Amniotic Membrane Transplantation
Erik Letko
C. Stephen Foster
Amniotic membrane transplantation (AMT) is indicated for numerous ocular surface disorders, the spectrum of which has been expanding recently (Table 58-1). The first written report on the use of amniotic membrane graft (AMG) in clinical medicine was made after Davis used both chorion and amnion in skin transplantation cases in 1910 (1). Since then, living (rather than preserved) amniotic membrane has been used for various purposes (2). The first report on the use of amniotic membrane for ocular disorders was by DeRoth in 1940; he used both amnion and chorion in the treatment of conjunctival defects (3). After a half century of lack of written reports on amnion use for ocular surface disorders, Battle and Perdomo introduced, in 1993, the idea of amniotic membrane transplantation to North America (4). Two years later, Kim and Tseng published their observations that preserved amniotic membrane facilitated corneal surface healing in rabbits after epithelial removal and limbal lamellar keratectomy (5). Since then, the use of amniotic membrane has been explored in a growing number of ocular surface disorders.
BASIC SCIENCE
Human amnion is the innermost layer of the placenta. It is composed of five layers that can be distinguished histologically (6). These include a single epithelial layer, a basement membrane, and an avascular connective tissue that is composed of three layers: the compact layer attached to the basement membrane, the fibroblast layer, and the spongy layer (6). The thickness of human amnion is approximately 0.02 to 0.50 mm (6). The epithelium consists of one layer of cells, the shape of which varies between cuboidal in the amnion reflectum, to columnar over the placental surface, and squamous over the cord amnion (7). The basement membrane consists of reticular fibers with processes to the basal region of the epithelial cells (6). The compact layer is thought to be the strongest layer, with significant tensile strength (6). The fibroblast layer is the thickest layer of the amnion and contains a loose fibroblast network and reticulum (6). The spongy layer is the outermost layer of the amnion. It contains wavy bundles of reticulin and scattered fibroblasts (6).
The mechanisms by which amniotic membrane promotes healing of the ocular surface are not fully understood. The previously proposed mechanism of action through oxygen permeability, epithelial hydration, and mechanical protection (8) may be too simplistic because soft contact lenses have these characteristics, too (9), yet there are numerous reports on cases where AMT resulted in epithelialization of epithelial defects after bandage soft contact lens therapy failed. There is growing evidence that biologic factors, including collagens and cytokines, may make a major difference between a soft contact lens and amniotic membrane. The amniotic membrane is believed to provide a superior substrate for migration of epithelial cells (10), reinforce adhesion of basal epithelial cells (11,12), promote epithelial differentiation (13, 14, 15, 16), prevent apoptosis of epithelial cells (17,18), and reduce neovascularization and fibrosis by reduction of inflammatory cell infiltration (19, 20, 21).
Both collagens IV and VII, components of corneal and conjunctival epithelial basement membrane, are present in the basement membrane of amniotic membrane (22). In addition, amniotic membrane contains collagen III (another collagen component of conjunctival epithelial basement membrane), collagens I, II, and V, fibronectin, and laminin (22, 23, 24, 25). The composition of amniotic basement membrane may be closer to conjunctival than to corneal basement membrane based on the presence of type IV collagen and laminin subchains (22).
The presence of cytokines expressed in the amniotic membrane has been investigated recently. The experiments show that cytokines that promote epithelialization and reduce inflammation, scarring, and neovascularization are present in preserved (26) as well as epithelium-denuded amniotic membrane (27). These cytokines are found in both the epithelium and stroma of the amniotic membrane, but are believed to be synthesized predominantly by the epithelium (28). The cytokines are present in cryopreserved amniotic membrane (26) and, surprisingly, approximately 50% of the epithelial cells of amniotic membrane
cryopreserved for several months can still be viable (29). Hence, it is possible that the amnion epithelial cells continue production of cytokines after transplantation. Nerve growth factor, epidermal growth factor, keratocyte growth factor, and hepatocyte growth factor, all present in the amnion, may play a role in promoting the epithelialization of the ocular surface after AMT (27,28).
cryopreserved for several months can still be viable (29). Hence, it is possible that the amnion epithelial cells continue production of cytokines after transplantation. Nerve growth factor, epidermal growth factor, keratocyte growth factor, and hepatocyte growth factor, all present in the amnion, may play a role in promoting the epithelialization of the ocular surface after AMT (27,28).
TABLE 58-1. EXAMPLES OF CLINICAL APPLICATION OF AMNIOTIC MEMBRANE GRAFTING | ||||||||||||
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The mechanisms by which amniotic membrane may reduce scarring, inflammation, and angiogenesis are more complex. Amniotic membrane matrix uniquely suppresses transforming growth factor-β signaling, resulting in reduction in fibroblasts and scarring (30). The AMGs trap inflammatory cells of monocyte-macrophage lineage that show signs of apoptosis (31). Fresh human amniotic epithelial and mesenchymal cells express potent antiangiogenic agents, interleukin-1 receptor antagonist, all four matrix metalloproteinase inhibitors, collagen XVIII, interleukin-10, and thrombospondin-1 (26). Similar observations were made in residual epithelial cells and stroma of cryopreserved amniotic membranes (26).
Unlike other allograft transplantations, AMT does not require administration of systemic immunosuppressive therapy to prevent immune rejection. The amniotic membrane typically dissolves within 3 to 5 weeks. It is not clear whether alloreactive immune mechanisms are involved in the process of amniotic graft dissolution after placement onto the ocular surface. The earlier hypotheses about lack of immune rejection response proposed that this phenomenon is a result of lack of human leukocyte antigens (HLA) in a cryopreserved amniotic membrane, but recent studies suggest that the issue is more complex. It is believed that amnion is an immune-privileged tissue (29). The epithelium, stroma, and fibroblasts of cryopreserved amniotic membrane contain HLA class I and II antigens, but the
amnion does have the ability to suppress alloreactive T cells in vitro (32). Furthermore, amnion expresses HLA-G, a nonclassic major histocompatibility complex class I molecule expressed in the extravillous cytotrophoblast at the fetomaternal interface, that protects the fetus from maternal cellular immunity. This antigen, along with some other immunoregulatory molecules (e.g., Fas ligand), plays a role in suppressing the infiltration of CD4+ and CD8+ T cells into amnion (29).
amnion does have the ability to suppress alloreactive T cells in vitro (32). Furthermore, amnion expresses HLA-G, a nonclassic major histocompatibility complex class I molecule expressed in the extravillous cytotrophoblast at the fetomaternal interface, that protects the fetus from maternal cellular immunity. This antigen, along with some other immunoregulatory molecules (e.g., Fas ligand), plays a role in suppressing the infiltration of CD4+ and CD8+ T cells into amnion (29).
PREPARATION AND STORAGE OF AMNION
The vast majority of clinical experience comes from the use of cryopreserved AMGs, but the use of fresh amniotic membrane has been previously reported (33, 34, 35, 36). Both in vivo and in vitro studies suggest that there is very little or no difference between fresh and cryopreserved amniotic membranes. The amniotic membrane shows little morphologic change after cryopreservation in 50% glycerol (37). Fresh amnion may be a good source of AMGs in countries where preserved tissue is not accessible, but the risk of blood-borne infections seems to be elevated (33,34). The cryopreserved amniotic membrane is typically stored in −80°C on a nitrocellulose paper in 50% glycerol with the epithelium side up. The epithelium side is smooth and can be distinguished from the sticky stromal side by touching it with a Weck-cel sponge (Edward Weck & Co, Inc., Research Triangle Park, NC). The AMGs can be stained before removal from the nitrocellulose paper with lissamine green B 1% that is not toxic to corneal epithelium and allows for better visualization of edges and folds or wrinkles during the surgical procedure. The color of the amniotic membrane returns to normal within 120 minutes of application of the dye.
The preparation and preservation techniques for AMGs were first described by Lee and Tseng (38). Amnion is obtained from placentas of donors undergoing elective caesarean section to eliminate contamination from vaginal delivery (39). Donor screening to exclude the presence of human immunodeficiency virus (HIV)-1 and HIV-2 (HIV-1 and HIV-2 antibodies), hepatitis B (surface antigen and core antibody), hepatitis C (antibody), human T-lymphocyte virus 1 and 2 (antibodies), and syphilis (rapid plasma reagin) in the donor’s serum are performed before harvesting placenta, and this is repeated 6 months later to guard against transmission of blood-borne pathogens. The placenta is transported under sterile conditions to a lamellar-flow hood and cleaned of blood clots with sterile Earle’s balanced saline solution (Life Technologies, Inc., Gaithersburg, MD) containing 50 μg/mL of penicillin, 50 μg/mL of streptomycin, 100 μg/mL of neomycin, and 2.5 μg/mL of amphotericin (Bio-Tissue Form #H-005.0, Miami, FL). The amnion is separated from chorion by blunt dissection and placed onto nitrocellulose paper with a pore size of 0.45 μm (Bio-Rad, Gainesville, FL) with the epithelium side up. The nitrocellulose paper with amnion is then cut into pieces typically not larger than 4 cm × 4 cm and stored at −80°C in a sterile vial containing Dulbecco’s modified Eagle medium (Life Technologies, Inc.) and glycerol (Baxter Healthcare Corporation, Stone Mountain, GA) at the ratio of 1:1.
Preserved human AMGs can be prepared as previously described, provided the availability of trained personnel, access to the ingredients, equipment necessary for processing the tissue, and storage equipment exist. Preserved amnion is also commercially available, either cryopreserved (Bio-Tissue, Miami, FL) or low-heat-dehydrated and sterilized (OKTO Ophtho, Costa Mesa, CA). Unlike low-heat-dehydrated amnion, cryopreserved amnion requires storage at −80°C if stored longer than 4 weeks. It can be stored at −20°C for as long as 4 weeks before use (Bio-Tissue, AmnioGraft Information Summary). The cryopreserved amnion is thawed before its use in the operating room. The low-heat-dehydrated amnion is stored at room temperature and is rehydrated with saline solution minutes before use. Unlike cryopreserved amnion, low-heat-dehydrated amnion is supplied without a carrier sheet.
SURGICAL TECHNIQUE
Corneal Epithelial Defect (with or without Stromal Ulcer)
After retrobulbar anesthesia, the base of epithelial defect or stromal ulcer is debrided with a microsponge and the surrounding poorly adherent epithelium is removed. The cryopreserved amniotic membrane is peeled from the nitrocellulose paper and placed on the surface of the defect in an overlay or inlay fashion.
Overlay Technique
In the overlay technique (Fig. 58-1), the amniotic membrane is placed epithelium side down to cover the entire corneal, limbal, and perilimbal surfaces. The graft is secured with interrupted 9-0 polyglactin sutures to the conjunctiva. A running 10-0 nylon suture can be added at the surgeon’s discretion to secure the AMG to the corneal stroma. It is preferable that the knots not be buried to prevent tears in the AMG.
Inlay Technique
The inlay technique (Fig. 58-2) can be used in the presence of stromal ulcer. One or multiple layers of the amniotic membrane, depending on the depth of the stromal defect, can be applied. In this case the amniotic membrane is placed epithelium side up onto the surface of stromal bed and secured by interrupted 10-0 nylon sutures to the edge
of the ulcer. The knots are then buried into the corneal stroma. The overlay technique can be added to cover the inlay AMG at the surgeon’s discretion (Fig. 58-3).
of the ulcer. The knots are then buried into the corneal stroma. The overlay technique can be added to cover the inlay AMG at the surgeon’s discretion (Fig. 58-3).
 FIGURE 58-1. Overlay amniotic membrane graft.(see color image) |
Fornix Reconstruction
After retrobulbar anesthesia, a 4-0 silk can be sutured to the tarsal plate of the eyelid to apply traction. The scarred conjunctiva is cut with scissors to release adhesions from the perilimbal area and then dissected from the underlying sclera towards the fornix. Excessive bleeding is controlled by Gelaspon (Chauvin Ankerpharm GmbH, Rudolstadt, Germany) or epinephrine applied subconjunctivally or topically. The amniotic membrane is removed from the nitrocellulose paper and placed epithelium side up onto the surface of bulbar and palpebral conjunctiva. The amniotic membrane is trimmed to fit the area of defect. The margin of the amniotic membrane is placed under the edge of conjunctiva and secured with 9-0 polyglactin sutures (Fig. 58-4). The amniotic membrane is then pulled and secured to the apex of the fornix with 9-0 polyglactin sutures. Alternatively, the amniotic membrane can be pulled and secured to the fornix through the eyelid with two or three double-armed 6-0 polyglactin using silicone bolsters or a symblepharon ring secured through the eyelid with 7-0 silk suture with bolsters.
 FIGURE 58-2. Inlay amniotic membrane graft. |
 FIGURE 58-3. Overlay and inlay amniotic membrane grafts. |
Conjunctival or Corneal Lesion/Pterygium Excision
After retrobulbar anesthesia, the lesion is dissected to bare sclera or corneal stroma. Application of mitomycin C at a concentration of 0.02% the surface for 3 minutes, followed by irrigation with balanced saline solution, may be used. The amniotic membrane is placed on the area of dissection with the epithelium side up and trimmed to the size of defect. The membrane is secured with 8-0 polyglactin suture to the sclera and with 10-0 nylon suture to the corneal stroma. After AMT, a limbal autologous transplant (3 × 5 mm) or autologous limbal stem cell transplant (5 × 5 mm), obtained from the upper bulbar conjunctiva or from the fellow eye, may be placed on the amniotic membrane and secured with 10-0 and 8-0 polyglactin sutures to the corneal stroma and sclera, respectively.
POSTOPERATIVE CARE
A hydrophilic bandage contact lens is placed on the ocular surface at the end of procedure. Alternatively, a central tarsorrhaphy can be performed in cases with poor fit of the bandage contact lens. Leaving the ocular surface without a bandage contact lens may lead to an early detachment of the amniotic membrane and lack of epithelialization (40). A combination
of topical antibiotics and corticosteroid drops is used over the period of 4 weeks after the surgery. The amniotic membrane typically dissolves within 3 to 6 weeks. The bandage contact lens is preferably kept in place until the AMG is completely dissolved. The use of topical antibiotics is discontinued after removal of the bandage contact lens, provided that the epithelium is healed. Topical corticosteroids are used until the inflammation subsides. The 10-0 nylon suture, if used, can be removed after complete dissolution of the AMG.
of topical antibiotics and corticosteroid drops is used over the period of 4 weeks after the surgery. The amniotic membrane typically dissolves within 3 to 6 weeks. The bandage contact lens is preferably kept in place until the AMG is completely dissolved. The use of topical antibiotics is discontinued after removal of the bandage contact lens, provided that the epithelium is healed. Topical corticosteroids are used until the inflammation subsides. The 10-0 nylon suture, if used, can be removed after complete dissolution of the AMG.
 FIGURE 58-4. Symblepharon in a patient with ocular cicatricial pemphigoid before (A) and 1 month after (B) conjunctival fornix reconstruction with amniotic membrane graft.(see color image) |
CLINICAL EXPERIENCE
Corneal Epithelial Defect (with or without Stromal Ulcer)
The amniotic membrane became yet another tool in treatment of persistent epithelial defects of the cornea associated with a spectrum of ocular and systemic conditions. The reports show that AMT is effective and safe in treatment of persistent epithelial defects of the cornea (19,20,38,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53) (Table 58-2). Although the efficacy of AMT has not been compared in controlled studies with other therapeutic interventions, amniotic membrane was shown to be able to promote healing of persistent epithelial defects in cases where other treatments, including topical lubricants, bandage contact lens, and tarsorrhaphy, failed. The success rate measured by the percentage of eyes with epithelialization of corneal defects after AMT ranges between 58% and 100%, according to the different reports that vary greatly in number of cases studied, spectrum of patient population, etiologic factors, size and severity of epithelial defects, stromal thinning, comorbid factors, and duration of follow-up period. Patients whose epithelial defect does not heal after the first AMT may benefit from repeated grafting (41). The success rate in treatment of persistent epithelial defects seems to be unrelated to the technique used (41). Multiple layers of amnion transplanted using the inlay technique may be superior to single-layer graft in case of deep stromal ulcers (1,20). The disadvantage of placing the amnion in an inlay fashion is a decrease in corneal transparency that may persist for many months. Once the amniotic membrane is reabsorbed, it is replaced by a new fibrotic stroma (54). The rate of recurrences of epithelial defects after successful AMT has been reported as between 0% and 29%. The success rate in cases with persistent epithelial defects associated with an underlying
systemic autoimmune disease is lower compared with those with persistent epithelial defects associated with a condition limited to the eye (41,43).
systemic autoimmune disease is lower compared with those with persistent epithelial defects associated with a condition limited to the eye (41,43).
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