Bovine Amniotic Membrane Research Articles

Preparation of Extra Cellular Matrix Scaffold from Bovine Amniotic Membrane

Preparation of Extra Cellular Matrix Scaffold from Bovine Amniotic Membrane

Potential use of bovine amniotic membrane in periodontal tissue engineering: A literature review

Potential use of bovine amniotic membrane in periodontal tissue engineering: A literature review

Antibacterial and cytotoxic effects of fresh bovine amniotic membrane with hydroxyapatite (BAM-HA): a laboratory experiment

Introduction: Bacterial infections, particularly by Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) and Porphyromonas gingivalis (P. gingivalis), can worsen alveolar bone resorption after tooth extraction. The capability of Bovine Amniotic Membrane-Hydroxyapatite (BAM-HA) biocomposite to reduce this resorption has been explored. However, before clinical use, cytotoxicity testing is imperative to ensure its biocompatibility. The aim of the study was to assess both the antibacterial effects and cytotoxicity of the BAM-HA biocomposite to ensure its suitability for clinical use biocompatibility of the BAM-HA biocomposite before its clinical application. Methods: The laboratory-based research involved testing BAM combined with HA powder in 4:1 and 4:2 ratios via freeze-drying and underwent antibacterial tests against A. actinomycetemcomitans and P. gingivalis, using the plate count method. Cytotoxicity tests were performed on HGF cells, including negative control, positive control, BAM-HA (4:1), and BAM-HA (4:2) groups, with statistical analysis conducted using One-Way ANOVA and Post Hoc Bonferroni and Tukey tests. Results: Antibacterial tests against A. actinomycetemcomitans revealed significant reduction in colony count with BAM-HA ratios 4:1 (129.0 ± 12.7 CFU/mL) and 4:2 (77.3 ± 15.5 CFU/mL) compared to the negative control (186.6 ± 27.5 CFU/mL). Similar reductions were observed for P. gingivalis, with BAM-HA ratios 4:1 (51.3 ± 6.6 CFU/mL) and 4:2 (3.1 ± 1.5 CFU/mL) compared to the negative control (117.3 ± 22.0 CFU/mL). Cytotoxicity tests showed no significant differences in HGF cell viability and IC50 values between the negative control and BAM-HA (4:1) or BAM-HA (4:2) groups. Conclusion: The BAM - HA biocomposite shows antibacterial effects against A. actinomycetemcomitans and P. gingivalis. Moreover, BAM - HA ratios of 4:1 and 4:2 do not induce cytotoxic effects on human gingival fibroblasts, suggesting potential biocompatibility for clinical applications.KeywordA. actinomycetemcomitans, antibacterial effects, BAM-HA biocomposite, cytotoxicity, P. gingivalis

The Role of Bovine Amniotic Membrane and Hydroxyapatite for the Ridge Preservation.

Ridge preservation is an important technique for maintaining the dimensions of the alveolar bone following tooth extraction, which is crucial for successful tooth rehabilitation. The combination of bovine amniotic membrane and hydroxyapatite has shown promise as a scaffold material containing growth factors that can stimulate osteogenic-related factors such as bone morphogenetic protein 2 (BMP2), Runt-related transcription factor 2 (RUNX2), and osteocalcin. This stimulation leads to collagen production and osteoblast proliferation, resulting in new bone formation. In this study, bovine amniotic membrane-hydroxyapatite (BAM-HA) composites were prepared using three different ratios of bovine amniotic membrane and hydroxyapatite (2 : 3, 3 : 7, 7 : 13). Thirty Sprague-Dawley rats had their first incisors extracted, and different types of BAM-HA were applied for ridge preservation. The control group received no treatment, while the positive control group was given xenograft. After 14 and 28 days, the animals were sacrificed, and immunohistochemical analysis was performed to evaluate the expression of BMP2, RUNX2, and osteocalcin. Additionally, a histological examination was conducted to analyse collagen thickness and osteoblast cell proliferation. The results demonstrated that the application of BAM-HA significantly increased collagen density, osteoblast cell proliferation, and the expression of BMP2, RUNX2, and osteoclacin compared to the control group (p < 0.05) on both days 14 and 28. Furthermore, increasing the hydroxyapatite content in the composite was found to enhance collagen thickness, osteoblast cell proliferation, and the expression of osteogenic-related factors. These preliminary findings suggest that the combination of BAM-HA can be used for ridge preservation to prevent further bone resorption following tooth extraction.

Bovine amniotic membrane stem cell-conditioned medium improves the culture conditions for in vitro produced ovine embryos

Despite the progress in in vitro embryo production technology, the quality of embryos produced in vitro remains inferior to that of embryos produced in vivo. There have been reports highlighting the beneficial impacts of stem cells and their conditioned medium on the in vitro maturation of oocytes and the in vitro culture of embryos. In this study, we investigated the effects of adding bovine amniotic membrane stem cell-conditioned medium (bAMSC-CM) to the IVC medium of ovine embryos. Moreover, the effects of bAMSC-CM were compared with the effects of charcoal-stripped fetal bovine serum (CSS). In the first experiment, culture media refreshment was performed on the third day of IVC (day 0 = IVF). The addition of bAMSC-CM and CSS to the IVC medium was conducted in three different ways: from day 1–3 of IVC, from day 1–7 of IVC, and from day 3–7 of IVC. In the second experiment, no culture medium renewal was conducted, and CSS and bAMSC-CM were added from the first day of IVC until the seventh day (CSS and CM groups). In both experiments, a control group (Con) was included that did not receive any supplementation. The data of embryonic cleavage of various treatments was aggregated. The cleavage rate of the embryos cultured in the CM-containing medium from the first day of IVC (CM1) was higher compared with the cleavage rate of the embryos cultured in the Con medium (P < 0.05). In the first experiment, the addition of bAMSC-CM from the onset of IVC resulted in a significant improvement in the blastocyst rate, hatching rate, and blastocyst cell numbers compared with the Con group (P < 0.05). The inclusion of CSS from the first day of IVC resulted in a significant reduction in both blastocyst rate and quality (P < 0.05). The addition of CSS on the third day of IVC resulted in a significant increase in the blastocyst rate compared with the Con group (P < 0.05). In the second experiment, the blastocyst rate and blastocyst cell count in the CM group showed a significant increase compared with the CSS and Con groups (P < 0.05). In conclusion, supplementing ovine IVC medium with bAMSC-CM from the start of IVC increased blastocyst rate and improved the quality of the resulting blastocysts. Furthermore, the need to refresh the culture medium on the third day of the culture has been eliminated.

THE EFFECT OF APPLICATION BOVINE AMNIOTIC MEMBRANE ON OSTEOBLASTS, OSTEOCYTES, AND COLLAGEN

Introduction: Tooth extraction can result in loss of alveolar bone. The application of biomaterials Bovine Amniotic Membrane (BAM) that containing growth factors and collagen is expected to play a role in socket preservation. This study aimed to observe the effect of BAM application on the growth of osteoblasts, osteocytes, and collagen in histological preparate of the alveolar bone socket of Sprague Dawley rats (SD). Methods: 16 male SD rats were used in this group. SD rats were extracted on the mandible central incisivus. Afterward, it was divided into two groups, specifically the control group (without treatment) and the treatment group (BAM application). After extraction, the treatment group was applied with BAM with a size of 1.5 mm x 10 mm in socket alveolar bone. On the 14th and 28th day, the rats were euthanized and the alveolar bone socket of mandibular tissue was taken and stained with Hematoxylin Eosin (HE) and Masson Trichrome (MT). Results: The results of all groups showed that the number of osteoblasts and collagen thickness was higher on day 14th compared to day 28th, while osteocytes continued to increase from day 14th to 28th. The thickness of collagen, osteoblasts, and osteocytes in groups with BAM application was higher than without application. Statistical results showed that there were significant differences between groups (p&lt;0.05). Conclusion: The application of BAM can act as material preservation socket. This biomaterial can accelerate recovery by increasing collagen thickness, osteoblasts, and osteocytes.

Characterisation of Bovine Amniotic Membrane with Hydroxyapatite Bio-Composite

The fabrication of bio-composite-derived bovine amniotic membrane (BAM) with hydroxyapatite (HAp) is an approach to combining organic and inorganic bio-material to improve the properties of both materials. This research aims to combine, fabricate and characterise the bio-composite of BAM–HA. The combination of bio-composite is made from BAM and HAp in a ratio of 30:70, 35:65, and 40:60. Dried BAM is immersed in saline and then blended until it forms an amniotic slurry with a jelly-like consistency. At this stage, HAp is added so that it can bind to BAM. After the mixture is homogeneous, the freeze-drying process is carried out. After fabrication, all the bio-composites were characterised using Fourier transform infrared spectrometry (FTIR), X-ray diffraction (XRD), scanning electron microscopy (SEM) and porosity analysis, and biological activity was conducted using fibroblasts. The bio-composite has functional groups of amides I, II, III, A, B, OH, CO32− and PO43− according to the results of the FTIR. The XRD analysis showed the presence of HAP crystals. This functional group and the crystal HAP indicate that these two materials are bound. An SEM examination revealed a variety of porous patterns on the surface area. The bio-composite with BAM and HAp at a ratio of 35:65 has a higher mean pore size of 155.625 µm with mean porosity of 89.23% and can maintain the fibroblast viability of 95.14%. In conclusion, the study successfully combined both bio-materials BAM and HAp, which have potential synergistic effects on soft and hard tissue regeneration. The ratio of 35:65 showed good characteristics and was non-toxic.

Structure and Strength of Bovine and Equine Amniotic Membrane.

Simple SummaryThin, strong scaffold materials are needed for surgical applications. There is a limited selection of available materials and new materials are required. Amnionic membrane from cattle and horses were investigated for this purpose. The structure of these materials was characterized with synchrotron techniques and the strength was measured. A possible relationship between the structure and strength was identified. These amnion materials from animal sources are strong, thin, and elastic materials, although weaker than some other collagen tissues. They may be suitable for use in surgery as an alternative to material from human donors.Thin, strong scaffold materials are needed for surgical applications. New materials are required, particularly those readily available, such as from non-human sources. Bovine amniotic membrane (antepartum) and equine amniotic membrane (postpartum) were characterized with tear and tensile tests. The structural arrangement of the collagen fibrils was determined by small-angle X-ray scattering, scanning electron microscopy, and ultrasonic imaging. Bovine amnion had a thickness-normalized tear strength of 12.6 (3.8) N/mm, while equine amnion was 14.8 (5.3) N/mm. SAXS analysis of the collagen fibril arrangement yielded an orientation index of 0.587 (0.06) and 0.681 (0.05) for bovine and equine, respectively. This may indicate a relationship between more highly aligned collagen fibrils and greater strength, as seen in other materials. Amnion from bovine or equine sources are strong, thin, elastic materials, although weaker than other collagen tissue materials commonly used, that may find application in surgery as an alternative to material from human donors.

Freeze-dried bovine amniotic membrane as a cell delivery scaffold in a porcine model of radiation-induced chronic wounds.

BackgroundLocoregional stem cell delivery is very important for increasing the efficiency of cell therapy. Amnisite BA (Amnisite) is a freeze-dried amniotic membrane harvested from bovine placenta. The objective of this study was to investigate the retention of cells of the stromal vascular fraction (SVF) on Amnisite and to determine the effects of cell-loaded Amnisite in a porcine radiation-induced chronic wound model.MethodsInitially, experiments were conducted to find the most suitable hydration and incubation conditions for the attachment of SVF cells extracted from pig fat to Amnisite. Before seeding, SVFs were labeled with PKH67. The SVF cell-loaded Amnisite (group S), Amnisite only (group A), and polyurethane foam (group C) were applied to treat radiation-induced chronic wounds in a porcine model. Biopsy was performed at 10, 14, and 21 days post-operation for histological analysis.ResultsRetaining the SVF on Amnisite required 30 minutes for hydration and 1 hour for incubation. A PKH67 fluorescence study showed that Amnisite successfully delivered the SVF to the wounds. In histological analysis, group S showed increased re-epithelialization and revascularization with decreased inflammation at 10 days post-operation.ConclusionsSVFs had acceptable adherence on hydrated Amnisite, with successful cell delivery to a radiation-induced chronic wound model.

Determining the efficacy of the bovine amniotic membrane homogenate during the healing process in rabbits' ex vivo corneas.

To evaluate the efficacy of bovine amniotic membrane homogenate (BAMH) on wounded ex vivo rabbit corneas. Eighteen corneas obtained from normal rabbit eyes were wounded equally using a 6mm trephine and cultured into an air-liquid interface model. Corneas were treated with phosphate-buffered saline (PBS) (n=6, control group), 0.2% ethylenediaminetetraacetic acid (EDTA; n=6), or BAMH (n=6). All treatments were applied topically 6times/day. Each cornea was macrophotographed daily with and without fluorescein stain to assess epithelialization and haziness. After 7days, corneal transparency was evaluated, and the tissues prepared for histologic analysis of viability, and total and epithelial thickness. The mean epithelialization time was 6.2±0.82days for the control group, 6.2±0.75days for the EDTA-treated group, and 5.1±0.40days for the BAMH-treated group, demonstrating a significant difference between the BAMH and the other groups. The corneas that received EDTA had better transparency compared with the other groups. Histologically, all corneas had adequate morphology and architecture after healing. Analysis of corneal and epithelial thickness revealed no significant difference among groups. Bovine amniotic membrane homogenate is an effective and promising treatment for stromal and epithelial ulcers.

Determining MMP-2 and MMP-9 reductive activities of bovine and equine amniotic membranes homogenates using fluorescence resonance energy transfer.

Matrix metalloproteinases (MMPs)-2 and -9 are present in corneal ulcers, and an imbalance between MMPs and tissue inhibitors of metalloproteinases (TIMPs) leads to further corneal degradation. Amniotic membrane homogenate (AMH) has proteolytic properties beneficial for corneal healing, but it is unknown whether AMH possesses TIMPs or effectively inhibits MMP-2 and MMP-9 activity. To determine if bovine and equine AMH reduce in vitro MMP-2 and MMP-9 activities associated with the presence of TIMPs. Undiluted and diluted twofold series (0-fold to 16-fold dilutions) of equine amniotic membrane homogenates (EAMH, n=8) and bovine amniotic membrane homogenates (BAMH, n=8) were subjected to fluorescence resonance energy transfer, and the fluorescence emitted was recorded over time. Average fluorescence was calculated versus recombinant concentration. Enzyme-linked immunosorbent assays for TIMPs 1-4 were applied to quantify TIMPs in the samples. AMH from both species were able to inhibit MMP-2 and MMP-9 activities in vitro, and the inhibition efficacy decreased gradually with dilution. BAMH was significantly more effective than EAMH at inhibiting MMP-2 and MMP-9 in vitro. TIMPs -2 and -3 were present in EAMH and BAMH. TIMP-1 was detected only in BAMH, and TIMP-4 was not detected in any samples. Both EAMH and BAMH directly inhibited MMP-2 and MMP-9 in vitro without dilution, and BAMH showed better inhibition of MMP-2 and MMP-9 before and after dilution compared to EAMH.

Proteome Composition of Bovine Amniotic Membrane and Its Potential Role in Corneal Healing.

PurposeTo investigate the protein profile of bovine amniotic membranes (bAM) and to determine putative associations between protein composition in bAM and known corneal healing pathways.MethodsThe bAM were acquired from normal full-term births (n = 10), processed, and stored at −80°C for two days. Subsequently, the frozen membranes were thawed at room temperature and prepared for proteomic exploration using high-resolution liquid chromatography–mass spectrometry, followed by bioinformatics analysis. Recently identified corneal healing pathways were contrasted with protein profiles and pathways present in bAM.ResultsThe analyses identified 2105 proteins, with an interactive network of 1271 nodes (proteins) and 8757 edges (interactions). The proteins with higher betweenness centrality measurements include microfibril-associated protein 4, HSD3B1, CAPNS1, ATP1B3, CAV1, ANXA2, YARS, and GAPDH. The top four pathways in Kyoto Encyclopedia of Genes and Genomes were ribosome, metabolic pathway, spliceosome, and oxidative phosphorylation. The bAM and cornea shared abundant proteins, genome ontology, and signaling pathways.ConclusionsThe high-throughput proteomic profile of the bAM demonstrated that numerous proteins present in the cornea are also present in this fetal membrane. Our findings collectively demonstrate the similarity between bAM and the cornea's protein composition, supporting our hypothesis that bAM can be used to treat corneal diseases.

Bovine sponge amnion stimulates socket healing: A histological analysis.

Bovine sponge amnion (BSA) is made from bovine amniotic membrane, which contains several growth factors with the ability to suppress inflammation and stimulate the healing process. The healing process of the socket is clinically marked by the proliferation of fibroblasts, formation of new blood vessels, and closure of the wound site. Fibroblasts and new blood vessels are the common histological indicators of tissue regeneration. Twenty-four lower anterior teeth were extracted from the rats. The socket was then applied with BSA and sutured, while a control group was only sutured. The animals were scarified 3, 7, and 14 days after application, and the mandibular was isolated. Histological analysis of socket tissue used the staining of hematoxylin and eosin to analyze the new blood vessels and fibroblasts. An independent t-test was used to analyze the fibroblasts and new blood vessels in each group, with P < 0.05 considered as significant. The number of fibroblasts is higher in the BSA group compared others groups at 3, 7, and 14 days (P < 0.05). The new blood vessel count is higher compared to the control group at only 3 and 7 days (P < 0.05). BSA aids the regeneration of the socket after tooth extraction by stimulating fibroblast proliferation and formation of new blood vessels.

Embryo co-culture with bovine amniotic membrane stem cells can enhance the cryo-survival of IVF-derived bovine blastocysts comparable with co-culture with bovine oviduct epithelial cells.

Culture conditions have a profound effect on the quality of in vitro-produced embryos. Co-culturing embryos with somatic cells has some beneficial effects on embryonic development. Considering the ability of stem cells to secrete a broad range of growth factors with different biological activities, we hypothesized that bovine amniotic membrane stem cells (bAMSCs) might be superior to bovine oviduct epithelial cells (BOECs) in supporting embryonic development and enhancing their cryo-survival. Bovine abattoir-derived oocytes were matured and fertilized in vitro. The resultant presumptive zygotes were then cultured up to the blastocyst stage in the following groups: (i) co-culture with bAMSCs, (ii) co-culture with BOECs, and (iii) cell-free culture (Con). Embryos that reached the blastocyst stage were vitrified and warmed, and their post-warming re-expansion, survival and hatching rates were evaluated after 72 h culture. Results showed that the cleavage, blastocyst, and 2 h post-warming re-expansion rates of embryos did not differ between groups. However, their survival rates in BOEC and bAMSC groups were significantly higher compared with the control (72.7, 75.6 and 37.5%, respectively, P < 0.05). In conclusion, our results showed that the cryo-survivability of IVF-derived bovine embryos could be improved through co-culturing with bAMSCs. Moreover, considering the possibility to provide multiple passages from bAMSCs compared with BOECs, due to their stemness properties and their ability to produce growth factors, the use of bAMSCs is a good alternative to BOECs in embryo co-culture systems.

A Sponge-Like Double-Layer Wound Dressing with Chitosan and Decellularized Bovine Amniotic Membrane for Promoting Diabetic Wound Healing.

The diabetic wounds do not heal easily in part because they are susceptible to infection due to environmental influences. Wound dressing is crucial to wound healing, as it can basically protect the wound from external damages and provide a suitable microenvironment for tissue regeneration. In this study, a double-layer membrane that consists of chitosan sponge and decellularized bovine amniotic membrane (dBAM) has been developed by freeze-casting method. The results showed that the porous structure of the sponge layer improved the performances of blood coagulation and swelling. The dense dBAM can optimize the mechanical property of wound dressing. In vitro studies revealed that the bilayer membrane had favorable biocompatible, especially for human foreskin fibroblast cells (HFF-1) cell adhesion and proliferation. Moreover, the full-thickness skin defects of diabetic model mice that treated with bilayer membrane showed over 80% closure in 8 days. Our findings imply that the double-layer dressing has great potentials to be used in diabetic patients.

Role of Bovine Amniotic Membrane in Repair of Abdominal Muscle Defects in Dogs

Role of Bovine Amniotic Membrane in Repair of Abdominal Muscle Defects in Dogs

Effectiveness of polypropylene mesh coated bovine amniotic membrane with adhesion barrier (polyethylene glycol) in repair of abdominal wall hernias in rats

This study was undertaken to investigate the effectiveness of polypropylene (PP) mesh coated bovine amniotic membrane (BAM) with 5% polyethylene glycol (PEG) 4000 as adhesion barrier in the repair of experimental 2 × 2 cm of abdominal hernias in rats. Rats (32) were divided into 4 groups. A 2 cm × 2 cm defect was created in the full thickness of abdominal muscle on the anterior abdominal wall. PP mesh was implanted in the abdominal cavity (Gr 1 to 4). BAM covered the abdominal face of the graft (Gr 3 and 4). It was given before the abdominal closure 5 ml of 5% PEG 4000 (Gr 2 and 4) and 5 ml of 0.9% NaCl (Gr 1 and 3). After 21 days following the operations, 32 rats were euthanized. Macroscopic and microscopic evaluations were performed according to the scoring systems. The differences between the groups was evaluated by Mann-Whitney U test and Kruskal Wallis analysis of variance. Highest adhesion percentage was observed in Gr 1 and lowest in Gr 4. Inflammatory reaction was observed in Gr 1 and 2. According to the results of this study, the combined use of BAM and 5% PEG 4000 was helpful to prevent the complications of PP mesh.

Isolation, culture, characterization and cryopreservation of stem cells derived from amniotic mesenchymal layer and umbilical cord tissue of bovine fetuses

ABSTRACT: Stem cells are undifferentiated cells with a high proliferation potential. These cells can be characterized by their in vivo ability to self-renew and to differentiate into specialized cell lines. The most used stem cell types, in both human and veterinary fields, are the mesenchymal stem cells (MSC) derived from bone marrow and adipose tissue. Nowadays, there is a great interest in using stem cells derived from fetal tissues, such as amniotic membrane (AM) and umbilical cord tissue (UCT), which can be obtained non-invasively at delivery time. Due to the scarcity of studies in bovine species, the aim of this study was to isolate, characterize, differentiate and cryopreserve MSC derived from the mesenchymal layer of amniotic membrane (AM), for the first time, and umbilical cord tissue (UCT) of dairy cow neonates after assisted delivery (AD) and from fetus at initial third of pregnancy (IT) obtained in slaughterhouse. Cells were isolated by enzymatic digestion of the tissue fragments with 0.1% collagenase solution. Six samples of AM and UCT at delivery time and six samples of AM and UCT at first trimester of pregnancy were subjected to morphology evaluation, imunophenotype characterization, in vitro osteogenic, adipogenic and chondrogenic differentiation and viability analysis after cryopreservation. All samples showed adherence to plastic and fibroblast-like morphology. Immunocytochemistry revealed expression of CD 44, NANOG and OCT-4 and lack of expression of MHC II in MSC from all samples. Flow cytometry demonstrated that cells from all samples expressed CD 44, did not or low expressed CD 34 (AM: IT-0.3%a, AD-3.4%b; UCT: 0.4%, 1.4%) and MHC II (AM: IT-1.05%a, AD-9.7%b; UCT: IT-0.7%a, AD-5.7%b). They were also capable of trilineage mesenchymal differentiation and showed 80% viability after cryopreservation. According to the results, bovine AM and UCT-derived cells, either obtained at delivery time or from slaughterhouse, are a painless and non-invasive source of MSC and can be used for stem cell banking.

Microbiological viability of bovine amniotic membrane stored in glycerin 99% at room temperature for 48 months

ABSTRACT The medium for storing biological tissues is of great importance for their optimal use in surgery. Glycerin has been proven efficient for storing diverse tissues for prolonged time, but the preservation of the bovine amniotic membrane in glycerin 99% at room temperature has never been evaluated to be used safely in surgical procedures. This study evaluated the preservation of 80 bovine amniotic membrane samples stored in glycerin 99% at room temperature. The samples were randomly divided evenly into four groups. Samples were microbiologically tested after 1, 6, 12 and 48 months of storage. The presence of bacteria and fungi in the samples was evaluated by inoculation on blood agar and incubation at 37 ºC for 48 hours and on Sabouraud agar at 25 ºC for 5 to 10 days. No fungal or bacterial growth was detected in any of the samples. It was concluded that glycerin is an efficient medium, regarding microbiology, for preserving pre-prepared bovine amniotic membrane, keeping the tissue free of microorganisms that grow in the media up to 48 months at room temperature.

A new surgical technique to treat corneal perforations using amniotic membrane and surgical adhesive

The objectives of this study were to compare surgical techniques and the effects of using n-butyl 2-cyanoacrylate and bovine amniotic membrane to repair perforated lesions in corneas. Penetrating keratoplasty was performed in sixty New Zealand White rabbits under general anesthesia. Group 1 (G1) was treated with n-butyl 2-cyanoacrylate, group 2 (G2) received a fragment of amniotic membrane through the anterior chamber and application of n-butyl 2-cyanoacrylate over the lesion, group 3 (G3) was treated with the same technique as G2 with the addition of an amniotic membrane bandage covering the cornea and sutured in the limbus region, and group 4 (G4) was treated with an amniotic membrane sutured to the lesion and an amniotic membrane bandage sutured in the limbus region. Clinical, histological and histomorphometric examinations of the corneas were performed. The membrane acted as a barrier for aqueous humor in G2 and G3, thereby keeping the surface dry for adhesive application; it also prevented the adhesive from contacting intraocular structures. The groups treated with amniotic membrane and surgical adhesive showed better results than the groups treated with either material alone. Thus, the combination of the membrane with the adhesive is recommended for this type of lesion.