WO2009044408A1

WO2009044408A1 - A treated amniotic membrane and method of treating amniotic membrane

Info

Publication number: WO2009044408A1
Application number: PCT/IN2008/000405
Authority: WO
Inventors: Soma Guhathakurta; Mammen Cherian Kotturathu
Original assignee: International Centre For Cardio Thoracic And Vascular Diseases (A Unit Of Frontier Lifeline Pvt. Ltd); Department Of Biotechnology (A Department Of Science And Technology, Government Of India)
Priority date: 2007-10-03
Filing date: 2008-06-25
Publication date: 2009-04-09

Abstract

The invention relates to a method of treating amniotic membrane and the use of treated amniotic membrane as dressing, as graft and for transplantation. It provides the method and compositions to actively control the whole process of skin regeneration. The uniqueness of the invention lies in the decellularisation and cross linking of the membrane prior to the use, which makes the membrane adaptable for adhering to the wound area and thereafter enhancing the growth of the neighbouring tissue which further allows the foreign membrane placed on the wound area to merge with the neighbouring skin.

Description

"A TREATED AMNIOTIC MEMBRANE AND METHOD OF TREATING AMNIOTIC MEMBRANE"

FIELD OF INVENTION:

Many have used a fresh fetal membrane (ie both amnion and chorion) as a graft for conjunctival surface reconstruction with limited success. There are cases of the successful use of amniotic membrane as a patch graft in the treatment of acute ocular burns.

PRIOR ART:

The use of various biological dressings to cover burn wounds goes back to ' centuries. A variety of techniques have been attempted in order to reduce wound sepsis, and variable results have been reported. Initially heterografts from different animal sources ranging from lizard to porcine skins were used.

Biological coverages should have the following characteristic.

It shall be such as to be adherent to (and grow well on) the bum surface. It shall maintain low bacterial growth or prevent subsequent bacterial contamination of the injured surface. It shall reduce the loss of fluids, microelements and proteins from the injured surface. It shall have good fluid or gas permeability from the surrounding tissue to the wound surface. It shall be easy to handle, i.e. when placing on or removing from the injured surface, and also fit closely to incised anatomical regions. - It shall relieve pain, and promote care of the injury. It shall decrease the possibility of scar or keloid formation during the hpaling process. It shall be available in sufficient quantity and should be affordable.

However the Biological dressings have the following disadvantages also - They exhibit an early graft rejection and a rapid, acute bacterial colonization of the injured area.

There are different types of Biological skin dressings known in prior art.

1 Allograft

2. Xenograft: bovine, ovine, canine, porcine; cheap and readily available; used fresh, frozen or lyophilized.

3. Cultured allograft: good results, but quite an expensive method .

4. Collagen products: gel, sponges or other different plaques.

5. Amniotic membrane

The nature of above biological skin dressings is different from each other

Allograft and xenografts possess numerous positive characteristics, but their use is sometimes limited, especially where tissue banks have not been developed.

Amniotic membrane has been used with variable success as a material for burn injury coverage.

The amnion is a unique membrane, composed of a single layer of cells, which completely line the cavity in which the foetus grows and develops. It is formed from the same small group of cells, which are formed when the ovum fertilised by the sperm begins to divide. These cells have the potential to develop into all the cells of the human body and under the action of growth factors, and by a process which is as yet undefined, the cells will differentiate into either muscles, bones, heart, liver or whatever structure is required. We now have reason to believe that the cells which go to form the amnion retain this pluripotential characteristic of being able to differentiate, at least in part, into other tissues.

Amniotic membrane can be used for superficial bums, deep burns, after necrectomy, (excision of the necrotic tissue) on extensive granulating wound surfaces, on autografts, in donor regions, and after dermoabrasion. Amniotic ■ membrane is readily available and does not present immunological problems. It does not cause allergic responses and reduces water loss. The risk of the transmission of some viral infections is there. Bacterial examinations performed with burn wounds covered with amniotic membrane showed low or no bacterial colonization of the burn surface. It is concluded that amniotic membrane should be more widely used in this particular aspect of burn treatment.

With the healthcare taking priority amongst public, recently there have been great developments in wound care, with allografts, amniotic membranes, artificial dermis, and synthetic and semi-synthetic dressing materials playing an important role. In superficial burns, amniotic membrane adheres after application and remains so until ^■ epithelialization is complete. In deep and deep dermal burns, the burned tissue needs to be removed by total burn excision or tangential excision, and the wound is covered with an autograft. At the same time, in burn patients who are not suitable for early excision, the raw surface is covered with an allograft, amniotic membrane, or other dressing material in order to prevent external contamination, with loss of fluid and electrolytes. At a later stage the allograft and the amniotic membrane need to be replaced by an autograft. Human foetal membrane was first used as a biological skin substitute or dressing about 90 years ago.1 FMs are also known as amniotic membrane.2 Anatomically, FMs consist of two loosely connected layers, an inner one of amnion and an outer one of chorion. The inner amnion layer is composed of cuboidal, flattened epithelial cells and mesenchymal connective tissue. The outer chorion is composed of fairly thick transitional epithelium. Amnion is thin and shiny in contrast to chorion, which is less homogeneous and dull.3 Foetal membrane can be used into to (amnion + chorion) or only as amnion (epithelium + base membrane).4 Amniotic membrane is also comparable with honey, for its good healing effect.

Another unique feature of the amniotic membrane is the complete lack of expression of surface antigens responsible for mounting an immune reaction. Thus, the amnion does not induce an immune response when transplanted into a "foreign" site, a feature which is of major importance to the foetus. Some groups found immunogenicity may be in lower form in homologous amniotic membrane.

There are favourable reasons to use Amniotic membrane

- It is readily available in sufficient quantity.

- The application is not associated with immunological problems.

- It is available in large size.

- It is simple to prepare and sterilize.

- It exhibits no allergic reactions in some cases.

- It eliminates up to 15% reduction of water losses in wounds.

- It exhibits histological structure similar to that of skin. -low much ever, be the advantages, simultaneously there exists disadvantage of the use of amniotic membrane is that there is some risk of viral infection transmission, e.g. hepatitis, syphilis and AIDS.

There are two varieties of amniotic membrane are mainly used in the clinical procedures.

1. The combination (amnion + chorion) on deep burns.

2. Or the amnion alone (epithelium + basic membrane) on superficial burns.

Amniotic membrane have been use by surgeons, during preparation of deep burns for necrectomy(excision of necrotic tissue), to keep necrotic areas dry or after necrectomies leaving raw areas of flesh - frequently in combination with other temporary biological skin coverages and/or autografts.

Survival of burn patient is largely dependent upon prompt and efficient wound healing. This not only limits wound contracture but also reduces the incidence of wound infection. So it is very popular to use amniotic membrane as a temporary coverage for burn wounds.

Human amniotic membrane is derived from the fetal membranes which consist of the inner amniotic membrane made of single layer of amnion cells fixed to collagen-rich mesenchyme 6 to 8 cells thick loosely attached to chorion. It is composed of three layers: a single epithelial layer, thick basement membrane, and avascular stroma. Human amniotic membrane has been shown to contain collagen types III and V. It also contains collagen types IV and VIl similar to corneal epithelial basement membrane as well as fibronectin and laminin. Additionally, it contains fibroblast and other growth factors.

Generally human amniotic membrane is believed to be nonimmunogenic. Antibodies or cell-mediated immune response to amniotic membrane have not been demonstrated by few groups, suggesting low antigenicity. Therefore, the use ofsystemic immunosuppressives in AMT is not required.

Foetal membranes have been put to a variety of surgical membranes for skin coverage in the past. Amnion has been used service early nineteenth century for treating burns and skin ulcers and reported relief of pain, increased rate of epithelialisation and no infection. There have been several use of amnion as a temporary dressing for ulcers, burns and other denuded areas. Stored and desiccated membranes have not been found to offer any significant improvements over other more conventional sterile dressings. But when applied fresh or following preservation at 4°centigrade in isotonic saline, the amnion appears to have advantages which have been reported to include reduction in bacterial counts, relief of pain and hastened healing. The amnion has been postulated as producing factors which promote granulation tissue formation, neovascularisation and re- epithelialisation although this is a controversial area.

The basic properties of amniotic membrane can be understood from the basic properties of human foetal membranes.

There is an absence of immunological rejection and the healing effects of FMs may be due to: a. Antibacterial factors b. Biological factors c. The biomechanical characteristics of FMs

From the discussion above, it will be appreciated that amniotic cells were known to be pluripotential in their natural state i.e. during formation of the foetus. It was also known that human allografts of amniotic cells generated no significant immunological problems though substantially it is substituted.

In addition to its physical properties in reducing water and heat loss, the mechanism responsible for the rapid healing observed is due to the inhibition of the proteinase activity, thus reducing the inflammatory responses by reducing the infiltration of polymorphonuclear leukocytes.

Human amniotic epithelial cells do not express on their surfaces HLA-A, B, C, and DR antigens, or beta 2- microglobulin, which could further contribute to the lower inflammatory responses and relatively delayed rejection of this type of biological dressing.

Generally conventionally dressing method of burn areas were cleansed with diluted povidone iodine in normal saline solution and a thick layer of nitrofurazone ointment was applied and covered with a cotton bandage. Inspection of the wounds for any possible infection was made on daily basis for up to 10 days. In cases where infection was suspected, the patients were hospitalized for debridement of the wounds and treated with appropriate systemic and local antibiotics. The amniotic membrane, which surrounds the fetus in the womb, is a unique source of 'biological dressing' that has been used in wound healing for decades. According to the researchers, these membranes appear to contain large amounts of growth factors which encourage tissue regeneration, as well as anti-bacterial and anti- scarring properties. They are especially useful as tissue grafts because they do not encourage autoimmune rejection as happens in organ transplant.

The amniotic membrane-treatment method means the patient will be similarly treated as in above described conventional method by cleansing of the bum wounds with diluted povidone iodine in normal saline and covered with a layer of the amniotic membrane with the amnion side down over the whole of the affected areas. The wounds were similarly inspected.

All these dressings had one major drawback of rejection of the graft, and only served as a temporary dressing. Therefore, they needed to be replaced regularly, and ultimately a permanent autograft, i.e. the patients own skin, was usually needed for deep burns. But before this is achievable, the wounds need to be primed with a neovascularized tissue for such purpose until the granulation tissues are ready for accepting the autograft.

Then attempts were made to go beyond merely dressing the wound to using the same for transplant. The first reported use of fetal membranes in skin transplantation was by Davis. The use of amniotic membrane on burned skin and ulcerated skin surfaces was done. There was a lack of infection, marked decrease in pain, and increased rate of re-epithelialization of traumatized skin surface. An ideal graft for use in skin surface reconstruction would not only, to a maximum, promote healing while minimizing scarring, but would also yield cosmetically acceptable results and be relatively easy to perform. Human amniotic membrane clearly has shown promise along these lines. It can be used for surgical reconstruction of the skin surfaces involving transplantation of amniotic membrane.

Human amniotic membrane is one of the most effective biological dressings that is used in burn treatment. Using human amniotic membrane incorporating silver nitrate gives a better therapeutic effect than plain amniotic membranes. Silver nitrate incorporated into the membranes increases their maneuverability, provides easier application to the burned area and creates a bactericidal effect, therefore redudnq the risk of contamination and infection. But re-epithelialization is hindered. One of the main advantages of wound coverage with only amniotic membrane is that it does not appear to discourage re-epithelization, reduces fluid, protein, heat and energy loss, increases mobility and most important this may be the ideal wound cover next to the patient's own skin. Therefore, it is highly recommended for the use of silver nitrate- , incorporated amniotic membrane, since it is readily available and freely obtainable, has low preparation and storage costs that make it an ideal dressing to use, especially in countries where economic factors prevent the purchase of other types of dressings. However the silver nitrate is found to be toxic in nature and thereby is not enhance the growth eventhough it may be useful when it is used as part of dressing only. It is not useful when amniotic membrane is used for implantation.

The main obstacle encountered in organ transplantation is the immune rejection of the transplant. The rejection phenomenon originates from antigenic differences between the cells of the recipient and the transplant, as well as from the natural immune response of the organism towards "non-self antigens. Attempts to prolong the survival of allografts and xenografts, both in experimental models and in medical practice, have been mainly aimed at the supression of the immune apparatus of the recipient. This has been achieved by means of cytotoxic drugs, antimetabolites, corticosteroids and antilymphocytic serum. The generalized immunosuppression, however, is accompanied by undesirable toxic effects, decreased resistance to infection and reduction in the level of the haemopoietic stem cells. Another possibility is to attenuate, or to abolish completely, the antigenicity of the graft with preservation of its biological functions. The advantage of this approach is that the immune capacity of the recipient is not affected. Investigations along this line, have been, for the most part, unsuccessful. Most studies were performed with animals and have been subjected to clinical evaluation. Using laboratory animals, the treatment of allografts or xenografts in vitro, with cortisone, thalidomide or urethane prolonged their retention time by a factor of about two. The amount of drug locally applied to the skin was smaller than the amount required to achieve a similar effect by injecting the drug systemically. In attempts to modify the antigenic properties of grafts, the donor skin has been treated in vitro with streptokinase/streptodornase, or with RNA and DNA preparations of the recipient. Allograft survival was not prolonged by exposure of donor skin to transplantation antigens of the recipient. Minimal immune reaction was observed towards grafts in which cellular viability was destroyed in vitro treatment with formalin or cyanide or by freeze-drying. The majority of the dead grafts were retained by the host for a limited period of time. Amniotic membrane has been used to replace damaged tissue or as a dressing on top of existing tissue. It is attempted to use Grafts made of amniotic membrane — one of the protective layers surrounding the fetus — can be used to stimulate the regrowth of damaged skin.

It was followed by the use of amniotic membrane transplantation (AMT) in ophthalmology in the treatment of conjunctival epithelial defects after symblepharon (scarring and adhesions between palpebral and bulbar conjunctiva). Later patients with caustic burns of the conjunctiva with corneal involvement were also treated successfully using amniotic membrane.

The exact mechanisms by which amniotic membrane delivers its beneficial effects on the ocular surface is not fully understood or known. Amniotic membrane modulates levels of cytokines and growth factors and has also been shown to have unique properties, including pain reducing, fibrosis suppressing, antibacterial, and wound protecting. It is unclear whether amniotic membrane promotes limbal stem cell proliferation.

Amniotic membrane transplant was also successful in promoting the growth of corneal tissue in a patient whose scarring had caused the eyelid to adhere to the eye. The 'antiadhesive' compounds in amniotic membrane tissue appear to help prevent this type of scarring.

BRIEF DESCRIPTION OF INVENTION:

The invention in general relates to the use of amniotic membrane as dressing, as graft and for transplantation. The Uniqueness of the invention lies in the decellularisation and cross linking of the membrane prior to the use, which makes the membrane adaptable for adhering to the wound area and thereafter enhancing the growth of the neighbouring tissue which further allows the foreign membrane placed on the wound area to merge with the neighbouring skin.

The method begins with harvesting the membrane from caesarean section and mother is screened for infectious diseases before going for surgery. A membrane piece is sent for RT- PCR studies for retrovirus identification. It is collected in balance salt solution with antifungal medication to prevent fungal contamination.

In GMP laboratory under class 100 laminar air flow hood AM is dissected free of chorionic membrane and goes to antibiotic cocktail solution

50gms of tissue is treated with 100ml of the solution of 1 % Deoxy cholic acid for 20- 50 hours alongwith ribonuclei enzyme treatment.

It is then treated with heparin in glucose solution ( 5-25% Dextrose 500ml with 5000 units -50,000units of heparin to reorganize the collagen matrix, to get back the compactness of the tissue.

It is then crosslinked with photo oxidation (physico chemical) with methylene blue (.01 %-10%) and UV irradiation (wavelength of 125-525-nano-metre) For 10-20 hours. It is further additionally treated with heparin in glucose solution to reorganize the collagen matrix with heparin, which acts as an agent adapted to modify the surface properties of the tissue.

Preservation is obtained in 80% ethyl alcohol solution with glycerine and water.

Acellular amniotic membrane for clinical use in eyes as corneal replacement and limbal cell growth have been done with trypsinization, and being practised by different groups . Our preparation is different and we have found out that it is helping auto- logous cell and tissue growth wherever applied. Cell adhesion molecules like laminin cadherin are active by this processing. Application of this will transform a full thickness skin burn of large area into re replacement of patients own skin with any scarring . It will encourage the adjacent skin stem cell to migrate in it and form new skin, thereby avoiding skin grafting in future as well as deformity of scar formation.

Not only skin it will be helpful in healing nonhealed venous ulcers, diabetic foot ulcers, open sternal wound after sternotomy due to infection.

Other than the above mentioned usage corneal replacements it is useful and also as a strong biological membrane to cover the heart surface after open-heart surgery thus preventing adhesion to the adjacent structure. This will help the second heart surgery if required. In case of complex congenital heart diseases children very often require multiple surgeries and the entry becomes easier to the heart during subsequent surgeries. DESCRIPTION OF INVENTION:

Amniotic membrane has thus inherent properties that are helpful in wound healing. Among other benefits it is thought to inhibit blood vessei growth in adjacent tissues, decrease cell death, reduce inflammation, and reduce scar tissue, which is vital for use in skin transplantation.

In all the previous successful cases, the amniotic membrane graft "dissolved" away within few months, leaving healed corneal tissue in its stead whereas in the invention, we are attempting to regenerate a damaged skin with an application of treated amniotic membrane in the process described in the invention.

The current consensus is that the intimate adherent property of the biological dressing to an open wound in some way suppresses bacterial proliferation and helps to eliminate existing bacteria. A number of factors may contribute to this effect. In a clean surgical wound, the collagen of the graft or biological dressing, via its haemostatic properties, will help to stop bleeding and thus prevent subsequent haematomas, which would provide opportunities for bacterial proliferation. In addition, the very close bonding between graft and wound eliminates dead space in which serous exudates could collect and encourage bacterial growth. When wounds are contaminated, grafts or biological dressings can not only suppress bacterial growth but also reduce the existing microbial population density. It may be due to the bonding of the graft to the wound bed by fibrin: the bacteria are trapped in the thin fibrin matrix linking the collagen fibres of the graft with the collagen of the wound )ed. The fibrin matrix provides an ideal substratum for migration of phagocytes and ensures that all the bacteria are within reach of the phagocytes.

Thereby, amniotic membrane transplantation can reduce inflammation, promote healing, and decrease irritation in surface problems.

.The amniotic membrane consists of a single layer of cuboidal epithelial cells, a thick basement membrane and an avascular stromal matrix, loosely attached to the chorion. Amniotic membrane so processed has been found to exhibit the following features:

It facilitates epithelialization.

It maintains a normal epithelial phenotype.

It reduces inflammation.

It reduces scarring, and

It reduces the adhesion of tissues.

^•A number of cytokines, growth factors and protease inhibitors, such as IL-4, 6 and 10; EGF, FGF, TGF, HGF, and 2-macrobulin, have been found in cryopreserved amniotic membranes. The presence, concentration and action of these substances may account for most of the observed clinical effects and its mechanisms of action such as:

an exclusion of inflammatory cells with anti-protease activities, suppression of TGF-signalling system and myofibroblast differentiation of normal fibroblasts, prolongation of the life span and clonogenicity of epithelial progenitor cells, promotion of non-goblet cells epithelial differentiation, and promotion of goblet cell differentiation when combined with conjunctival fibroblast.

Amniotic membrane can be used in a number of indications, either as a 'substrate' to replace the damaged ocular tissue or as a 'patch' (biological dressing), or a combination of both. Even it can be used as a covering for the heart when pericardium is lost due to surgery. By this covering, the second surgery of the heart becomes easier because of its inflammatory function. It is also very much useful for healing non-healing access diabetic foot ulcer and gangrenous foot ulcer.

The human body has considerable capacity for regeneration. The skin with high rates of cell turnover are regenerated continually through out life.

The present invention provides methods and compositions to actively control the whole process of skin regeneration. During this process, cells, the smallest unit of life, are stimulated, propagate, differentiate, integrate with each other to physiologically repair the damaged skin or to regenerate the skin destroyed in various courses, such as trauma and diseases. These nascent skin then conjoin together to form a fully functional skin.

To achieve this result in an adult, specific, active human intervention is needed. The general guidance for this intervention revealed in the present invention is that 1) preserving the injured or damaged skin, the viable cells in the remaining tissues should be preserved to a maximum extent; 2) removing necrotic cells or skin as early as possible; 3) activating and propagating the regenerative cells in an environment mimicking the their own native physiological conditions; and 4) administering regulators for cell growth and differentiation to the regenerating organ to direct proper, physiological repair of tissues.

Neutrophiles, then macrophages, migrate into the wound, characterizing acute inflammation. These inflammatory cells provide phagocytosis of bacteria and debridement of injured tissue. This is proceeded by chronic inflammation where lymphocytes and monocytes infiltrate the wound site. The latter become macrophages, which are considered the main coordinators of adult wound healing.

Neutrophils phagocytize contaminating bacteria and digest the fibrin matrix in preparation for new tissue. They also secrete vasodilatory mediators and cytokines •that activate fibroblasts and keratinocytes and attract macrophages to the injury site.

Macrophages phagocytize potential pathogens, debride the wound, and secrete cytokines and growth factors such as fibroblast growth factors (FGF), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), tumor necrosis factor (TNF-a), interleukin-1 (IL-1) and interferon-gamma (IFN-. gamma.). These chemical messengers also stimulate the infiltration, proliferation, and migration of fibroblasts and endothelial cells, resulting in angiogenesis.

Amnion becomes incorporated into, and becomes part of, the host In the following description, reference is made to the use of human amnion, although it is to be understood that the invention is not restricted to the use only of human amnion. The human amnion is easily obtained in the fresh state from caesarean sections in the maternity wards, and can be kept in organ culture. We have further found that the amnion induces healing of burnt skin and in time is indistinguishable from the original skin. Accordingly, in one aspect the present invention provides the use of human or animal amniotic cells to regenerate or replace diseased or damaged skin.

However, if the assumption is accepted that the transplanted dead amniotic membrane pieces function as inert biocompatible non-immunogenic and non-viable foreign bodies then one would expect to see the process of organisation and eventual degradation which as most clearly not been seen.

WORKING OF THE INVENTION:

After the amniotic membrane has been placed on the patient, thereafter the fibroblasts enter the wound site to replace the existing fibrin matrix with glycosaminoglycans and proteoglycans. The healing ECM also contains many glycoproteins, including fibronectin, and tenascin. Fibronectin promotes substrate adhesion, whereas tenascin facilitates substrate migration by antagonizing fibronectin.

Further, fibroblasts and endothelial cells convert dissolved molecular oxygen to superoxide, which is important in resistance to wound infection as well as oxidative signaling in further stimulation of growth factor production.

Later in the subsequent days, a wide variety of adjacent cells from remnant of normal skin increase proliferation and migrate to the wound, including macrophages, lymphocytes, fibroblasts, epithelial cells (i.e., keratinocytes), and endothelial cells for constructing blood vessels. During the migratory and proliferation processes, these cells that are recruited into the healing wound undergo rapid mitosis and begin to define the ultimate structure of the scar.

During this phase, a process known as epithelization occurs in order to re- epithelialize the wound edges. During the epithelization process, an epidermal covering composed predominantly keratinocytes begins to migrate and undergo stratification and differentiation to reconstitute the barrier function of epidermis. This process also promotes extracellular matrix (ECM) production, growth factor and cytokine expression and angiogenesis through the release of growth factors such as keratinocyte growth factor (KGF). Keratinocytes stimulate angiogenesis by releasing basic fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF). They also secrete TGF-.alpha. which acts as a chemoattractant and mitogen, and PDGF which is involved in matrix production. Further migration and proliferation of fibroblasts lead to the replacement of proteoglycan in the ECM with collagen deposition. In addition, endothelial cell proliferation creates neovascularization, i.e., angiogenesis.

The amniotic membrane processed by the present invention if placed on a skin wound, acts as a bioactive scaffold which attracts adjacent normal skin's stem cells from the basement membrane to migrate.

After migration with the milieu of the skin environment and by virtue of its ceil adhesion property, they differentiate into essential components of skin, eg. keratinocytes, hair follicles, and epithelial surface (stratified) thus slowly the scaffold of processed amniotic membrane becomes an autologous skin graft.

Claims

CLAIMS:

1. A process for diminishing the antigenicity of derived amniotic membrane when intended to be used as a transplant, for rendering such membranes substantially resistant to infection, and for rendering such membranes liable to be stored for a prolonged period of time, comprising:

a. cleansing the harvested tissue in balanced salt solution with antifungal agent;

b. treating the tissue in a solution of Deoxycholic acid in an aqueous solvent of suitable pH along with ribonucleic enzyme treatment;

c. reorganizing the collagen matrix with heparin in glucose solution;

d. cross linking with photooxidation physico chemical with methylene blue and UV radiation for a duration;

e. preserving with alcohol solution; and

f. transplanting the treated tissue onto an injured skin needing repair.

2. The process according to claim 1 , wherein said treating step is effected in a 1 percent solution of deoxycholic acid with RBN enzyme sequentially for 20 to 50 hours.

3. The process according to claim 1 , wherein said treating step is effected during 20-50 hours.

4. The process according to claim 1 , wherein said reorganizing step is effected in 5-25% dextrose solution, with 5,000 to 50,000 ml. of heparin per 100 ml. of glucose.

5. The process according to claim 1 , wherein said step of crosslinking is effected in the 0.01-10% range of methylene blue.

6. The process according to claim 1 , wherein said step of crosslinking further effected with UV radiation of wavelength 125-525 nanometers.

7. The process according to claim 1 , wherein said step of crosslinking further effected for 10-20 hours.

8. The process according to claim 1 , wherein said step of preserving further effected with 80% ethyl alcohol solution.

9. The process according to claim 1 , wherein said step of preserving further effected with glycerin and water.

10. The process according to ciaim 1 , further including the step of additional reorganizing the collagen matrix with heparin, which acts as an agent adapted to modify the surface properties of the tissue.

11. The process according to claim 10, wherein said agent is adapted to attach negative or positive charges to the surface of the tissue.

12. The process according to claim 1 , wherein the tissue used is placental membrane and said transplanting step is a skin graft.

13. The process according to claim 1 , wherein the tissue used is human placental membrane and said transplanting step is a skin graft.

14. A process according to claim 1 , wherein the treated tissue is an animal placental membrane, and said transplanting step is a skin graft.

15. A process according to claim 1 , wherein the treated tissue is transplanted onto a human skin and was derived from an animal species other than a human.

16. A process according to claim 1 , wherein the treated tissue is transplanted onto a human skin and was derived from another human.

17. A treated amniotic membrane as claimed in claim 1 is intended to be used as a transplant, the said membrane substantially resistant to infection and liable to be stored for a prolonged period of time before implantation.

18. A method of regenerating damaged skin which comprises implanting into the burnt skin an effective amount of viable human or animal amniotic membrane.

19. A method as claimed in claim 18, wherein said amniotic membrane is acellular comprising substantially of type 4 collagen tissue.

20. A method as claimed in claim 18, wherein (i) fresh amniotic membranes are harvested from ceaseran section (ii) the harvested membranes are decellularised; and (iii) the decellularised membranes are crosslinked; and iv) crosslinked membranes are introduced onto the burnt skin.

21. A method as claimed in claim 18, wherein said amniotic membranes mounted onto biologically acceptable carriers prior to implantation into the burnt skin.

22. A method as claimed in claim 21 wherein said carriers is a bio-degradable scaffold.

23. A method as claimed in claim 18, wherein said amniotic membranes are introduced directly into the burnt skin .