Dehydrated Amniotic Membrane Use in Wound Management
Human amniotic membrane forms the lining of the fetal environment during gestation, separating the developing fetus from the mother in utero. The material used for surgical wound allografts is isolated from the membranous sac surrounding the infant to the point where it adjoins the placenta at the chorionic plate. On gross examination, the amniotic membrane is composed of a number of layers that can be seen and appreciated with simple handling and the naked eye. The material easily splits into an amnion layer and chorion layer, separated by a jelly-like, intermediate layer, apparent on separation of the 2 layers. The amnion or fetal side of the membrane is further coated with a layer of epithelial cells, which can be gently removed with simple cell scraping techniques to reveal a translucent underlying membrane. The amnion and chorion layers are in turn each composed of a basement membrane and stromal layer as outlined in Figure 1.
(Enlarge Image)
Figure 1.
Layers of the amniotic membrane. Used as a wound allograft, the entire membrane is used, placed with the chorion side down against the wound and the epithelial surface upright.
On microscopic examination, the overlying epithelial cells are clearly apparent, while the underlying architecture is defined by the various layers visible in an H/E stain (Figure 2). Overall, the amniotic membrane is composed principally of 3 types of material: structural collagen and extracellular matrix, biologically active cells, and a large number of important regenerative molecules.
(Enlarge Image)
Figure 2.
H/E Stain of human amniotic membrane tissue. Note the epithelial cell later visible through the presence of the epithelial cells and their nuclei. Amnion and chorion layers are characterized by a layered appearance.
Extracellular matrix materials form the structural components of the architecture of the membrane and contain a variety of specialized proteins including fibronectin, proteoglycans, glycosaminoglycans, laminins, and other similar materials. Collagens type IV, V, and VII create an important substrate, which is not only important for the structural integrity of the membrane, but also to create advanced wound healing and ingrowth cells. There is clear evidence that many of these molecules interact with one another in a highly complex milieu of bio-regulation that requires the presence of membranes, individual growth factors, and interactions that up-regulate and down-regulate the various regenerative processes of healing. Metalloproteinases for example, are counterbalanced by the tissue inhibitors of these molecules (TIMPS); growth factors, such as fibroblast growth factor, may need the presence of the extracellular matrix components for some functions, and so forth.
Cellular material includes the epithelial lining of the amnion facing the infant, but also pluripotential stem cells important in regenerating new cellular materials within the membrane lining. Epithelial stem cells, in particular, have also been isolated from the epithelial layer of the amniotic membranes. Fibroblasts are also present and provide lining and strengthening of tissues. The epithelial cells are also biologically active in the healing process through various receptors on the cell surface. The role of hematogenous, mesenchymal, and closely located stem cells is also affected by the interactions among various components of the membrane.
Finally, regenerative biomolecules important in the healing and growth process are concentrated in the amniotic membrane. These include epidermal growth factor, transforming growth factor (TGF) beta, fibroblast growth factors, platelet-derived growth factors, metalloproteinases, and TIMPS. Interestingly, there is a lack of HLA-A, -B, and -C antigens, and beta 2-microglobulin. This, combined with the presence of immunosuppressive cytokines interleukin-4, interleukin-10, and TGF, results in a relatively "immunologically privileged" material that does not present itself as foreign material to either the mother or the infant, and which is likely responsible for its observed lack of rejection in patients treated externally with amniotic membrane.
The amniotic membrane is also home to a variety of unique molecules with specific functions. Defensins, for example, are a group of molecules that assist in conferring antibacterial properties to the material and are known to increase near the time of delivery. Similarly, both matrix metalloproteinases, which are significant in the role of membrane development, are balanced by TIMPS, which simultaneously confer on the material the ability to both heal and control tissue breakdown.
Stains of the amniotic membrane in both natural and dehydrated human amniotic membrane (dHAM) clearly show the presence of these various growth factors and cytokines, which have been well-documented in the literature. Similarly, a large number of cytokines have actually been measured in both natural human amniotic membrane and dHAM in various concentrations, presumably accounting for their biological properties. The presence of these molecules can be confirmed through immunohistochemical staining, which shows various amounts of the materials at different levels in the membrane (See Figure 3).
(Enlarge Image)
Figure 3.
Immunohistochemical staining for some representative important biomolecules. (Stain is brown.) Note that the amount and general distribution of the growth factors are different among the various types.
Amniotic Membrane Structure and Function
Human amniotic membrane forms the lining of the fetal environment during gestation, separating the developing fetus from the mother in utero. The material used for surgical wound allografts is isolated from the membranous sac surrounding the infant to the point where it adjoins the placenta at the chorionic plate. On gross examination, the amniotic membrane is composed of a number of layers that can be seen and appreciated with simple handling and the naked eye. The material easily splits into an amnion layer and chorion layer, separated by a jelly-like, intermediate layer, apparent on separation of the 2 layers. The amnion or fetal side of the membrane is further coated with a layer of epithelial cells, which can be gently removed with simple cell scraping techniques to reveal a translucent underlying membrane. The amnion and chorion layers are in turn each composed of a basement membrane and stromal layer as outlined in Figure 1.
(Enlarge Image)
Figure 1.
Layers of the amniotic membrane. Used as a wound allograft, the entire membrane is used, placed with the chorion side down against the wound and the epithelial surface upright.
On microscopic examination, the overlying epithelial cells are clearly apparent, while the underlying architecture is defined by the various layers visible in an H/E stain (Figure 2). Overall, the amniotic membrane is composed principally of 3 types of material: structural collagen and extracellular matrix, biologically active cells, and a large number of important regenerative molecules.
(Enlarge Image)
Figure 2.
H/E Stain of human amniotic membrane tissue. Note the epithelial cell later visible through the presence of the epithelial cells and their nuclei. Amnion and chorion layers are characterized by a layered appearance.
Extracellular matrix materials form the structural components of the architecture of the membrane and contain a variety of specialized proteins including fibronectin, proteoglycans, glycosaminoglycans, laminins, and other similar materials. Collagens type IV, V, and VII create an important substrate, which is not only important for the structural integrity of the membrane, but also to create advanced wound healing and ingrowth cells. There is clear evidence that many of these molecules interact with one another in a highly complex milieu of bio-regulation that requires the presence of membranes, individual growth factors, and interactions that up-regulate and down-regulate the various regenerative processes of healing. Metalloproteinases for example, are counterbalanced by the tissue inhibitors of these molecules (TIMPS); growth factors, such as fibroblast growth factor, may need the presence of the extracellular matrix components for some functions, and so forth.
Cellular material includes the epithelial lining of the amnion facing the infant, but also pluripotential stem cells important in regenerating new cellular materials within the membrane lining. Epithelial stem cells, in particular, have also been isolated from the epithelial layer of the amniotic membranes. Fibroblasts are also present and provide lining and strengthening of tissues. The epithelial cells are also biologically active in the healing process through various receptors on the cell surface. The role of hematogenous, mesenchymal, and closely located stem cells is also affected by the interactions among various components of the membrane.
Finally, regenerative biomolecules important in the healing and growth process are concentrated in the amniotic membrane. These include epidermal growth factor, transforming growth factor (TGF) beta, fibroblast growth factors, platelet-derived growth factors, metalloproteinases, and TIMPS. Interestingly, there is a lack of HLA-A, -B, and -C antigens, and beta 2-microglobulin. This, combined with the presence of immunosuppressive cytokines interleukin-4, interleukin-10, and TGF, results in a relatively "immunologically privileged" material that does not present itself as foreign material to either the mother or the infant, and which is likely responsible for its observed lack of rejection in patients treated externally with amniotic membrane.
The amniotic membrane is also home to a variety of unique molecules with specific functions. Defensins, for example, are a group of molecules that assist in conferring antibacterial properties to the material and are known to increase near the time of delivery. Similarly, both matrix metalloproteinases, which are significant in the role of membrane development, are balanced by TIMPS, which simultaneously confer on the material the ability to both heal and control tissue breakdown.
Stains of the amniotic membrane in both natural and dehydrated human amniotic membrane (dHAM) clearly show the presence of these various growth factors and cytokines, which have been well-documented in the literature. Similarly, a large number of cytokines have actually been measured in both natural human amniotic membrane and dHAM in various concentrations, presumably accounting for their biological properties. The presence of these molecules can be confirmed through immunohistochemical staining, which shows various amounts of the materials at different levels in the membrane (See Figure 3).
(Enlarge Image)
Figure 3.
Immunohistochemical staining for some representative important biomolecules. (Stain is brown.) Note that the amount and general distribution of the growth factors are different among the various types.
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