Akanksha Gupta,1 Suresh D. Kedige,1 and Kanu Jain2
1Department of Periodontics, Maharishi Markandeshwar College of Dental Sciences and Research, Mullana, Ambala,
Haryana 133207, India
2Department of Oral Pathology, Jaipur Dental College, Jaipur, Rajasthan 303805, India
Correspondence should be addressed to Kanu Jain; kanu firstname.lastname@example.org
Received 10 September 2015; Accepted 8 November 2015
Academic Editor: Sean Peel
Copyright © 2015 Akanksha Gupta et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
The periodontal therapy usually aims at elimination of disease causing bacteria and resolution of inflammation. It involves either
resective or regenerative surgery to resolve the inflammation associated defects. Over the years, several methods have been used
for achievement of periodontal regeneration. One of the oldest biomaterials used for scaffolds is the fetal membrane. The amniotic
membranes of developing embryo, that is, amnion (innermost lining) and chorion (a layer next to it), have the properties with
significant potential uses in dentistry. This paper reviews the properties, mechanism of action, and various applications of these
placental membranes in general and specifically in Periodontics.
The ultimate goal of periodontal treatment using regenerative techniques is to restore the supporting tissues lost as sequelae of inflammatory periodontal disease . Different methods have been used in the past for the achievement of this goal. These procedures include root planing, softtissue curettage, and flap surgeries.The latter procedures have often been used along with grafting of bone, guided-tissue regeneration, incorporation of biomaterials like derivatives and substitutes of bone, and biologic factors like enamel matrix proteins . One of the new materials which has
also been tried recently includes placental membranes. The placental allografts possess antibacterial and antimicrobial properties being tissues with immune privilege and are thus quite different from cadaveric allograft, xenograft, and alloplast barrier membranes used in periodontal therapy . They reduce inflammation and provide a matrix highly rich in protein and thereby facilitate migration of cells at the area of defect . Applications of amnion membrane include chemical or thermal burns, correction of corneal epithelial defects, neurotrophic corneal ulcers, leaking blebs after glaucoma surgery, reconstruction of conjunctival and ocular surfaces, ocular cicatricial pemphigoid or Stevens-Johnson syndrome, and bullous keratopathy . In Periodontics, these membranes have also been used in furcation defects, intrabony defects, and gingival recession coverage (Figure 1). In this literature review, the various properties of the placental membranes are discussed in light of their potential uses in the field of Periodontics.
2. Historical Background
Human amniotic membranes have been used successfully for a wide range of applications for over 70 years. The use of fetal membrane in skin transplantation was first reported by Davis in 1910 . Description of the use of human amniotic membrane for burned and ulcerated skin surfaces was given by Stern in 1913 . They evaluated the accelerative effect of the membrane on epithelialization and the reduction in pain by its application on burned or ulcerated sites . In 1940, De R¨oth  first reported use of fetal membranes in the ocular surface. He used fresh amnion and chorion as a biological dressing material for management of conjunctival defects. Kim and Tseng gave the preservation method for maximal maintenance of biologic properties of membranes which is still the best way . The amniotic membrane has gained importance specifically because of various factors. Firstly, it reduces scarring and inflammation and enhances wound healing. Secondly, it serves as a scaffold for proliferation and differentiation of cells owing to its antimicrobial properties. Thirdly, its extracellular matrix and its components, such as growth factors, suggest it to be an excellent biomaterial to act as a native scaffold for tissue engineering. Lastly, it can be easily procured, processed, and transported.
3. Anatomy and Histology
Placental membranes have their origin from extraembryonic tissue. This tissue is composed of a fetal component (the chorionic plate) and a maternal component (the deciduas).
The fetal component includes the amnion and chorion membranes which separate the fetus from the endometrium. The structure of amniotic membrane has three parts which are epithelial monolayer, a thick basement membrane, and an avascular stroma.
Epithelial monolayer consists of a single layer of cells which are arranged uniformly on the basement membrane. It is the innermost layer, lies nearest to the fetus, and is also called the amniotic epithelium. The amniotic epithelial cells have an active secretory and transport functions as suggested by their ultrastructure . This epitheliumis firmly fixed to a basement membrane which is in turn attached to a condensed acellular layer.This layer is composed of collagen types I, II, and V . Blood vessels or nerves are absent in amniotic membrane. It derives its nutrition directly by diffusion out of the amniotic fluid. The basement membrane is quite remarkable as it is one of the thickest membranes found in all human tissues and provides support to the fetus throughout gestation . It is similar to that of conjunctiva in its distribution of collagen type IV subchains.
The main fibrous skeleton of amniotic membrane is formed by the compact layer of stroma lying adjacent to basement membrane. Next layer that is fibroblastic layer containing mesenchymal cells is responsible for secretion of different types of collagens. Predominant types are interstitial collagens (types I and III) which formparallel bundles to maintain the mechanical integrity of the membrane. Filamentous connections between interstitial collagens and epithelial basement membrane are provided by collagens types V and VI. The last layer which is known as intermediate layer or spongy layer or zona spongiosa lies adjacent to the chorionic membrane and contains a meshwork of mostly type III collagen . Its spongy appearance is the result
of presence of abundant content of proteoglycans and glycoproteins. Chorion is composed of reticular layer, basement membrane, and trophoblasts (Figure 2) (Table 1).
4. Principle of Therapeutic Tissue
The preparation of placental extract was described by Russian ophthalmologist, Professor VP Filatov. Though its use was popular in Europe and parts of Asia, primarily China, Korea, and Japan, for over a century, there was no documentation on its therapeutic efficacy prior to Filatov’s research. An increased surge in research on human placental extract took place after his description. Filatov started research on grafting human corneas by using the principle of transplantation of preserved material. He observed that animal or vegetable tis- sues undergo a biochemical readjustment after their isolation from the organism and subjection to environmental factors that inhibit their vital processes. As a result, the tissues start developing substances to stimulate their vital processes. These substances were termed as biogenic stimulators by Filatov .
After many experiments of Filatov, he was convinced that any human or animal tissue which may not necessarily correspond histologically to pathological tissue could be used to obtain curative effect. He extended this concept to general medicine which later gave birth to principle of therapeutic tissue. He confirmed the process to be just as valid for other human tissues .
5. Properties of Membranes (Table 2)
5.1. Biomechanical Properties. Thickness of normal amniotic membrane lies between 0.02 and 0.5 millimetres which includes around 6–8 layers of cells. An average surface area
of this membrane is about 1600 square centimetres . An important property of amniotic membrane is its resistance to various proteolytic factors owing to the presence of interstitial collagens . Elastin present in amnion is responsible for providing elasticity. It has multiple metabolic functions such as its role in water and soluble material transportation and production of bioactive peptides, growth factors, and cytokines .
5.2. Promotion of Epithelialization. Amniotic membrane facilitates migration of epithelial cells , reinforces basal cell adhesion , promotes epithelial differentiation , prevents epithelial apoptosis , and promotes epithelial- ization in healing of wounds . Various growth factors produced by amniotic membrane can stimulate epithelializa- tion . It can also promote expansion and maintenance of epithelial progenitor cells in vivo  and can produce endothelin-1 and parathyroid hormone related protein. Brain natriuretic peptide and corticotrophin releasing hormone are also produced by membrane epithelial cells which play roles in increasing cellular proliferation and calcium metabolism . Expression of mRNA for epidermal growth factor, hepatocyte growth factor receptor, and keratocyte growth factor receptor was demonstrated by Koizumi et al. in 2000 in cryopreserved amniotic membrane . Its basement membrane serves as a safe and suitable bed for the growth of epithelial cells. Sufficient oxygenation for epithelial cells is provided by its good permeability in contrast to other synthetic materials. Thus, amniotic membrane is an ideal tissue which facilitates the growth of epithelial cells, helping in their migration and differentiation [26, 27].
5.3. Inhibition of Fibrosis. The amniotic membrane possesses antifibrosis property. Fibroblasts are naturally responsible for scar formation during wound healing and are activated by transforming growth factor 𝛽.
Amniotic membrane reduces the risk of fibrosis by down- regulation of transforming growth factor 𝛽 and its receptor expression by fibroblasts. Therefore, scaffold of an amniotic membrane modulates wound healing by promoting recon- struction of tissues rather than promoting formation of scar tissue [28, 29].
5.4. Inhibition of Inflammation and Angiogenesis. The exact mechanism of the anti-inflammatory properties of amniotic membrane is not clear. It is hypothesized that it decreases influx of inflammatory cells to the wound area and con- sequently reduces inflammatory mediators by serving as a barrier. It entraps T lymphocytes when it is applied as a patch in vivo . Matrix metalloproteinases released by infiltrating neutrophils and macrophages are taken care of by inhibitors of matrix metalloproteinases found in the amniotic membrane. Presence of various tissue inhibitors of metallo- proteinases 1, 2, 3, and 4, interleukin-10, and interleukin-1 receptor antagonists and endostatin which inhibit endothelial cell proliferation, angiogenesis, and tumor growth has also been observed in amniotic membrane . The presence of proteinase inhibitors may facilitate wound healing . Thrombospondin-1, secreted by the amniotic epithelium, acts an antiangiogenic factor. Two very potent proinflammatory mediators, interleukin-1𝛼 and interleukin-1𝛽, are suppressed by matrix of stroma of amniotic membrane . Shimmura et al. in 2001 reported that amniotic membrane reduces inflam- mation through entrapment of inflammatory cells .
A high molecular-weight glycosaminoglycan, hyaluronic acid, present in large quantities in amniotic membrane acts as a ligand for CD44 which is expressed on inflammatory cells. It plays an important role in adhesion of inflammatory cells including lymphocytes, to the amniotic membrane stroma .
Other substances expressed in the amniotic membrane are low-molecular-mass elastase inhibitors which include secretory leukocyte proteinase inhibitor and elafin [35, 36]. These inhibitors have antimicrobial actions in addition to their anti-inflammatory properties . They protect related surfaces from infection, thereby acting as components of the innate immune system . Both antimicrobial and anti- inflammatory properties can also be induced into amniotic membranes by their treatment with both lactoferrin and interleukin-1 receptor . Lactoferrin, a globular multifunc- tional protein, has both antimicrobial and anti-inflammatory qualities. It serves as an antioxidant and an iron chelator in tissues  and suppresses the production of interleukin-6 in the amniotic fluid during amniotic infection. Interleukin-1 receptor antagonist on the contrary reduces inflammation as it is a potent inhibitor of interleukin-1 which is a mediator of inflammation .
5.5. Lack of Immunogenicity. Occurrence of acute rejection after transplantation of amniotic membranes is negated by the fact that amniotic epithelial cells do not express HLA-A, HLA-B, HLA-D, and HLA-DR antigens but express HLA- G on their surfaces . Presence of interferon 𝛾 and other immunologic factors has also been observed in the amniotic membrane. It seems that amniotic membrane may induce immunologic reactions in the presence of viable epithe- lial cells. One study revealed that transplantation of fresh amniotic membrane is associated with a mild inflammatory response. This could be probably due to expression of HLA- I antigens by viable epithelial cells . However, immuno- genicity of cryopreserved amniotic membrane is less than that of fresh amniotic membrane as epithelial cells are lost in cryopreservation . T lymphocytes in allografted limbus cells are suppressed by amniotic membrane. This implies immunosuppressive properties of amniotic membrane which can increase the chances of successful grafting . As tissue grafts of placental membrane materials present a low risk of immune rejection, they are considered to be bestowed with “immune privilege” [45, 46].
5.6. Antimicrobial and Antiviral Properties. The risk of infec- tion is reduced by amniotic membrane due to its antimi- crobial and antiviral properties . Microorganisms upon their entry into the body are eliminated by our immune system through an adaptive immune response, 𝛽-defensins, a major group of antimicrobial peptides and an integral part of the innate immune system, which are expressed at surfaces of mucosa by epithelial cells and leukocytes [48, 49]. Amniotic membranes also have the ability to produce 𝛽- defensins  with the predominant type present in amniotic epithelium being 𝛽3-defensin . Kjaergaard et al. in 2001 have also shown in vitro antimicrobial effects of the amnion and chorion against certain microorganisms. Its antiviral properties are exhibited by presence of cystatin E, the ana- logue of cysteine proteinase inhibitor . There is still fur- ther need for studies to verify these properties of the amniotic membrane . Amniotic membrane may prevent infiltration and adhesion of microorganisms to wound surfaces by acting as a barrier. The hemostatic property of collagen fibers of amniotic basement membrane prevents hematoma forma- tion in clean surgical wounds. This reduces bacterial load and risk of infection by preventing accumulation of microbes. Another mechanism of action against infection by mem- branes is through their adhesion to the wound surface. This attachment prevents formation of dead space and accumula- tion of serous discharge. Furthermore, bacterial entrapment and stimulation of migration of phagocytes also occur by fib- rin filaments formed during wound healing. These filaments cause adhesion of the wound bed to amniotic membrane col- lagens. There is a report that bacterial proliferation is reduced even in contaminated wounds by amniotic membrane .
5.7. Cell Differentiation Property. The fetal placental tissues have the potential to transform into different cell lineages. The hematopoietic lineage is found in the chorion, allantois, and yolk sac; and the mesenchymal lineage is found in both the chorion and amnion. The cells isolated from the chorion are good sources of cells of hematopoietic and mesenchymal lineages as they possess these properties. It is considered that the amniotic membrane can maintain pluripotent stem cell potential for cell differentiation.
Amniotic membrane can be used for transplantation either as a temporary graft or as a permanent graft. It can be used alone or in conjunction with other surgical procedures when employed as permanent graft. In temporary grafting procedure, it is sutured to both healthy host tissue and site of interest at the same time as a bandage or dressing, or patch. This is so done to promote healing of host epithelial lining lying underneath. The membrane is invariably dissolved once epithelialization is complete. The removal is carried out in a period varying from 2 to 6 weeks. When used for permanent grafting, for example, in cornea or conjunctiva, it is sutured to fill in the tissue defect so that host cells proliferate into it and a sound integration with the host tissue is achieved.
7. Applications of Placental Membranes Based on Their Properties
- The physical properties of amniotic membrane have proven it to be compatible with corneal surface of the eye. The eye and placental membranes have so much similarity in their immune modulatory properties that they have been referred to as “parallel universes” .
- The use of human amniotic membrane as a surgical wound dressing in treatment of leg ulcers, skin loss in Stevens-Johnsons diseases, reconstruction of the pelvic floor, vaginal epithelialization, replacement of normal mucosa in Rendu-Osler-Weber diseases, and ear surgery has been described . Amniotic mem- brane acts as a biologic dressing resulting in signif- icant pain relief in burns owing to adhesion to the wound surface and coverage of dermal nerve endings. It also prevents wound surface drying, which acceler- ates wound healing, and thus can be used to manage wounds in the oral cavity like that of tongue, buccal mucosa, vestibule, palatal mucosa, and floor of the mouth [55, 56].
- Amniotic membrane is considered as an important potential source for scaffolding material that must easily integrate with host tissue and provide an excel- lent environment for cell growth and The extracellular matrix components of the basement membrane have a great role in cell adhesion during the cell seeding protocol.
- Human amniotic membrane is also used as a potential dressing that accomplishes four major goals:
- Reduction of water loss through evaporation, by acting as water barrier, and providing a moist environment for cell survival and
- Acting as barrier to microbial colonization and
- Reduction in
- Talmi et in 1990 have reported the use of human amnion for overlying epithelial defects after flap necrosis following surgery in the head and neck region with good results .
- Florian et al. in 2013 tried amniotic membrane as an interpositional material for the reconstruction of TMJ ankylosis as it has antifibrotic properties and thus can prevent reankylosis .
- Owing to its antimicrobial properties, amniotic mem- brane can be even used as a carrier for local delivery of the various drugs .