ( P < 0.05) between control group 1 (0.679 ± 0.048 kgf) and control group 2 (0.585 ± 0.092 kgf). These results revealed that the 15 min of CDB adhesive time had adhesive properties as strong as suture group. Conclusions and Clinical Relevance: CDB provides a satisfactory level of adherence to ex vivo suture-less attachment of equine amniotic membrane transplantation to the porcine cornea. [ABSTRACT FROM AUTHOR]

Purpose/Aim: To determine whether amniotic membrane transplantation (AMT) at trabeculectomy affects intrableb structures and which intrableb parameters are significantly correlated with the long-term intraocular pressure (IOP) control. Materials and Methods: Enrolled were 64 eyes of 56 glaucoma patients who underwent trabeculectomy without (36 eyes) or with (28 eyes) AMT. Bleb structure was evaluated by ultrasound biomicroscopy (UBM) at least one year after surgery. IOP control was defined as good when the eyes had a more than 30% decrease in the preoperative IOP and an IOP value <18 mmHg if the preoperative IOP was higher than 21 mmHg. Logistic regression analyses were conducted to identify factors significantly associated with IOP control. Results: Intervals between surgery and the timing of the UBM examinations (median; 2.5 years) and the overall frequency of good IOP control (28/36 in the eyes without AMT and 17/28 in those with AMT; chi-square test, P == 0.2276) were similar in the two groups. The eyes with AMT had a significantly lower number of type H (high reflective) or L (low reflective) blebs and a higher number of type E (encapsulated) blebs compared to those without AMT ( P < 0.0001). Among independent variables, which included age, sex, glaucoma type, lens status, the number of ocular hypotensives, and previous intraocular surgeries, only type F (flattened) bleb in eyes without AMT ( P == 0.0008, odds ratio [OR] == 0.0256) and no or limited intrableb fluid-filled space in eyes with AMT were significantly associated with poor IOP control ( P == 0.0026, OR == 0.0111, and 0.0071, 0.0167, respectively). Conclusions: Intrableb structures after trabeculectomy alone versus AMT-assisted trabeculectomy were distinct. The bleb wall reflectivity in the former and the extent of the subconjunctival fluid-filled space in the latter were factors associated with long-term IOP control. [ABSTRACT FROM AUTHOR]

Purpose: To histopathologically evaluate the influence of lyophilized human amniotic membrane transplant on the development of postoperative adhesions and fibrosis after extraocular muscle surgery. Methods: Ten albino rabbits were used. The superior rectus muscle was resected 4 mm in both eyes. In right eyes, the superior rectus muscle was wrapped with lyophilized (air-dried, freeze-dried) human amniotic membrane (group AM). In left eyes, the superior rectus muscle was not wrapped with amniotic membrane, and served as a control group (group C). The rabbits were sacrificed and the eyes were enucleated 6 weeks after surgery to perform histopathological examination. Results: Two rabbits died 1 week after surgery and were excluded. Histopathological evaluation of both eyes of the remaining 8 rabbits was done. The amniotic membrane was not seen in all eyes. On comparing eye pairs of each rabbit, right eyes showed significantly less conjunctival inflammation ( p == 0.034), but insignificantly less foreign body inflammation ( p == 0.625), adhesions between sclera and muscle ( p == 0.206), muscle fibrosis ( p == 1.000), and conjunctival hyperemia ( p == 0.059) as compared to left eyes. Conclusions: Lyophilized amniotic membrane insignificantly reduces postoperative adhesions and fibrosis, when used to wrap the operated upon extraocular muscles, limiting the benefit of this membrane in strabismus surgery. [ABSTRACT FROM AUTHOR]

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/cm2 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. [ABSTRACT FROM AUTHOR]

Purpose: Membrane-associated mucins are important components of the ocular tear film. Our objective was to characterize the expression of membrane-associated mucins MUC1, MUC4, and MUC16 in healthy cornea, healthy conjunctiva, fibrovascular tissue (pannus) covering the corneal surface in limbal stem cell deficiency (LSCD), human limbal epithelial cells cultivated on intact amniotic membrane (HLEC-AM), and corneal epithelium after transplantation of cultivated limbal epithelium (TCLE). Methods: Total RNA was isolated from six samples of healthy human cornea, healthy conjunctiva, pannus, limbal epithelial cell cultures, and four corneal samples after TCLE. The expression of MUC1, MUC4, and MUC16 was evaluated by real-time polymerase chain reaction (PCR), Western blotting, and immunofluorescence. Results: Conjunctiva showed a significantly higher expression of MUC1 and MUC4 than cornea, but the expression of MUC16 was similar in both tissues. The expression of MUC1 in HLEC-AM increased, MUC16 decreased, and MUC4 showed no significant difference when compared to cornea. The expression of all studied mucins in conjunctiva was significantly higher than in HLEC-AM. Pannus revealed a similar expression pattern of membrane-associated mucins to conjunctiva. The expression of all studied mucins in pannus was significantly higher than in cornea or HLEC-AM. A corneal expression pattern of membrane-associated mucins was found in all four samples of cornea after TCLE. Conclusion: A tissue-specific expression of MUC1 and MUC4 but not MUC16 was found in human cornea and conjunctiva. LSCD led to a conjunctival expression pattern of mucins on the corneal surface. A corneal phenotype could be restored after TCLE, confirming the potential of this method to reconstruct the corneal surface. [ABSTRACT FROM AUTHOR]

Purpose: In order to investigate the potential underlying neurotrophic mechanisms of human amniotic membrane (AM) in the treatment of neurotrophic corneal ulcers, we evaluated whether or not there are significant differences in the neuritic growth of neuronal cell cultures on different surfaces of AM. Methods: Neurite outgrowth of dorsal root ganglia neurons was examined under two separate conditions: (1) in serum-free medium consisting of minimal essential medium (MEM), glucose, and L-glutamine, (2) in same medium additionally supplemented with horse serum, chick embryonic extract, and nerve growth factor. Neuritic outgrowth was labeled with antibodies against neurofilaments and tubulin and screened by confocal laser scanning microscopy. Moreover, Western blot analysis was performed with antibodies to neuronal cell adhesion molecule (NCAM), L1, pan-cadherin, semaphorin-3F, as well as various ephrins. Results: The basement membrane and the stromal surface promoted the outgrowth of an extensive neuritic network, whereas the epithelial surface did not. Interestingly, these differences of neuritic growth were evident in cell cultures treated with serum-free medium lacking neurotrophic factors and in standard medium containing neurotrophic factors. Western blot analysis revealed an abundant expression of ephrin A4 in intact AM but not in epithelium-denuded AM, and no significant difference of pan-cadherin and NCAM expression was found in intact AM compared with denuded AM. Additionally, no expression of L1, semaphorin-3F, and the ephrins A1, A2, B1, and B3 was detected. Conclusions: This study shows that AM exhibits location-dependent neuritic growth-promoting effects that might influence the clinical outcome of (amniotic membrane transplantation) in neurotrophic conditions. [ABSTRACT FROM AUTHOR]

Purpose: To investigate the change of tissue inhibitor of metalloproteinase (TIMP) in cryopreserved amniotic membranes (AM) according to preservation time, and to evaluate the expression of TIMP in freeze-dried AM. Methods: Cryopreserved or fresh AMs were incubated in dispase II for two hours at 37°C and their epithelial cells were scraped with a cell scraper. Remaining stromal AM was minced and frozen in liquid nitrogen, and then treated with 0.1% diethyl pyrocarbonate. The mRNA levels of TIMP-1 and -2 were determined by reverse transcription-polymerase chain reaction (RT-PCR) in epithelial and stromal cells of fresh AM, AMs cryopreserved for 6 and 12 months, and freeze dried AM, respectively. Western blot analysis and immunohistochemical staining were performed to assess the expression of TIMP-1 in fresh, cryopreserved, and freeze dried AMs. Results: RT-PCR revealed that mRNAs of TIMP-1 and -2 were expressed in the amniotic epithelial cells of both fresh and cryopreserved AMs, while the stromal cells of fresh or cryopreseved AMs and freeze-dried AM showed higher expression of TIMP-1 than TIMP-2 mRNA. On Western blot analysis, the level of TIMP-1 was more in fresh AMs than in cryopreserved or freeze-dried AM, but it was not statistically significant. Conclusion: TIMP-1 was expressed in cryopreserved AMs until 12 months, and the amount of expression was comparable to that in fresh AMs. [ABSTRACT FROM AUTHOR]

Purpose : To investigate adhesion complex formation in cultivated human limbal epithelium after transplantation into the limbal deficient model. Methods : Cultivated epithelium on amniotic membrane was transplanted into limbal deficient rabbits. The transplanted rabbits and the controls were sacrificed at 1, 2, 3, and 4 weeks. The adhesion complex was examined by electron microscopy and immunohistochemistry. Results : Morphologically identifiable hemidesmosomes appeared at 1 week, and matured adhesion complex was found at 3 weeks. Collagen VII was partly stained after transplantation. The mean numbers of hemidesmosomes/2.25 μ m were 2.3 ± 0.9, 2.5 ± 0.5, 5.2 ± 1.0, and 4.0 ± 0.9 at 1, 2, 3, and 4 weeks, and all they were smaller than those in the control, respectively (p< 0.05). Conclusions: Assembly of adhesion complex in cultivated epithelium transplanted in limbal deficient rabbit might recover to the level of that in thehuman after 3 weeks, although it was delayed compared with that in normal wound healing of the rabbit. [ABSTRACT FROM AUTHOR]

Purpose: To evaluate ultraviolet (UV) A and B transmittance by human limbal epithelial cells cultured on human amniotic membranes. Methods: Human limbal epithelial cells were taken from the limbus of donor corneas and were cultured on human amniotic membranes with inactivated 3T3 fibroblasts for 2 to 4 weeks. Then, the cultured cells were examined histologically. Next, cells from different culture periods were irradiated with UV-A (365 nm) or UV-B (302 nm) at energy levels ranging from 50 to 800 µW/cm2, and UV transmittance was measured with a UV light meter. Results: Histological examination revealed a monolayer of corneal epithelial cells on the amniotic membrane after 2 weeks of culture, and a layer of 3–4 cells was formed after 4 weeks. Transmittance of UV-A and UV-B was highest by the amniotic membrane alone, followed in decreasing order by limbal epithelial cells cultured on amniotic membranes for 2 weeks, 3 weeks, and 4 weeks. Conclusions: These results indicate that UV absorbance increases in proportion to the number of limbal epithelial cell layers in cultures on amniotic membranes. Limbal epithelial cells may need to be cultured until 3–4 layers are formed in order to prevent ocular damage by UV light after transplantation. [ABSTRACT FROM AUTHOR]

PURPOSE. To determine if laminin-5 is retained in the matrix of cryopreserved human amniotic membrane tissue prepared for ocular surgeries. METHODS. Amniotic membrane was solubilized in urea/SDS buffer. Constituent proteins were resolved by SDS-PAGE and laminin-5 content was determined by Western blot analysis using a panel of antibodies directed against the α3, β3 or γ2 chains of the molecule. Human corneal epithelial cells were seeded on amniotic membrane and cultured in the presence or absence of EGF. The cell-membrane construct was examined for laminin-5 content using Western blot analysis and immunofluorescence microscopy. RESULTS. In preserved amniotic membrane the laminin-5 α3 chain is present in both the unprocessed (190-kDa) and processed (160-kDa) forms. The β3 chain is found in the 145-kDa form. The γ2 chain appears to be predominantly in the processed (105-kDa) form. Very little of the unprocessed form of the γ2 chain (155-kDa) could be detected using immunoblot analysis. A similar distribution of laminin-5 was also present in extracts of corneal epithelial cells cultured on amniotic membrane. Immunofluorescence analysis of cells cultured on the membrane demonstrated polarization of laminin-5 at the cell-membrane interface. CONCLUSIONS. The presence of both the unprocessed and processed forms of laminin-5 α3 and γ2 chains in preserved human amniotic membrane suggests that when used as a substrate in ocular surgeries, this membrane may be capable of promoting corneal epithelial cell motility and adhesion. Regulation of the motile or adhesive function may lie with factors secreted by the corneal epithelium that populates the membrane following surgery. [ABSTRACT FROM AUTHOR]

PURPOSE. When used as an alternative substrate following bare sclera removal of pterygium and other ocular surface diseases, amniotic membrane transplantation can reduce scarring on the reconstructed conjunctival surface. This study was carried out to determine if the amniotic membrane (AM) suppresses the expression of the TGFb signaling system in cultured normal conjunctival (HCF) and pterygial body fibroblasts (PBF). METHODS. HCF and PBF were cultured on AM and plastic wells in serum-containing and serum-free DMEM with or without TGF-β1. Total RNA was extracted and subjected to Northern hybridization with probes of TGF-β1, b2 and b3; TGF-β receptors (TGF- βR) type I, II and III; a-smooth muscle actin (α-SM), b1-integrin, CD44, fibroblast growth factor receptor 1 (FGF-R1/ flg) and platelet-derived growth factor receptor b (PDGFR-β); and GAPDH as a loading control. MTT assay was used for cell proliferation. RESULTS. Amniotic membrane markedly suppressed the transcript expression of TGF-β2, b3 and all three types of TGF-β receptors by both fibroblasts as compared to their cultures on plastic surface. In addition, expression of CD44 transcript was also markedly suppressed while that of b1 integrin, a-SM actin, and FGFR1/flg was mildly suppressed. In contrast, expression of TGF-β1 and PDGFR-β remained largely unchanged. The cell proliferation of HCF and PBF grown on AM was also significantly suppressed. CONCLUSIONS. Amniotic membrane matrix uniquely suppresses TGF- β signaling in both types of fibroblasts. It may also suppress signaling via CD44, b1 integrin and FGFR1/flg. As a result, the phenotype may become less mitogenic, contractile and fibrogenic. These data support in part why amniotic membrane transplantation has an anti-scarring effect for conjunctival surface reconstruction. [ABSTRACT FROM AUTHOR]