Amniotic Membrane Transplantation for Ocular Surface Reconstruction

The use of amniotic membrane (or amnion) for transplantation as graft in ocular surface reconstruction is reviewed. This technique has become widespread because of the availability of the amnion, convenience and ease of use, and high and reproducible success rates. The mechanisms of action of the transplantation are varied and include the prolongation and clonogenic maintenance of epithelial progenitor cells, promotion of goblet and non-goblet cell differentiation, exclusion of inflammatory cells with anti-protease activities, suppression of Transforming Growth Factor signaling and myoblast differentiation of normal fibroblasts. The observed clinical effects include facilitation of epithelialization, maintenance of normal phenotypes, and reduction of inflammation, vascularization and scarring. Amniotic membrane transplantation is being increasingly used as graft for various conjunctival and corneal diseases and as a patch in cases of chemical and thermal burns, refractory and recalcitrant keratitis, and most recently as an excellent substrate for expanding epithelial stem cells ex vivo.     

Augmented Dried versus Cryopreserved Amniotic Membrane as an Ocular Surface Dressing

Purpose

Dried amniotic membrane (AM) can be a useful therapeutic adjunct in ophthalmic surgery and possesses logistical advantages over cryopreserved AM. Differences in preservation techniques can significantly influence the biochemical composition and physical properties of AM, potentially affecting clinical efficacy. This study was established to investigate the biochemical and structural effects of drying AM in the absence and presence of saccharide lyoprotectants and its biocompatibility compared to cryopreserved material.

Methods

AM was cryopreserved or dried with and without pre-treatment with trehalose or raffinose and the antioxidant epigallocatechin (EGCG). Structural and visual comparisons were assessed using electron microscopy. Localisation, expression and release of AM biological factors were determined using immunoassays and immunofluorescence. The biocompatibility of the AM preparations co-cultured with corneal epithelial cell (CEC) or keratocyte monolayers were assessed using cell proliferation, cytotoxicity, apoptosis and migration assays.

Results

Drying devitalised AM epithelium, but less than cryopreservation and cellular damage was reduced in dried AM pre-treated with trehalose or raffinose. Dried AM alone, and with trehalose or raffinose showed greater factor retention efficiencies and bioavailability compared to cryopreserved AM and demonstrated a more sustained biochemical factor time release in vitro. Cellular health assays showed that dried AM with trehalose or raffinose are compatible and superior substrates compared to cryopreserved AM for primary CEC expansion, with increased proliferation and reduced LDH and caspase-3 levels. This concept was supported by improved wound healing in an immortalised human CEC line (hiCEC) co-cultured with dried and trehalose or raffinose membranes, compared to cryopreserved and fresh AM.

Conclusions

Our modified preservation process and our resultant optimised dried AM has enhanced structural properties and biochemical stability and is a superior substrate to conventional cryopreserved AM. In addition this product is stable and easily transportable allowing it to be globally wide reaching for use in clinical and military sectors.     

Preserved Amniotic Membrane Transplantation for Conjunctival Surface Reconstruction

Objective. To study the efficacy of preserved human amniotic membrane in the reconstruction of conjunctival defect created during surgical removal of conjunctival lesions or symblepharon lysis. Methods. Preserved human amniotic membrane transplantation was performed in 93 eyes of 85 patients for reconstruction of various conjunctival surface problems. The indications for surgery were (1) pterygium excision (54 eyes), (2) conjunctival tumors excision (23 eyes), lysis of symblepharon (13 eyes), and covering a scleral graft (three eyes). Results. Success was noted in 69.9% (65/93) eyes, partially success in 22.6% (21/93) eyes, and failure in 7.5% (7/93) eyes with a mean follow-up of 8.9 months (1–28 months). In pterygium, conjunctival tumor, symblepharon, and scleral graft group, the success rate in each group was 70.3%, 78.3%, 53.8%, and 66.7% respectively. No serious immediate post-operative complications or graft rejection occurred. Conclusion. Amniotic membrane transplantation can be considered an alternative treatment for difficult ocular surface problems, and is effective in promoting epithelial healing, and reducing inflammation and scarring.     

Application of Preserved Human Amniotic Membrane for Corneal Surface Reconstruction

Objective: To evaluate the efficacy of preserved human amniotic membrane transplantation for reconstruction of the corneal surface diseases. Methods: Preserved human amniotic membrane transplantations were performed in 84 eyes of 78 patients for corneal surface reconstruction. The indications were limbal stem cell deficiency from Steven–Johnson syndrome, chemical burn and herpes keratitis (27 eyes), bullous keratopathy (26 eyes), persistent epithelial defect and dellen (17 eyes), band keratopathy (11 eyes), preparing for prosthesis (1 eye), corneal ulcer (1 eye) and acute chemical burn (1 eye). Results: Success was noted in 83.3% (70/84) eyes, partial success in 13.1% (11/84) eyes, and failure in 3.6% (3/84) eyes for an average follow-up of 10.5 months (3 – 29 months). No patient developed major immediate post-operative complications. Conclusion: Amniotic membrane transplantation can reduce inflammation, promote corneal epithelial healing, and decrease irritation in corneal surface problems.     

Ocular Complications of Chloroquine Therapy

Long-term therapy with chloroquine can lead to irreversible retinal damage and serious loss of visual acuity and visual field. Not only are the retinal changes irreversible but they may continue to progress after discontinuance of the drug. Work is proceeding on the development of electrophysiological techniques for early detection of toxic effect before significant retinal damage occurs. Another possibility for minimizing the toxic effect of chloroquine is the use of other drugs which increase the excretion of chloroquine from the body.     

Ocular Complications of Chloroquine Therapy

Ocular complications of long-term chloroquine therapy were observed in 18 of 45 patients so treated. This therapy was used in patients with rheumatoid arthritis, lupus erythematosus, sarcoidosis, discoid lupus and other chronic “collagen disease”. Thirteen patients had reversible corneal opacifications, and seven had irreversible retinal changes, with visual loss and visual field defects. Pathological evidence of chloroquine retinopathy was obtained in one patient. Physicians are therefore warned to use this drug only after careful consideration. If it is used, repeated ocular examinations should include assessment of visual acuity, visual fields on a tangent screen and fundus examination through a dilated pupil.     

New Amniotic Membrane Based Biocomposite for Future Application in Reconstructive Urology

Objective

Due to the capacity of the amniotic membrane (Am) to support re-epithelisation and inhibit scar formation, Am has a potential to become a considerable asset for reconstructive urology i.e., reconstruction of ureters and urethrae. The application of Am in reconstructive urology is limited due to a poor mechanical characteristic. Am reinforcement with electrospun nanofibers offers a new strategy to improve Am mechanical resistance, without affecting its unique bioactivity profile. This study evaluated biocomposite material composed of Am and nanofibers as a graft for urinary bladder augmentation in a rat model.

Material and Methods

Sandwich-structured biocomposite material was constructed from frozen Am and covered on both sides with two-layered membranes prepared from electrospun poly-(L-lactide-co-E-caprolactone) (PLCL). Wistar rats underwent hemicystectomy and bladder augmentation with the biocomposite material.

Results

Immunohistohemical analysis (hematoxylin and eosin [H&E], anti-smoothelin and Masson’s trichrome staining [TRI]) revealed effective regeneration of the urothelial and smooth muscle layers. Anti-smoothelin staining confirmed the presence of contractile smooth muscle within a new bladder wall. Sandwich-structured biocomposite graft material was designed to regenerate the urinary bladder wall, fulfilling the requirements for normal bladder tension, contraction, elasticity and compliance. Mechanical evaluation of regenerated bladder wall conducted based on Young’s elastic modulus reflected changes in the histological remodeling of the augmented part of the bladder. The structure of the biocomposite material made it possible to deliver an intact Am to the area for regeneration. An unmodified Am surface supported regeneration of the urinary bladder wall and the PLCL membranes did not disturb the regeneration process.

Conclusions

Am reinforcement with electrospun nanofibers offers a new strategy to improve Am mechanical resistance without affecting its unique bioactivity profile.     

Improved Fixation of Cellulose-Acetate Reverse-Osmosis Membrane for Scanning Electron Microscopy

Fixation of cellulose-acetate membranes with either glutaraldehyde-osmium tetroxide or glutaraldehyde-ruthenium tetroxide resulted in extensive electron beam damage. Beam damage was eliminated and the bacterial surface structure was preserved, however, when cellulose-acetate membranes were fixed with glutaraldehyderuthenium tetroxide and treated successively with thiocarbohydrazide and osmium tetroxide.     

Progress in Clinical Gene Therapy for Cardiac Disorders

Molecular Diagnosis & Therapy - Despite significant advances in novel treatments and approaches, cardiovascular disease remains the leading cause of death globally. Gene therapy is a promising...     

Amniotic Membrane Grafts for the Prevention of Esophageal Stricture after Circumferential Endoscopic Submucosal Dissection

Background and Aims

The prevention of esophageal strictures following circumferential mucosal resection remains a major clinical challenge. Human amniotic membrane (AM) is an easily available material, which is widely used in ophthalmology due to its wound healing, anti-inflammatory and anti-fibrotic properties. We studied the effect of AM grafts in the prevention of esophageal stricture after endoscopic submucosal dissection (ESD) in a swine model.

Animals and Methods

In this prospective, randomized controlled trial, 20 swine underwent a 5 cm-long circumferential ESD of the lower esophagus. In the AM Group (n = 10), amniotic membrane grafts were placed on esophageal stents; a subgroup of 5 swine (AM 1 group) was sacrificed on day 14, whereas the other 5 animals (AM 2 group) were kept alive. The esophageal stent (ES) group (n = 5) had ES placement alone after ESD. Another 5 animals served as a control group with only ESD.

Results

The prevalence of symptomatic strictures at day 14 was significantly reduced in the AM group and ES groups vs. the control group (33%, 40% and 100%, respectively, p = 0.03); mean esophageal diameter was 5.8±3.6 mm, 6.8±3.3 mm, and 2.6±1.7 mm for AM, ES, and control groups, respectively. Median (range) esophageal fibrosis thickness was 0.87 mm (0.78–1.72), 1.19 mm (0.28–1.95), and 1.65 mm (0.7–1.79) for AM 1, ES, and control groups, respectively. All animals had developed esophageal strictures by day 35.

Conclusions

The anti-fibrotic effect of AM on esophageal wound healing after ESD delayed the development of esophageal stricture in our model. However, this benefit was of limited duration in the conditions of our study.     

Ophthalmic Applications of Preserved Human Amniotic Membrane: A Review of Current Indications

Preserved human amniotic membrane (AM) is currently being used for a wide spectrum of ocular surface disorders. The AM has a basement membrane, which promotes epithelial cell migration and adhesion. The presence of a unique avascular stromal matrix reduces inflammation, neovascularization and fibrosis. The basic tenets of amniotic membrane transplantation (AMT) are to promote re-epithelialization, to reconstruct the ocular surface and to provide symptomatic relief from surface aberrations. AMT is a useful technique for reconstruction of surface defects resulting from removal of surface tumors and symblephara. AMT has effectively restored a stable corneal epithelium in eyes with, persistent epithelial defects and corneal ulcers. In the setting of acute ocular burns and SJS, AMT has satisfactorily reduced scarring and inflammation. AMT alone may be an effective alternative for partial limbal stem cell deficiency. However remarkable improvements in surface stability have resulted from concurrent AMT and limbal stem cell transplantation, wherein the limbal grafts are obtained from the normal fellow eye, living relative or cadaveric eye. In severe or bilateral cases, well being of the donor eye is a major concern. Currently, the most unique application of preserved human AM in ophthalmology is its use as a substrate for ex-vivo expansion of corneal and conjunctival epithelium. In this novel technique of tissue engineering, epithelial stem cells can be safely harvested and expanded on denuded AM. The resultant composite cultured tissue has been successfully transplanted to restore vision, as well as the structure and function of damaged ocular surfaces.     

The Lipid Layer: The Outer Surface of the Ocular Surface Tear Film

The outer layer of the tear film—the lipid layer—has numerous functions. It is a composite monolayer composed of a polar phase with surfactant properties and a nonpolar phase. In order to achieve an effective lipid layer, the nonpolar phase, which retards water vapor transmission, is dependent on a properly structured polar phase. Additionally, this composite lipid layer must maintain its integrity during a blink. The phases of the lipid layer depend on both lipid type as well as fatty acid and alcohol composition for functionality. Surprisingly, the importance of the composition of the aqueous layer of the tear film in proper structuring of the lipid layer has not been recognized. Finally, lipid layer abnormalities and their relationship to ocular disease are beginning to be clarified.     

Effect of Amniotic Membrane Proteins in Human Cancer Cell Lines: An Exploratory Study

Human amniotic membrane (hAM) has recently drawn attention as an upcoming anti-cancer therapy. Regarding the strategies which have already investigated, little is known about hAM protein extracts (hAMPE) effect on cancer. So, this work aims to study the effect of hAMPE in metabolic activity of several human cancer cell lines. hAMPE were mechanically obtained, thus avoiding the effect of detergents and other reagents commonly used in protein extraction under the cell lines studied. After quantification of proteins in hAMPE, their effect on the metabolic activity of 21 human cancer cell lines was assessed by 3-(4,5-dimethylthia-zolyl-2)2,5-diphenyltetrazolium bromide (MTT) assay. Our results indicate that there is an inhibition of metabolic activity until 25 and 50 % in two and seven cell lines, respectively. Five cell lines proved to be very sensitive to hAMPE, being its metabolic activity more than 50 % inhibited. Our results show that hAMPE can inhibit the metabolic activity of some human cancer cell lines. However, research about this cell line-dependent response to hAMPE becomes indispensable.     

Autologous Transplantation of Oral Mucosal Epithelial Cell Sheets Cultured on an Amniotic Membrane Substrate for Intraoral Mucosal Defects

The human amniotic membrane (AM) is a thin intrauterine placental membrane that is highly biocompatible and possesses anti-inflammatory and anti-scarring properties. Using AM, we developed a novel method for cultivating oral mucosal epithelial cell sheets. We investigated the autologous transplantation of oral mucosal epithelial cells cultured on AM in patients undergoing oral surgeries. We obtained specimens of AM from women undergoing cesarean sections. This study included five patients without any history of a medical disorder who underwent autologous cultured oral epithelial transplantation following oral surgical procedures. Using oral mucosal biopsy specimens obtained from these patients, we cultured oral epithelial cells on an AM carrier. We transplanted the resultant cell sheets onto the oral mucosal defects. Patients were followed-up for at least 12 months after transplantation. After 2–3 weeks of being cultured on AM, epithelial cells were well differentiated and had stratified into five to seven layers. Immunohistochemistry revealed that the cultured cells expressed highly specific mucosal epithelial cell markers and basement membrane proteins. After the surgical procedures, no infection, bleeding, rejection, or sheet detachment occurred at the reconstructed sites, at which new oral mucous membranes were evident. No recurrence was observed in the long-term follow-up, and the postoperative course was excellent. Our results suggest that AM-cultured oral mucosal epithelial cell sheets represent a useful biomaterial and feasible method for oral mucosal reconstruction. However, our primary clinical study only evaluated their effects on a limited number of small oral mucosal defects.     

抗青光眼药物长期使用对眼表的影响

目的：研究长期使用青光眼药物对患者眼表结构的影响,为正确选择青光眼治疗药物提供依据。方法：将采用青光眼药物治 疗≥ 3 个月的青光眼患者40 例（62 只眼）设为病例组,以同期健康志愿者30 例（30 只眼）设为对照组,比较两组基础泪液分泌长 度(ST)、泪膜破裂时间(BUT)、角膜荧光染色评分（FB）及结膜印记细胞评级（IC）。结果：病例组ST、BUT 分别为（6.12± 3.63）mm、 （5.21± 2.13）s,均短于对照组的（10.72± 4.62）mm、（11.45± 5.12）s（P<0.01);病例组FB 评分为（2.19± 0.42）分,高于对照组（0.62± 0.04）分（P<0.01）;病例组IC 评级为Ⅱ~Ⅲ级比例为54.84%,高于对照组的5.00%（P<0.01)。结论：青光眼患者长期使用青光眼药物 会导致泪液分泌减少,泪膜稳定性下降,并加快结膜细胞鳞状上皮化进程,对泪膜、角膜及结膜均有损伤,应引起重视。