Claims:We claim,
1. Biological composite sheet comprising of an amniotic membrane, wherein the amniotic membrane is coated/enriched with mixture of chorionic membrane, umbilical cord matrix and placenta disc particles/extract.
2. Biological composite sheet comprising of an amniotic membrane, wherein the amniotic membrane is coated one or two side in a scaffold, layer or as coating with mixture of chorionic membrane, umbilical cord matrix and placenta disc particles/extract, with or without crosslinking agent.
3. Biological composite sheet comprising of two layers of an amniotic membrane, wherein the mixture of chorionic membrane, umbilical cord matrix and placenta disc particles/extract is placed in between the layers;
wherein the arrangement of the amniotic membrane is such that the chorion side of first amniotic membrane is positioned up, the particles/extract is placed on chorionic side of first amniotic membrane and the second amniotic membrane is placed from the lower epithelial side onto the particles.
4. Biological composite sheet comprising of an amniotic membrane, wherein the amniotic membrane is attached to the layer made up of chorionic membrane, umbilical cord matrix and placenta disc particles/extract.
5. Biological composite sheet comprising of an amniotic membrane, wherein the amniotic membrane is attached to chorionic membrane, umbilical cord matrix and placenta disc particles/extract and micronized to less 500 µm diameter.
6. Biological composite sheet comprising of an amniotic membrane, wherein each side of the amniotic membrane is coated/enriched by layer made up mixture of chorionic membrane, umbilical cord matrix and placenta disc particles/extract.
7. Biological composite sheet comprises epidermal layer and dermal layer;
wherein the epidermal layer comprises of combination of chorionic membrane, umbilical cord matrix and placenta disc particles/extract with nanofiber or microfiber or nanoparticle or microparticles, which is coated/enriched on denuded amniotic membrane; and
wherein the dermal layer comprises scaffolding mixture of chorionic membrane, umbilical cord matrix and placenta disc particles/extract and polymers such as collagen, gelatin, hyaluronic acid and chitosan with fibroblast cell on the chorionic surface of the amniotic membrane.
8. Biological composite sheet comprises of:
a. an amniotic membrane, wherein the amniotic membrane is coated/enriched with mixture of chorionic membrane, umbilical cord matrix and placenta disc particles/extract; and/or
b. an amniotic membrane, wherein the amniotic membrane is coated one or two side in a scaffold, layer or as coating with mixture of chorionic membrane, umbilical cord matrix and placenta disc particles/extract, with or without crosslinking agent; and/or
c. two layers of an amniotic membrane, wherein the mixture of chorionic membrane, umbilical cord matrix and placenta disc particles/extract is placed in between the layers;
wherein the arrangement of the amniotic membrane is such that the chorion side of first amniotic membrane is positioned up, the particles/extract is placed on chorionic side of first amniotic membrane and the second amniotic membrane is placed from the lower epithelial side onto the particles; and/or
d. an amniotic membrane, wherein the amniotic membrane is attached to the layer made up of chorionic membrane, umbilical cord matrix and placenta disc particles/extract; and/or
e. an amniotic membrane, wherein the amniotic membrane is attached to chorionic membrane, umbilical cord matrix and placenta disc particles/extract and micronized to less 500 µm diameter; and/or
f. an amniotic membrane, wherein each side of the amniotic membrane is coated/enriched by layer made up mixture of chorionic membrane, umbilical cord matrix and placenta disc particles/extract; and/or
g. epidermal layer and dermal layer;
wherein the epidermal layer comprises of combination of chorionic membrane, umbilical cord matrix and placenta disc particles/extract with nanofiber or microfiber or nanoparticle or microparticles, which is coated/enriched on denuded amniotic membrane; and
wherein the dermal layer comprises scaffolding mixture of chorionic membrane, umbilical cord matrix and placenta disc particles/extract and polymers such as collagen, gelatin, hyaluronic acid and chitosan with fibroblast cell on the chorionic surface of the amniotic membrane.
9. The biological composite sheet of claims 1 to 8 wherein, the different ratio of mixture of chorionic membrane, umbilical cord matrix and placenta disc particles/extract is used based on the wound type.
10. The biological composite sheet of claims 1 to 8 wherein, the different concentration of mixture of chorionic membrane, umbilical cord matrix and placenta disc particles/extract is used based on the wound type.
11. The biological composite sheet of claims 1 to 8 wherein, the adhesion of mixture of chorionic membrane, umbilical cord matrix and placenta disc particles/extract to the surface of the amniotic membrane is by crosslinking.
12. The biological composite sheet of claim 11 wherein, the crosslinking is carried out by chemical cross-linking agents or physical cross linking.
13. The biological composite sheet of claim 12 wherein, the cross linking agents are selected from the group consisting of chemical crosslinker consists of aldehydes (e.g., glutaraldehyde), carbodiimides (e.g., 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide [EDC]) or isocyanates (e.g., hexamethylene diisocyanate [HMDI]); photo initiator agents (e.g., rose Bengal, riboflavin); carbohydrates (e.g., ribose, glucose); plant extracts (e.g., oleuropein, genipin, and Myrica rubra); polyethylene glycol (PEG) polymers; and/or physical crosslinker consists of dehydrothermal; UV irradiation; and/or biological crosslinker consists of (e.g., transglutaminase) or combination thereof.
14. The biological composite sheet of claim 9 or 10 wherein, the wound type consists of full thickness burns; full thickness diabetic foot ulcers; partial-thick wounds; dentistry; ophthalmology and bilayer skin substitute or combination thereof.
15. The biological composite sheet of claim 9 wherein, the mixture of placenta disc, umbilical cord matrix and chorionic membrane is used in ratio of 8:4:2, 8:6:4, 8:4:4, 8:6:6, 8:5:3 or 8:3:3 for full thickness burns.
16. The biological composite sheet of claim 9 wherein, the mixture of chorionic membrane, placenta disc and umbilical cord matrix is used in ratio of 8:4:2, 8:6:4, 8:4:4, 8:6:6, 8:5:3 or 8:3:3 for full thickness diabetic foot ulcers.
17. The biological composite sheet of claim 9 wherein, the mixture of placenta disc, chorionic membrane and umbilical cord matrix is used in ratio of 4:1:4, 4:2:4, 4:1:3, 3:1:4, 3:2:4, 2:1:4, 2:2:4 or 2:0:4 for partial-thick wounds.
18. The biological composite sheet of claim 9 wherein, the mixture of placenta disc, chorionic membrane and umbilical cord matrix is used in ratio of 4:1:2, 4:2:3, 4:1:3, 4:1:1, 4:2:2 or 4:0:1 for dentistry.
19. The biological composite sheet of claim 9 wherein, the mixture of placenta disc, chorionic membrane and umbilical cord matrix is used in ratio of 4:0:3, 4:0:2, 4:0:2 or 4:0:0 for ophthalmology.
20. The biological composite sheet of claim 9 wherein, the mixture of placenta disc, umbilical cord matrix and chorionic membrane is used in ratio of 4:1:4, 4:2:4, 4:1:3, 3:1:4, 3:2:4, 2:1:4, 2:2:4 or 2:0:4 for epidermal layer of bilayer skin substitute;
wherein the mixture of chorionic membrane, placenta disc and umbilical cord matrix is used in ratio of 8:4:2, 8:6:4, 8:4:4, 8:6:6, 8:5:3 or 8:3:3 for dermal layer of bilayer skin substitute.
21. The biological composite sheet of claim 1 to 8 comprises its use for the treatment of acute and chronic wounds.
22. The biological composite sheet of claim 1 to 8 comprises its use for the treatment of diabetic foot ulcers, bed sores, burn wounds, venous leg ulcer or surgical wounds.
23. Biological composite sheet comprising of an amniotic membrane for use in treatment of acute and chronic wounds, wherein the amniotic membrane is coated/enriched with mixture of chorionic membrane, umbilical cord matrix and placenta disc particles/extract.
24. Biological composite sheet comprising of an amniotic membrane for use in treatment of diabetic foot ulcers, bed sores, burn wounds, venous leg ulcer or surgical wounds, wherein the amniotic membrane is coated/enriched with mixture of chorionic membrane, umbilical cord matrix and placenta disc particles/extract.
25. A method of preparation of biological composite sheet comprises:
a. screening of donor for placenta preparation;
b. identification of donor, taking consent of donor and evaluation of donor;
c. sterilizing the placenta collected from caesarean section;
d. primary packaging and labelling operation for donor identification based on the type of tissues;
e. storage and transportation of placenta to the production site;
f. separation of the amniotic membrane, chorionic membrane, umbilical cord and placenta disc from the placenta;
g. a microbial sample is taken from tissue and environment where the placenta is located and sent to the quality control department;
h. all tissues are stored in a refrigerator at 4-8 °C for 24 hours;
i. after all the serological tests have been performed, the tissues enter the production room;
j. decontamination and separation of tissue components;
k. decellularization by enzymatic and mechanical methods;
l. powdered the umbilical cord matrix, chorion membrane and placenta disc by cryogenic method or by sonication;
m. incubation of tissues in preservative solution for overnight;
n. the composition of the powder is made by a specified ratio and specified weight;
o. determination of the size of amniotic membrane among 3×3 cm2, 2×4 cm2, 5×5 cm2, 6×6 cm2 or 10×10 cm2 and cut accordingly;
p. coating the surface of the amniotic membrane with particles or extract obtained in step (n);
q. treating component of step (p) with cryoprotectant and lyoprotectant agent;
r. dehydration by lyophilization;
s. packaging.
26. A method of preparation of biological composite sheet comprises:
a. combining the particles or extract in a relatively defined ratio according to the type of wound;
b. treating the particles of step (a) with crosslinking agent;
c. coating the surface of the amniotic membrane with particles or extract of step (b);
d. treating component of step (c) with cryoprotectant and cytoprotectant agent;
e. dehydration by lyophilization;
f. packaging.
27. A method of preparation of biological composite sheet comprises:
a. combining the particles or extract in a relatively defined ratio according to the type of wound;
b. treating the particles of step (a) with or without crosslinking agent;
c. moulding the particles with use of silicon or teflon mould;
d. treating component of step (c) with cryoprotectant and cytoprotectant agent;
e. dehydration by lyophilization;
f. connecting the foam layer made in step (e) with the amniotic membrane layer;
g. dehydration by lyophilization;
h. packaging.
28. A method of preparation of biological composite sheet comprises:
a. combining the particles or extract in a relatively defined ratio according to the type of wound;
b. treating the particles of step (a) with or without crosslinking agent;
c. coating the chorionic surface of amniotic membrane with particles or extract of step (b);
d. treating the amniotic membrane with crosslinking agent;
e. placing the amniotic layer on the epithelial side onto the sandwiched particles;
f. treating component of step (e) with cryoprotectant and cytoprotectant agent;
g. dehydration by lyophilization;
h. packaging.
29. A method of preparation of biological composite sheet comprises:
a. combining the particles or extract in a relatively defined ratio according to the type of wound;
b. treating the particles of step (a) with or without crosslinking agent;
c. coating the surface of the amniotic membrane with particles or extract of step (b);
d. treating component of step (c) with cryoprotectant and cytoprotectant agent;
e. grinding with cryogenic method;
f. dehydration by lyophilization;
g. packing in syringe.
30. A method of preparation of biological composite sheet comprises:
a. combining the particles or extract in a relatively defined ratio according to the type of wound;
b. treating the particles of step (a) with or without crosslinking agent;
c. coating the surface of other membranes, but are not limited to, allograft or xenograft acellular dermis, bladder acellular matrix allograft, allograft pericardium, collagen sheet, bio polymers or copolymers or copolymer films, small intestinal submucosa allograft and fascia lata with particles or extract;
d. treating component of step (c) with cryoprotectant and cytoprotectant agent;
e. dehydration by lyophilization;
f. packaging.
31. A method of preparation of biological composite sheet comprises:
a. combining the particles or extract in a relatively defined ratio according to the type of wound;
b. treating the particles of step (a) with or without crosslinking agent;
c. combining the particles or extract of step (b) with polymer cratose, alginate oxide, PLGA in a relatively defined ratio;
d. coating the surface of other membranes with particles or extract;
e. treating component of step (d) with cryoprotectant and cytoprotectant agent;
f. dehydration by lyophilization;
g. packaging.
32. A method of preparation of biological composite sheet comprises:
a. combining the particles and extract in a relatively defined ratio according to the type of wound;
b. Epidermal layer: denuded amniotic membrane coated with nanofiber or microfiber or nano particle or microparticle made of a polymer like as alginate oxide in concentration range of 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 1.5%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15% or 20%; cartos and placenta disc / umbilical Cord Matrix / chorionic membrane in ratio of 4:1:4, 4:2:4, 4:1:3, 3:1:4, 3:2:4, 2:1:4, 2:2:4 or 2:0:4, with or without keratinocyte cell culture;
c. Dermal layer: Scaffolding using a chorionic membrane / placenta disc/ umbilical cord matrix of 8:4:2, 8:6:4, 8:4:4, 8:6:6, 8:5:3 or 8:3:3 and polymers like as collagen, gelatin, hyaluronic acid and chitosan with fibroblast cell culture and connecting this scaffold to the chorionic surface of the amniotic membrane.
d. treating the two parts, epidermal layer of step (b) and dermal layer of step (c), with nontoxic and non-immunogenic crosslinking agent;
e. connecting of the layers;
f. treating above components with cryoprotectant agent;
g. preservation with cryopreservation method;
h. packaging.
33. The method of preparation of biological composite sheet according to claim 25 to 36 wherein, the adhesion of mixture of chorionic membrane, umbilical cord matrix and placenta disc particles/extract to the surface of the amniotic membrane is by crosslinking.
34. The method of preparation of biological composite sheet according to claim 33 wherein, the crosslinking is carried out by chemical cross-linking agents or physical cross linking.
35. The method of preparation of biological composite sheet according to claim 34 wherein, the cross linking agents are selected from the group consisting of chemical crosslinker consists of aldehydes (e.g., glutaraldehyde), carbodiimides (e.g., 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide [EDC]) or isocyanates (e.g., hexamethylene diisocyanate [HMDI]); photo initiator agents (e.g., rose Bengal, riboflavin); carbohydrates (e.g., ribose, glucose); plant extracts (e.g., oleuropein, genipin, and Myrica rubra); polyethylene glycol (PEG) polymers; and/or physical crosslinker consists of dehydrothermal; UV irradiation; and/or biological crosslinker consists of (e.g., transglutaminase) or combination thereof.
36. The method of preparation of biological composite sheet according to claim 25 to 36 wherein, the different ratio of mixture of chorionic membrane, umbilical cord matrix and placenta disc particles/extract is used based on the wound type.
37. The method of preparation of biological composite sheet according to claim 25 to 36 wherein, the different concentration of mixture of chorionic membrane, umbilical cord matrix and placenta disc particles/extract is used based on the wound type.
38. The method of preparation of biological composite sheet according to claim 36 or 37 wherein, the wound type consists of full thickness burns; full thickness diabetic foot ulcers; partial-thick wounds; dentistry; ophthalmology and bilayer skin substitute or combination thereof.
39. The method of preparation of biological composite sheet according to claim 36 wherein, the mixture of placenta disc, umbilical cord matrix and chorionic membrane is used in ratio of 8:4:2, 8:6:4, 8:4:4, 8:6:6, 8:5:3 or 8:3:3 for full thickness burns.
40. The method of preparation of biological composite sheet according to claim 36 wherein, the mixture of chorionic membrane, placenta disc and umbilical cord matrix is used in ratio of 8:4:2, 8:6:4, 8:4:4, 8:6:6, 8:5:3 or 8:3:3 for full thickness diabetic foot ulcers.
41. The method of preparation of biological composite sheet according to claim 36 wherein, the mixture of placenta disc, chorionic membrane and umbilical cord matrix is used in ratio of 4:1:4, 4:2:4, 4:1:3, 3:1:4, 3:2:4, 2:1:4, 2:2:4 or 2:0:4 for partial-thick wounds.
42. The method of preparation of biological composite sheet according to claim 36 wherein, the mixture of placenta disc, chorionic membrane and umbilical cord matrix is used in ratio of 4:1:2, 4:2:3, 4:1:3, 4:1:1, 4:2:2 or 4:0:1 for dentistry.
43. The method of preparation of biological composite sheet according to claim 36 wherein, the mixture of placenta disc, chorionic membrane and umbilical cord matrix is used in ratio of 4:0:3, 4:0:2, 4:0:2 or 4:0:0 for ophthalmology.
44. The method of preparation of biological composite sheet according to claim 36 wherein, the mixture of placenta disc, umbilical cord matrix and chorionic membrane is used in ratio of 4:1:4, 4:2:4, 4:1:3, 3:1:4, 3:2:4, 2:1:4, 2:2:4 or 2:0:4 for epidermal layer of bilayer skin substitute;
wherein the mixture of chorionic membrane, placenta disc and umbilical cord matrix is used in ratio of 8:4:2, 8:6:4, 8:4:4, 8:6:6, 8:5:3 or 8:3:3 for dermal layer of bilayer skin substitute.
45. The method of preparation of biological composite sheet according to claim 25 to 33 wherein, its use for the treatment of acute and chronic wounds.
46. The method of preparation of biological composite sheet according to claim 25 to 33 wherein, its use for the treatment of diabetic foot ulcers, bed sores, burn wounds, venous leg ulcer or surgical wounds.

, Description:Title:
Biological composite sheet comprising of enriched amniotic membrane
Field of the invention:
The present invention is related to biological composite sheet comprising of an amniotic membrane as a platform, wherein the amniotic membrane is coated and enriched with mixture of chorionic membrane, umbilical cord matrix and placenta disc particles/extract. The invention further related to the method of preparation of biological composite sheet and its use in would healing.
Background of the invention:
A chronic wound can be defined as one that has failed to proceed through an orderly and timely reparative process to produce anatomic and functional integrity within a period of 3 months or that has proceeded through the repair process without establishing a sustained, anatomic and functional result. The nomenclature is far from agreed upon, and these wounds are sometimes referred to as hard-to-heal or difficult-to-heal wounds/ulcers, and the time span required for chronicity has been defined in the range 4 weeks up to more than 3 months. Based on the causative aetiologies, the Wound Healing Society classifies chronic wounds into four categories: pressure ulcers, diabetic ulcers, venous ulcers and arterial insufficiency ulcers. Chronic wounds are often termed ulcers and can be defined as wounds with a full thickness in depth and a slow healing tendency. It is estimated that 1 to 2 % of the population experience a chronic wound during their lifetime in developed countries.
Diabetic patients are at risk for spontaneous foot ulcers, chronic wounds, infections, and tissue necrosis. The development and progression of diabetic foot ulcers are mainly caused by arteriosclerosis and peripheral neuropathy. Tissue necrosis plays a primordial role in the progression of diabetic foot ulcers. Hyperglycemia increases the susceptibility to limb necrosis in ischemic conditions. Prolonged or untreated ulcers can lead to wound infection, septicaemia or limb amputation. Statistics show that about 62 million people in India have diabetes and 25% of them have diabetic foot ulcers, of which about 50% become infectious and require hospitalization.
Human placental tissue has been used in various surgical procedures, including skin transplantation and ocular surface disorders, for over a century. The placenta is a fetomaternal organ consisting placental globe, umbilical cord, associated membranes (chorionic membrane and amniotic membrane). The chorionic membrane and the amniotic membrane are attached by loose connective tissue and make up the placental sac. The innermost membrane of the placental sac is the amniotic membrane, which comes into contact with the amniotic fluid that surrounds the fetus. The chorionic membrane is the outermost layer of the sac and is heavily cellularized. The placental membranes have an abundant source of collagen that provides an extracellular matrix (ECM) to act as a natural scaffold for cellular attachment in the body. According to Hossam et al., amniotic membrane (AM) is an attractive method of grafting for wounds as it has unique properties, including anti-inflammatory effects, bacteriostatic, wound protection, decreased scarring, and pain reduction properties. However, it has few drawbacks such as low mechanical strength, fast degradation time, inability to use in full thickness burns/ulcers, loss of biological factors due to processing methods/steps, and variation in results from patient to patient.
The compositions comprising a non-homogenized chorionic matrix, a homogenized amniotic matrix and a homogenized umbilical cord (UC) matrix, wherein the non- homogenized chorionic matrix comprises viable cells are disclosed in patent application no. EP3474869A1. The compositions composed of micronized particles derived from one or more components present in placental tissue in combination with one or more bone grafts are disclosed in patent application no. EP2904094A2.
In the present invention, the biological composite sheet comprising of an amniotic membrane as platform, wherein the amniotic membrane is coated and enriched with mixture of chorionic membrane, umbilical cord matrix and placenta disc particles/extract. The ratios/concentration of coating material varies according to type of wound. The enriched amniotic membrane has improved biological and physical properties.
Summary of the invention:
The present invention is related to the biological composite sheet comprising of an amniotic membrane as a platform wherein the amniotic membrane is coated/enriched with mixture of chorionic membrane, umbilical cord matrix and placenta disc particles/extract. The enriched amniotic membrane has improved biological and physical properties.
In one embodiment, the biological composite sheet comprising of an amniotic membrane as a platform wherein the amniotic membrane is coated/enriched with mixture of chorionic membrane, umbilical cord matrix and placenta disc particles/extract. The ratios/concentration of chorionic membrane, umbilical cord matrix and placenta disc varies based on the wound type. The wounds types are categories as follows: Full thickness burns, Full thickness diabetic foot ulcers, Partial-thick wounds, Dentistry, Ophthalmology, Bilayer skin substitute
In another embodiment, the biological composite sheet comprising of an amniotic membrane wherein the amniotic membrane is coated one or two side in a scaffold, layer or as coating with mixture of chorionic membrane, umbilical cord matrix and placenta disc particles/extract, with or without crosslinking agent.
In another embodiment, the biological composite sheet comprising of two layers of an amniotic membrane wherein the mixture of chorionic membrane, umbilical cord matrix and placenta disc particles/extract is placed in between the layers. The arrangement of the amniotic membrane is such that the chorion side of first amniotic membrane is positioned up, the particles/extract is placed on chorionic side of first amniotic membrane and the second amniotic membrane is placed from the lower epithelial side onto the particles.
In another embodiment, the biological composite sheet comprising of an amniotic membrane wherein the amniotic membrane is attached to the layer made up of chorionic membrane, umbilical cord matrix and placenta disc particles/extract.
In another embodiment, the biological composite sheet comprising of an amniotic membrane, wherein the amniotic membrane is attached to chorionic membrane, umbilical cord matrix and placenta disc particles/extract and micronized to less 500 µm diameter.
In another embodiment, the biological composite sheet comprising of an amniotic membrane, wherein each side of the amniotic membrane is coated/enriched by layer made up mixture of chorionic membrane, umbilical cord matrix and placenta disc particles/extract.
In another embodiment, the biological composite sheet used for by-layer skin substitute comprises epidermal layer and dermal layer, wherein the epidermal layer comprises of combination of chorionic membrane, umbilical cord matrix and placenta disc particles/extract with nanofiber or microfiber or nanoparticle or microparticles, which is coated/enriched on denuded amniotic membrane; and wherein the dermal layer comprises scaffolding mixture of chorionic membrane, umbilical cord matrix and placenta disc particles/extract and polymers such as collagen, gelatin, hyaluronic acid and chitosan with fibroblast cell on the chorionic surface of the amniotic membrane.
In another embodiment, the cross-linking agents are used for the adhesion of mixture of chorionic membrane, umbilical cord matrix and placenta disc particles/extract to the surface of the amniotic membrane.
In another embodiment, different ratio/concentration of coating/enriching material (mixture of chorionic membrane, umbilical cord matrix and placenta disc particles/extract) is used to coat on surface of amniotic membrane, based on the wound type.
The present invention includes the method of preparations of biological composite sheet.
The present invention further includes use of the biological composite sheet for the treatment of acute and chronic wounds. It further used for the treatment of diabetic foot ulcers, bed sores, burn wounds, venous leg ulcer, surgical wounds.
Brief description of drawings:
Fig. 1A to 1G describes different type of biological composite sheet comprising of an amniotic membrane as a platform wherein the amniotic membrane is coated/enriched with mixture of chorionic membrane, umbilical cord matrix and placenta disc particles/extract.
Fig. 2 describes the result report of the BCA (Bicinchoninic Acid) Protein Assay.
Fig. 3 shows SDS page staining with Coomassie blue
Fig. 4 shows fibroblast cell migration after scratching.
Fig. 5 describes the ratio of cell migration in scratch assay.
Fig. 6 describes report of irradiation and skin sensitization study on biological composite sheet
Fig. 7 shows wound created in the back of the animal using a surgical punch with dimensions of 2×2 cm2 for Full thickness dermal defect
Fig. 8 shows the H&E stained sections of post wounding
Fig. 9. shows the H&E stained after day 21 and re-epithelialization process was completed
Detailed description of the invention:
The present disclosure will now be described more fully hereinafter with reference to exemplary embodiments thereof. These exemplary embodiments are described so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art. Indeed, the present disclosure may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements.
Human placental tissue has been used in various surgical procedures, including skin transplantation and ocular surface disorders. The placenta is a fetomaternal organ consisting placental globe, umbilical cord, associated membranes (chorionic membrane and anmiotic membrane). It is known that the placenta is a temporary organ that supplies enough blood through the umbilical cord to the developing fetus to regulate waste disposal, nutrition, regulate hormonal and temperature balance, and the maturation of the immune system. These elements have little immunogenic effect due to the lack of HLA - A and -B tissue antigens that prevent T cell recognition and expression of HLA - C, -E, -F, -G antigens to prevent destruction. The placenta is by NK. HLA-C is the only classical MHC class I antigen that is specifically expressed and HLA-G does not differ between individuals and may even support antiviral, immunosuppressive, and non-immunological functions. Trophoblasts with the secretion of signals indoleamine-2,3-dioxygenase (IDO), vasoactive intestinal peptide (VIP), Fast ligand, phosphocholine, programmed death ligand 1 (PDL1) and progesterone T cells inhibit regulatory T, NK, T helper in native decidua. This feature has made it possible to use these tissues as allografts for wound healing and soft tissue repair.
Amniotic membrane
The amniotic membrane (AM) has been used for about a century as a biological dressing or tissue graft in ophthalmology, dermatology and surgery. The presence of extra cellular matrix (ECM) with storage of growth factors and thick and firm basal membrane is its unique characteristics. Amniotic membrane composite of the three layers include: Epithelial layer, basement membrane, intermediate layer contains: compact, mesodermal, sponge layers. Epithelial cells, fibroblasts, mesenchymal stem cells and macrophages are residual cells in amniotic membrane which produces various biological factors that are stored in the extra cellular matrix (ECM). These biological factors include: growth factors, immunomodulatory cytokines and chemokines and tissue inhibitors of metalloproteinases (TIMPs), such as PDGF-AA, PDGF-BB, TGF-a, TGF-ß, bFGF, EGF, VEGF, IL-10, IL-4, placental growth factor (PlGF), TIMP-1, TIMP-2 and TIMP-4, which possess important regulatory roles in regulating fetal development and pregnancy. Extracellular matrix (ECM) of amniotic membrane includes: Collagens I, III, IV, V, VI, elastin, Fibronectin, laminins, nidogen, Chondroitin, dermatan sulfate, hyaluronan, decorin, biglycan.
The amniotic membrane has been used for years to wounds healing. it has a number of characteristics that make it especially appropriate to wound healing, which includes: (1) Growth factors, such as: Epithelial Proliferation: EGF TGFa KGF HGF; Monocyte chemotaxis: PDGF FGF TGFb; Fibroblast Migration: PDGF FGF TGFb; Fibroblast Proliferation: PDGF FGF EGF TNF; Angiogenesis: VEGF Ang FGF; Collagen Synthesis: TGFb PDGF; Collagen secretion: PDGF FGF EGF TNF; (2) Reduces pain when applied to a wound; (3) Increases and enhances the wound healing process; (4) Antibacterial properties such as: Bactricidin, b-lysin, transferrin, 7S immunoglobulin, elafin, leukocyte proteinase inhibitor, human b3 defensin and cystatin E; Barrier against microbial inoculation; (5) non-immunogenic; (6) provides a biological barrier; (7) provides a matrix for migration and proliferation of cells; (8) Protection against loss of fluids and proteins; (9) reduces inflammation; (9) reduces scar tissue formation: Amniotic membrane also reduces protease activity via the secretion of TIMP’s (tissue inhibitors of metalloproteinases) and therefore has an anti-fibrotic effect; TGF-ß 1 is down regulated which is responsible for the activation of fibroblasts and prevent the adhesion of injured surfaces to each other.
Chorionic membrane
The chorionic membrane (CM) is the outermost layer of the sac and is heavily cellularized. It is formed by extracellular mesoderm and trophoblast bilayer, which surrounds the embryo and other membranes. The chorionic membrane is histologically composed of the following layers: reticular, basement membrane, and trophoblast. The reticular layer of the chorion membrane contains collagen types I, III, IV, V, VI and VII together with proteoglycans. The pseudo basement membrane is made of type IV collagen, fibronectins and laminins and attaches the trophoblast to the reticular layer by collagen IV, fibronectin and laminin (anchors). Biological factors HGF, TGF-b1, TGF-a, bFGF, VEGF, PDGF, PIGF, Interferon a, Defensins, TIMP-1, IL-6, IL-8, IL-4, SDF-1a, IL-10, GCSF exiting in chorionic membrane.
Umbilical cord
The umbilical cord (UC) consists of a vein that carries oxygen-rich blood and nutrients from the placenta to the fetus, and two arteries that return oxygen-free blood and waste material from the fetus to the placenta. The umbilical cord contains Wharton's jelly, a gelatinous substance made largely from mucopolysaccharides which protects the blood vessels inside. The cord matrix is a jelly-shaped extracellular scaffold composed of insoluble filamentous collagen such as collagen VI, hyaluronic acid and glycosaminoglycan, dermatan sulfate, chondroitin sulfate, keratin sulfate, and heparan sulfate, proteoglycans (PGs) I, III, V. The umbilical cord matrix also acts as a storage of growth factors, which are attached to high molecular weight extra cellular matrix (ECM) components. ubMSC secretes many exosomes that contain proteins that are associated with wound healing, such as ankyrin, smooth muscle fibronectin, vimentin, fibrillin, myosin, and desmin.
Placental disc
The placenta structure consists of an extracellular matrix made of large amounts of collagen I, III, IV and VI, fibronectin, decorin, fibrillin I, thrombospondin I, tenascin C, laminin, heparan sulfate, proteoglycans and elastin. Biological factors such as insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), fibroblast growth factor-2 (FGF-2), vascular endothelial growth factor (VEGF), and the transforming growth factor beta, TGF-b vitamins, glucose, amino acids, lipids and triglycerides are abundant in this tissue. In addition, it has biological properties including anti-inflammatory, antibacterial, immune, low anti-scar formation, making it an ideal candidate for soft tissue repair.
In one embodiment of the present invention, the biological composite sheet comprising of an amniotic membrane as a platform wherein the amniotic membrane is coated/enriched with mixture of chorionic membrane, umbilical cord matrix and placenta disc particles/extract. The enriched amniotic membrane has improved biological and physical properties. Fig. 1A describes this embodiment.
In another embodiment, the biological composite sheet comprising of an amniotic membrane as a platform wherein the amniotic membrane is coated/enriched with mixture of chorionic membrane, umbilical cord matrix and placenta disc particles/extract. The ratios/concentration of chorionic membrane, umbilical cord matrix and placenta disc varies based on the wound type. The wounds types are categories as follows: Full thickness burns, Full thickness diabetic foot ulcers, Partial-thick wounds, Dentistry, Ophthalmology, Bilayer skin substitute
In another embodiment, the biological composite sheet comprising of an amniotic membrane wherein the amniotic membrane is coated one or two side in a scaffold, layer or as coating with mixture of chorionic membrane, umbilical cord matrix and placenta disc particles/extract, with or without crosslinking agent. Fig. 1B describes this embodiment.
In another embodiment, the biological composite sheet comprising of two layers of an amniotic membrane wherein the mixture of chorionic membrane, umbilical cord matrix and placenta disc particles/extract is placed in between the layers. The arrangement of the amniotic membrane is such that the chorion side of first amniotic membrane is positioned up, the particles/extract is placed on chorionic side of first amniotic membrane and the second amniotic membrane is placed from the lower epithelial side onto the particles. Fig. 1C describes this embodiment
In another embodiment, the biological composite sheet comprising of an amniotic membrane wherein the amniotic membrane is attached to the layer made up of chorionic membrane, umbilical cord matrix and placenta disc particles/extract. Fig. 1D describes this embodiment.
In another embodiment, the biological composite sheet comprising of an amniotic membrane, wherein the amniotic membrane is attached to chorionic membrane, umbilical cord matrix and placenta disc particles/extract and micronized to less 500 µm diameter. Fig. 1E describes this embodiment.
In another embodiment, the biological composite sheet comprising of an amniotic membrane, wherein each side of the amniotic membrane is coated/enriched by layer made up mixture of chorionic membrane, umbilical cord matrix and placenta disc particles/extract. Fig. 1F describes this embodiment.
In another embodiment, the biological composite sheet used for by-layer skin substitute comprises epidermal layer and dermal layer, wherein the epidermal layer comprises of combination of chorionic membrane, umbilical cord matrix and placenta disc particles/extract with nanofiber or microfiber or nanoparticle or microparticles, which is coated/enriched on denuded amniotic membrane; and wherein the dermal layer comprises scaffolding mixture of chorionic membrane, umbilical cord matrix and placenta disc particles/extract and polymers such as collagen, gelatin, hyaluronic acid and chitosan with fibroblast cell on the chorionic surface of the amniotic membrane. Fig. 1G describes this embodiment.
In another embodiment, the cross-linking agents are used for the adhesion of mixture of chorionic membrane, umbilical cord matrix and placenta disc particles/extract to the surface of the amniotic membrane. The crosslinking is carried out using chemical cross-linking agents or physical cross linking. The non-limiting examples of crosslinking agents are as follow: the cross linking agents are selected from the group consisting of chemical crosslinker: aldehydes (e.g., glutaraldehyde), carbodiimides (e.g., 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide [EDC]) or isocyanates (e.g., hexamethylene diisocyanate [HMDI]); photo initiator agents (e.g., rose Bengal, riboflavin); carbohydrates (e.g., ribose, glucose); plant extracts (e.g., oleuropein, genipin, and Myrica rubra); polyethylene glycol (PEG) polymers; physical crosslinker: such as dehydrothermal; UV irradiation; or biological crosslinker: (e.g., transglutaminase) or combination thereof. Further, crosslinking agents includes sugar like materials consisting of D-ribose, altrose, ertheose, glucose, mannose, gulose, idose, allose, galactose, maltose, sucrose, gentibiose, melibiose, turanose, trehalose, isomaltose or carbodiimide or mixture thereof.
Based on the wound type, different ratio/concentration of coating/enriching material (mixture of chorionic membrane, umbilical cord matrix and placenta disc particles/extract) is used to coat on surface of amniotic membrane. The high volumes of collagen, growth factors, vitamins and food of the placenta disc are used depending on the type and extent of tissue damage with high amounts of hyaluronic acid-rich umbilical cord extracellular matrix, and mesenchymal stem cell-derived growth factors which is combined with the extracellular matrix of the chorion membrane, which is rich in angiogenic growth factors. It is coated/enriched one or two side of an amniotic membrane in a scaffold, layer or as a coating, with or without crosslinking agent. In another aspect, the different ratios/concentration of particles/extract based on the type and extend of tissue damage are described below:
a. Full thickness burns: Coating the chorionic surface of the amniotic membrane with a Placenta disc / umbilical Cord Matrix / chorionic membrane in ratios of 8:4:2, 8:6:4, 8:4:4, 8:6:6, 8:5:3 or 8:3:3.
b. Full thickness diabetic foot ulcers: Coating the chorionic surface of the amniotic membrane with a chorionic membrane / placenta disc/ umbilical cord matrix in ratios of 8:4:2, 8:6:4, 8:4:4, 8:6:6, 8:5:3 or 8:3:3
c. Partial-thick wounds: Coating of chorionic surface of amniotic membrane with placenta disc / chorionic membrane / umbilical cord matrix in ratios of 4:1:4, 4:2:4, 4:1:3, 3:1:4, 3:2:4, 2:1:4, 2:2:4 or 2:0:4
d. Dentistry: Coating of chorionic surface of amniotic membrane with placenta disc / chorionic membrane / umbilical cord matrix in ratios of 4:1:2, 4:2:3, 4:1:3, 4:1:1, 4:2:2 or 4:0:1
e. Ophthalmology: Coating of chorionic surface of amniotic membrane with placenta disc / chorionic membrane / umbilical cord matrix in ratios of 4:0:3, 4:0:2, 4:0:2 or 4:0:0
f. Bilayer skin substitute:
Epidermal layer: Denuded amniotic membrane coated with combination of nanofiber or microfiber or nanoparticle or microparticle made of a polymer like as alginate oxide in concentration range of 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 1.5%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%; cartos and placenta disc / umbilical Cord Matrix / chorionic membrane in ratio of 4:1:4, 4:2:4, 4:1:3, 3:1:4, 3:2:4, 2:1:4, 2:2:4 or 2:0:4, with or without keratinocyte cell culture.
Dermal layer: Scaffolding using a chorionic membrane / placenta disc/ umbilical cord matrix in ratio of 8:4:2, 8:6:4, 8:4:4, 8:6:6, 8:5:3 or 8:3:3 and polymers like as collagen, gelatin, hyaluronic acid and chitosan with fibroblast cell culture and attaching this scaffold to the chorionic surface of the amniotic membrane.
In another embodiment, the biological composite sheet is used for the treatment of acute and chronic wounds. It further used for the treatment of diabetic foot ulcers, bed sores, burn wounds, venous leg ulcer, surgical wounds.
Amniotic membrane placenta disc / chorionic membrane / umbilical cord matrix is the source of biological factors that are important in wound healing, inflammation modulation, and scar tissue formation. The amniotic basement membrane provides a natural coating and ECM scaffold for the wound. On the other hand, the grafts are made of amniotic membrane, chorion membrane, placenta and umbilical cord matrix with or without treatment with crosslinking agent.
In another embodiment of the present invention, the method of preparation of biological composite comprises:
a. screening of donor for placenta preparation;
b. identification of donor, taking consent of donor and evaluation of donor;
c. sterilizing the placenta collected from caesarean section;
d. primary packaging and labelling operation for donor identification based on the type of tissues;
e. storage and transportation of placenta to the production site;
f. separation of the amniotic membrane, chorionic membrane, umbilical cord and placenta disc from the placenta;
g. a microbial sample is taken from tissue and environment where the placenta is located and sent to the quality control department;
h. all tissues are stored in a refrigerator at 4-8 °C for 24 hours;
i. after all the serological tests have been performed, the tissues enter the production room;
j. decontamination and separation of tissue components;
k. decellularization by enzymatic and mechanical methods;
l. powdered the umbilical cord matrix, chorion membrane and placenta disc by cryogenic method or by sonication;
m. incubation of tissues in preservative solution for overnight;
n. the composition of the powder is made by a specified ratio and specified weight;
o. determination of the size of amniotic membrane among 3×3 cm2, 2×4 cm2, 5×5 cm2, 6×6 cm2 or 10×10 cm2 and cut accordingly;
p. coating the surface of the amniotic membrane with particles or extract obtained in step (n);
q. treat component of step (p) with cryoprotectant and lyoprotectant agent;
r. dehydration by lyophilization;
s. packaging
In another embodiment of the present invention, the method of preparation of biological composite comprises:
a. combining the particles or extract in a relatively defined ratio according to the type of wound;
b. treating the particles of step (a) with crosslinking agent;
c. coating the surface of the amniotic membrane with particles or extract of step (b);
d. treating component of step (c) with cryoprotectant and cytoprotectant agent;
e. dehydration by lyophilization;
f. packaging
In another embodiment of the present invention, the method of preparation of biological composite comprises:
a. combining the particles or extract in a relatively defined ratio according to the type of wound;
b. treating the particles of step (a) with or without crosslinking agent;
c. moulding the particles with use of silicon or teflon mould;
d. treating component of step (c) with cryoprotectant and cytoprotectant agent;
e. dehydration by lyophilization;
f. connecting the foam layer made in step (e) with the amniotic membrane layer;
g. dehydration by lyophilization;
h. packaging
In another embodiment of the present invention, the method of preparation of biological composite comprises:
a. combining the particles or extract in a relatively defined ratio according to the type of wound;
b. treating the particles of step (a) with or without crosslinking agent;
c. coating the chorionic surface of amniotic membrane with particles or extract of step (b);
d. treating the amniotic membrane with crosslinking agent;
e. placing the amniotic layer on the epithelial side onto the sandwiched particles;
f. treating component of step (e) with cryoprotectant and cytoprotectant agent;
g. dehydration by lyophilization;
h. packaging
In another embodiment of the present invention, the method of preparation of biological composite comprises:
a. combining the particles or extract in a relatively defined ratio according to the type of wound;
b. treating the particles of step (a) with or without crosslinking agent;
c. coating the surface of the amniotic membrane with particles or extract of step (b);
d. treating component of step (c) with cryoprotectant and cytoprotectant agent;
e. grinding with cryogenic method;
f. dehydration by lyophilization;
g. packing in syringe
In another embodiment of the present invention, the method of preparation of biological composite comprises:
a. combining the particles or extract in a relatively defined ratio according to the type of wound;
b. treating the particles of step (a) with or without crosslinking agent;
c. coating the surface of other membranes, but are not limited to, allograft or xenograft acellular dermis, bladder acellular matrix allograft, allograft pericardium, collagen sheet, bio polymers or copolymers or copolymer films, small intestinal submucosa allograft and fascia lata with particles or extract;
d. treating component of step (c) with cryoprotectant and cytoprotectant agent;
e. dehydration by lyophilization;
f. packaging
In another embodiment of the present invention, the method of preparation of biological composite comprises:
a. combining the particles or extract in a relatively defined ratio according to the type of wound;
b. treating the particles of step (a) with or without crosslinking agent;
c. combine the particles or extract of step (b) with polymer cratose, alginate oxide, PLGA in a relatively defined ratio;
d. coating the surface of other membranes with particles or extract;
e. treating component of step (d) with cryoprotectant and cytoprotectant agent;
f. dehydration by lyophilization;
g. packaging
In another embodiment of the present invention, the method of preparation of biological composite sheet used for by-layer skin substitute comprises,
a. combining the particles and extract in a relatively defined ratio according to the type of wound;
b. Epidermal layer: denuded amniotic membrane coated with nanofiber or microfiber or nano particle or microparticle made of a polymer like as alginate oxide in concentration range of 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 1.5%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15% or 20%; cartos and placenta disc / umbilical Cord Matrix / chorionic membrane in ratio of 4:1:4, 4:2:4, 4:1:3, 3:1:4, 3:2:4, 2:1:4, 2:2:4 or 2:0:4, with or without keratinocyte cell culture;
c. Dermal layer: Scaffolding using a chorionic membrane / placenta disc/ umbilical cord matrix of 8:4:2, 8:6:4, 8:4:4, 8:6:6, 8:5:3 or 8:3:3 and polymers like as collagen, gelatin, hyaluronic acid and chitosan with fibroblast cell culture and connecting this scaffold to the chorionic surface of the amniotic membrane.
d. treating the two parts, epidermal layer of step (b) and dermal layer of step (c), with nontoxic and non-immunogenic crosslinking agent;
e. connecting of the layers;
f. treating above components with cryoprotectant agent;
g. preservation with cryopreservation method;
h. packaging
The biological composite sheet is used as pharmaceutical composition and more particularly used as pharmaceutical film for the treatment of acute and chronic wounds.
Examples
Example 1 - Placental Tissue Collection
The placental tissue is collected form caesarean section of the volunteer in sterile manner after obtaining the consent of volunteer. The whole placenta and blood sample are stored in primary package containing isotonic or nutrient preservative solution (DMEM, M199) with antibiotics such as penicillin in concentration of 10,000 µg/mL; streptomycin in concentration of 10,000 µg/mL; and amphotericin B in concentration of 25 µg/mL. The packages are labelled for donor identification. The said packages are transported to the production site with refrigerating condition of 2 to 8 °C. The product is controlled for microbial, viral and fungal contamination. Hence, during the preservation of placental tissue, sterility test of a placenta sample is performed at regular intervals. Clinical-microbiological techniques are performed with in period of 14 days. The samples are cultured in an appropriate fluid enrichment media for aerobic and anaerobic bacteria, yeasts and fungi and any growth in the medium is monitored on a daily basis.
Example 2 - Decellularization, Decontamination and dissection placenta component
Processing of the tissue is performed in (clean room) class A area according to GMP guidelines. The sterile cloth, the sterile set containing tray, forceps, scissors and scalper are used for tissue processing. All devices have been sterilized by gamma-ray or autoclave and sterility tests have been performed to confirm the same. The placenta is placed in a tray containing 0.9% saline to adjust with the ambient room temperature and blood clots are removed. The amniotic membrane and the chorionic membrane are cut from the placenta with scissors and separated into two layers.
The amniotic membrane is forming an opaque layer on the chorion that is physically interconnected and easily separated. Amniotic membrane is harvested and washed extensively in phosphate-buffered saline (PBS) to wipe the blood and other cellular debris. Then amniotic tissue is placed in Glass Reagent Bottles containing hypotonic saline (used in concentration range of 0.75%, 0.65%, 0.60%, 0.55% or 0.5%) and protease inhibitors (used in concentration range of 0.2%, 0.4%, 0.6%, 0.8%, 0.1%, 1.2%, 1.4%, 1.6%, 1.8% or 2%). Glass Reagent Bottles is placed on a shaker and agitated for about 40 to 120 minutes. Then, the tissue is rinsed with deionized water. After washing the with deionized water, amniotic tissue is placed in Glass Reagent Bottles containing 3-[(3- cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) (used in concentration range of 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM,10 mM, 11 mM, 12 mM, 13 mM, 14 mM or 15 mM); EDTA (used in concentration range of 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, or 30 mM) and incubated for 12 to 24 hours. Then, the decontamination of amniotic is performed using peracetic acid (used in concentration range of 0.01% v/v, 0.1% v/v, 0.2% v/v, 0.3% v/v, 0.4% v/v, 0.5% v/v or 1% v/v). Then, the amniotic tissue is rinsed with deionized water to completely remove detergents.
Chorionic tissue is immersed in phosphate-buffered saline (PBS) for 10 to 30 minutes in order to wipe blood and cellular debris. Chorionic tissue is placed in Glass Reagent Bottles containing hypotonic saline (used in concentration range of 0.75%, 0.65%, 0.60%, 0.55% or 0.5%) and protease inhibitors (used in concentration range of 0.2%, 0.4%, 0.6%, 0.8%, 0.1%, 1.2%, 1.4%, 1.6%, 1.8% or 2%). Glass Reagent Bottles is placed on a shaker and agitated for about 40 to 120 minutes. Then, the tissue is rinsed with deionized water. After washing with deionized water, chorionic tissue is placed in Glass Reagent Bottles containing CHAPS (used in concentration range of 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM,10 mM, 11 mM, 12 mM, 13 mM, 14 mM or 15 mM); EDTA (used in concentration range of 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, or 30 mM) and incubated for 12 to 24 hours. The chorionic tissue is then split into smaller parts, grinded using cryogenic method and homogenized to achieve a micron size. The decontamination of chorionic tissue is performed using peracetic acid (used in concentration range of 0.01% v/v, 0.1% v/v, 0.2% v/v, 0.3% v/v, 0.4% v/v, 0.5% v/v or 1% v/v). Then, the chorionic tissue is rinsed with deionized water to completely remove detergents. The 2:1 chorionic tissue particle is mixed with deionized water and centrifuged at 1500 rpm for 7 minutes and the supernatant is discarded.
Placental disk is immersed in phosphate-buffered saline (PBS) for 10 to 30 minutes in order to wipe blood and cellular debris. The tissue is split into smaller parts with thickness less of 0.5mm using sterile scalpel. Then, placental tissue is placed in Glass Reagent Bottles containing hypotonic saline (used in concentration range of 0.75%, 0.65%, 0.60%, 0.55% or 0.5%) and protease inhibitors (used in concentration range of 0.2%, 0.4%, 0.6%, 0.8%, 0.1%, 1.2%, 1.4%, 1.6%, 1.8% or 2%). Glass Reagent Bottles is placed on a shaker and agitated for about 40 to 120 minutes. Then, the tissue is rinsed with deionized water. After washing with deionized water, placental tissue is placed in Glass Reagent Bottles contain 3-[(3- cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) (used in concentration range of 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM,10 mM, 11 mM, 12 mM, 13 mM, 14 mM or 15 mM); EDTA (used in concentration range of 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, or 30 mM); Sodium dodecyl sulfate (SDS) (used in concentration range of 1 mM,1.1 mM,1.2 mM,1.3 mM,1.4 mM,1.5 mM,1.6 mM,1.7 mM,1.8 mM,1.9 mM,2 mM,2.1 mM,2.2 mM,2.3 mM,2.4 mM, or 2.5 mM) and incubated for 12 to 24 hours. Grinding of the placental tissue is carried out using cryogenic method and homogenized to achieve a micron size. The decontamination of placental tissue is performed using peracetic acid (used in concentration range of 0.01% v/v, 0.1% v/v, 0.2% v/v, 0.3% v/v, 0.4% v/v, 0.5% v/v or 1% v/v). The, the Placental tissue is rinsed with deionized water to completely remove detergents. The 2:1 placental tissue particle is mixed with deionized water and centrifuged at 1500 rpm for 7 minutes and the supernatant is discarded.
The umbilical cord is cut into 3cm sections. The cord consists of four parts: vein, allantois, Wharton's jelly, and epithelial layer. Insert each part into vessel with sterile applicator and then make a vertical incision so that it does not tear the vessels and does not reach to the end. Then, by pushing the applicators upward, the vessels are easily separated from the matrix. The remnants of allantois are also cut and removed with a scissor. Using a scalper, the epithelium layer is cut and removed. Umbilical cord is immersed in phosphate-buffered saline (PBS) for 10 to 30 minutes in order to wipe blood and cellular debris. The tissue is split into smaller parts, then tissue is placed in Glass Reagent Bottles containing hypotonic saline (used in concentration range of 0.75%, 0.65%, 0.60%, 0.55% or 0.5%) and protease inhibitors. Glass Reagent Bottles is placed on a shaker and agitated for about 40 to 120 minutes. Then, the tissue is rinsed with deionized water. After washing with deionized water, umbilical cord matrix tissue is placed in Glass Reagent Bottles containing 3-[(3- cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) (used in concentration range of 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM,10 mM, 11 mM, 12 mM, 13 mM, 14 mM or 15 mM); EDTA (used in concentration range of 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, or 30 mM); Sodium dodecyl sulfate (SDS) (used in concentration range of 1 mM,1.1 mM,1.2 mM,1.3 mM,1.4 mM,1.5 mM,1.6 mM,1.7 mM,1.8 mM,1.9 mM,2 mM,2.1 mM,2.2 mM,2.3 mM,2.4 mM, or 2.5 mM) and incubated for 12 to 24 hours. Grinding of the umbilical cord matrix tissue is carried out using cryogenic method and homogenized to achieve a micron size. The decontamination of the umbilical cord matrix tissue is performed using peracetic acid (used in concentration range of 0.01% v/v, 0.1% v/v,0.2% v/v,0.3% v/v,0.4% v/v,0.5% v/v, or 1% v/v). Then, the umbilical cord matrix tissue is rinse with deionized water to completely remove detergents. The 2:1 tissue particle is mixed with deionized water and centrifuged at 1500 rpm for 7 minutes and the supernatant is discarded.
Example 3 - Preparation of micronized tissue particle
The tissues become thicker by less than 5 mm. Using a cryopreservation process, the tissues are treated with glycerol (used in concentration of 2%,5%,10%,15%,20%,25%, or 30%) or dimethyl sulphoxide (DMSO) and moulded into medical grade plastic bags, and the outer package is frozen with liquid nitrogen. It is micronized by mechanical grinding. The powdered tissue passes through the filter which is have pore size in the range of 70 to 350 µm.
Example 4 - Preparation of extract
Tissue powder is combined in Dulbecco's Modified Eagle Medium F-12 medium (DMEM-F12, 1:1) and sonicated (UP200S-Heilescher) 1,2,3,4,5-times in maximum power, 20%,25%,30%,35%,40% duty cycle for 5,10,15,20,25,30,35 min on ice. The homogenate was centrifuged at 4000 g for 10 min to remove cellular debris, and then, the supernatant is separated. The separated supernatant is again centrifuged at 15,000 g for 5 min to remove impurities. The supernatant is filtered through a 0.2 µm filter and is aliquoted and reserved in -80 °C refrigerator.
Example 5 - Tissues Treatment with a Cross-Linking Agent
The amniotic tissue and each of the micronized tissues are treated with crosslinking agent. The amount and concentration of tissue composition is determined according to the type of wound and then homogenized for 2 minutes. The homogenized tissues are treated with the crosslinking agent for a specified period. These agents can be one of the following factors or two of the following. The non-limiting example of cross linking agents are as follow: the crosslinking agent is selected from the group consisting of chemical crosslinker: aldehydes (e.g., glutaraldehyde), carbodiimides (e.g., 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide [EDC]) or isocyanates (e.g., hexamethylene diisocyanate [HMDI]); Photo initiator agents (e.g., rose Bengal, riboflavin); carbohydrates (e.g., ribose, glucose); plant extracts (e.g., oleuropein, genipin, and Myrica rubra); polyethylene glycol (PEG) polymers; physical crosslinker: such as dehydrothermal; UV irradiation; or biological crosslinker: (e.g., transglutaminase) or combination thereof.
Example 6 - Preparation of Tissue Graft
The amniotic membrane is placed on the surface of the epithelium which is placed on the surface of the drying fixture. The drying fixture is made of Teflon, Thermolon ceramic or Delrin. It’s intermediate layer is removed or Nat removed. With microneedle, fine pores are created on the surface having a diameter of 5-75 micrometers per cm2. Wharton jelly is first coated with a thin layer of amniotic membrane, then the particles/extract is dispersed as a single layer on the amniotic layer to cover its surface.
a. Full thickness burns
Coating material include: Placenta disc / umbilical Cord Matrix / chorionic membrane in the ratio of 8:4:2, 8:6:4,8:4:4,8:6:6, 8:5:3, or 8:3:3 with concentration selected from 0.5 mg /cm2, 1 mg /cm2,3 mg /cm2,5 mg /cm2,7 mg /cm2,10 mg/cm2,12 mg /cm2,15 mg /cm2, 20mg/cm2,25 mg /cm2, 30 mg/cm, 35 mg /cm2, 40 mg /cm2,45 mg /cm2,50 mg /cm2, 60 mg /cm2, 70 mg/cm2, 80mg/cm2, 90mg/cm2, 100 mg/cm, 150mg/cm2, 20mg/cm2, 300mg/cm2, 400mg/cm2,500mg/cm2 or 1000 mg/cm2 and ratio of total protein selected from 0.5 µg/dlit,0.7 µg/dlit,1 µg/dlit,1.2µg/dlit,1.5 µg/dlit,1.7 µg/dlit,2 µg/dlit,2.3 µg/dlit,2.5 µg/dlit,2.7 µg/dlit,3 µg/dlit,3.3 µg/dlit,3.5 µg/dlit,3.7 µg/dlit,4 µg/dlit,4.3 µg/dlit,4.5 µg/dlit,4.7 µg/dlit,5 µg/dlit,5.3 µg/dlit,5.5 µg/dlit,5.7 µg/dlit,6 µg/dlit,6.3 µg/dlit,6.5 µg/dlit,6.7 µg/dlit or 7 µg/dlit for cm2 placed on the chorionic surface of amniotic membrane and dispersed uniformly with cell spreaders that can be made from glass, plastic, or metal, in L or T shapes.
b. Full thickness diabetic foot ulcers
Coating material include: chorionic membrane / placenta disc/ umbilical cord matrix in ratio of 8:4:2, 8:6:4, 8:4:4, 8:6:6, 8:5:3, or 8:3:3 with concentration selected from 0.5 mg /cm2, 1 mg /cm2,3 mg /cm2,5 mg /cm2,7 mg /cm2,10 mg/cm2,12 mg /cm2,15 mg /cm2, 20mg/cm2,25 mg /cm2, 30 mg/cm, 35 mg /cm2, 40 mg /cm2,45 mg /cm2,50 mg /cm2, 60 mg /cm2, 70 mg/cm2, 80mg/cm2, 90mg/cm2, 100 mg/cm, 150mg/cm2, 20mg/cm2, 300mg/cm2, 400mg/cm2, 500mg/cm2 or 1000 mg/cm2; with thickness selected from 50 µm,100 µm,150 µm,200 µm,250 µm,300 µm,350 µm,400 µm,450 µm, or 500µm, placed on the chorionic surface of the amniotic membrane with crosslink method mentioned or with fibrin glue.
c. Partial-thick wounds
Coating material include: Placenta disc / umbilical Cord Matrix / chorionic membrane in ratio of 4:1:4, 4:2:4, 4:1:3, 3:1:4, 3:2:4, 2:1:4,2:2:4, or 2:0:4 with concentration selected form 0.5 mg /cm2, 1 mg /cm2,3 mg /cm2,5 mg /cm2,7 mg /cm2,10 mg/cm2,12 mg /cm2,15 mg /cm2, 20mg/cm2,25 mg /cm2, 30 mg/cm, 35 mg /cm2, 40 mg /cm2,45 mg /cm2,50 mg /cm2, 60 mg /cm2, 70 mg/cm2, 80mg/cm2, 90mg/cm2, 100 mg/cm, 150mg/cm2, 20mg/cm2, 300mg/cm2, 400mg/cm2,500mg/cm2 or 1000 mg/cm2 and in ratio of total protein selected from 0.5 µg/dlit,0.7 µg/dlit,1 µg/dlit,1.2µg/dlit,1.5 µg/dlit,1.7 µg/dlit,2 µg/dlit,2.3 µg/dlit,2.5 µg/dlit,2.7 µg/dlit,3 µg/dlit,3.3 µg/dlit,3.5 µg/dlit,3.7 µg/dlit,4 µg/dlit,4.3 µg/dlit,4.5 µg/dlit,4.7 µg/dlit,5 µg/dlit,5.3 µg/dlit,5.5 µg/dlit,5.7 µg/dlit,6 µg/dlit,6.3 µg/dlit,6.5 µg/dlit,6.7 µg/dlit, or 7 µg/dlit for cm2; is placed on the chorionic surface of amniotic membrane and dispersed uniformly with Cell spreaders that can be made from glass, plastic, or metal, in L or T shapes.
d. Dentistry
Coating material include: placenta disc / chorionic membrane / umbilical cord matrix in ratios of 4:1:2, 4:2:3, 4:1:3, 4:1:1, 4:2:2, or 4:0:1 with concentration selected from 0.5 mg /cm2, 1 mg /cm2,3 mg /cm2,5 mg /cm2,7 mg /cm2,10 mg/cm2,12 mg /cm2,15 mg /cm2, 20mg/cm2,25 mg /cm2, 30 mg/cm, 35 mg /cm2, 40 mg /cm2,45 mg /cm2,50 mg /cm2, 60 mg /cm2, 70 mg/cm2, 80mg/cm2, 90mg/cm2, 100 mg/cm, 150mg/cm2, 20mg/cm2, 300mg/cm2, 400mg/cm2,500mg/cm2 or 1000 mg/cm2 and in ratio of total protein selected from 0.5 µg/dlit,0.7 µg/dlit,1 µg/dlit,1.2µg/dlit,1.5 µg/dlit,1.7 µg/dlit,2 µg/dlit,2.3 µg/dlit,2.5 µg/dlit,2.7 µg/dlit,3 µg/dlit,3.3 µg/dlit,3.5 µg/dlit,3.7 µg/dlit,4 µg/dlit,4.3 µg/dlit,4.5 µg/dlit,4.7 µg/dlit,5 µg/dlit,5.3 µg/dlit,5.5 µg/dlit,5.7 µg/dlit,6 µg/dlit,6.3 µg/dlit,6.5 µg/dlit,6.7 µg/dlit or 7 µg/dlit for cm2; is placed on the chorionic surface of amniotic membrane and dispersed uniformly with Cell spreaders that can be made from glass, plastic, or metal, in L or T shapes.
e. Ophthalmology:
Coating material include: placenta disc / chorionic membrane / umbilical cord matrix in ratios of 4:0:3, 4:0:2, 4:0:2, or 4:0:0 with concentration selected from 0.5 mg /cm2, 1 mg /cm2,3 mg /cm2,5 mg /cm2,7 mg /cm2,10 mg/cm2,12 mg /cm2,15 mg /cm2, 20mg/cm2,25 mg /cm2, 30 mg/cm, 35 mg /cm2, 40 mg /cm2,45 mg /cm2,50 mg /cm2, 60 mg /cm2, 70 mg/cm2, 80mg/cm2, 90mg/cm2, 100 mg/cm, 150mg/cm2, 20mg/cm2, 300mg/cm2, 400mg/cm2,500mg/cm2 or 1000 mg/cm2 and in ratio of total protein selected from 0.5 µg/dlit,0.7 µg/dlit,1 µg/dlit,1.2µg/dlit,1.5 µg/dlit,1.7 µg/dlit,2 µg/dlit,2.3 µg/dlit,2.5 µg/dlit,2.7 µg/dlit,3 µg/dlit,3.3 µg/dlit,3.5 µg/dlit,3.7 µg/dlit,4 µg/dlit,4.3 µg/dlit,4.5 µg/dlit,4.7 µg/dlit,5 µg/dlit,5.3 µg/dlit,5.5 µg/dlit,5.7 µg/dlit,6 µg/dlit,6.3 µg/dlit,6.5 µg/dlit,6.7 µg/dlit, or 7 µg/dlit for cm2; is placed on the chorionic surface of amniotic membrane and dispersed uniformly with cell spreaders that can be made from glass, plastic, or metal, in L or T shapes.
f. Bilayer skin substitute
Epidermal layer: epithelial cell removed by mechanical and enzymatical method and intermediate layer is removed. Denuded amniotic membrane coated with combination of nanofiber or microfiber or nanoparticle or microparticle made of a polymer like as alginate oxide (used in concentration of 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 1.5%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, or 20%), cartos and placenta disc / umbilical cord matrix / chorionic membrane ratio of 4:1:4, 4:2:4, 4:1:3, 3:1:4, 3:2:4, 2:1:4, 2:2:4, or 2:0:4,with or without keratinocyte cell culture.
Dermal layer: Scaffolding using a chorionic membrane / placenta disc/ umbilical cord matrix in ratio of 8:4:2, 8:6:4,8:4:4,8:6:6, 8:5:3, or 8:3:3 and polymers like as collagen, gelatin, hyaluronic acid and chitosan with fibroblast cell culture and connecting this scaffold to the chorionic surface of the amniotic membrane.
Example 7 - Dehydration:
In preparation of tissue graft, tissues incubated in final solution PBS, Dextran (used in concentration range of 1mg/ml,2 mg/ml,3 mg/ml,4 mg/ml,5 mg/ml,6 mg/ml,7 mg/ml,8 mg/ml,9 mg/ml or 10 mg/ml); Sucrose (used in concentration range of 2 mg/ml, 4 mg/ml, 6 mg/ml, 8 mg/ml, 10 mg/ml, 12 mg/ml, 14 mg/ml, 16 mg/ml, 18 mg/ml or 20 mg/ml) and glycerol (used in concentration range of 2%,5%,7%,10%,12%,15%,20%,25%,30%,35%,40%,45%, or 50%) for 5 to 20 hours at 37 °C.
Biological composite sheet fabricates after amniotic membrane is coated with effective ingredients (particles/extract) on surface by drying fixture and stored in rate control freezer and temperature at 1°C/min decrease to -50°C.
Drying fixture placed in freeze-dryer with specific program as mentioned in table 1 below:
Step Temperature (°C) Duration
Loading -35 -
Freezing -50 2 hours
Freezing -27 30 – 60 min
Freezing -40 2 hours
Primary drying -27 10 hours
Secondary drying 10 4 hours
30 2 hours
Residual moisture %7-3
Example 8 - Packing:
Biological composite sheet is packaged in two pouches, the first pouch such as Tvyek®, manufactured by DuPont and second pouch such as aluminum foil.
Example 9 – Important features of biological composite sheet:
The present invention is an amniotic membrane coated and enriched with chorionic, placental and umbilical cord particles and extract and based on wound type. The addition of these elements to the amniotic membrane has improved the biological and physical properties of the amniotic membrane. Treatment of all components with crosslinking agent facilitates non-enzymatic bonding of macromolecules and proteins such as collagen and elastin etc. This structure having features such as safety, efficacy, biodegradable rate concomitant with wound contraction, terminal coverage. Free nerve and pain relief, prevent loss of body fluids, have ECM similar to skin, protect wound surface against bacteria, release beneficial growth factors to facilitate wound healing progression can be used as a tissue graft in wound healing and other soft tissues.
The biological composite sheet has below featured:
• They must be safe for the patient
• Clinically effective and efficient
• Ease of transport vulnerability
• Biodegradable
• Physical and mechanical properties similar to skin
• Reduce pain
• Cost effective
• Wound exudate management
• Protect the wound surface against bacteria
Diabetic wounds, can cause many problems for the patient, such as acute and chronic, full and partial thickness wounds, venous leg ulcers, arterial ulcers, pressure ulcers, post-surgical or post traumatic wound dehiscence, burns wounds.
Transplantation of this graft onto the diabetic wound due to the high adhesion of the tissue to the wound surface results in its full coverage and reduces the bacterial load on it by secreting its antibacterial factors such as human neutrophil peptides 1, 2 and, lysozyme [6], bactericidal/permeability-increasing protein (BPI), LL-37, calprotectin (MRP8/14) and ubiquitin, bactericidin, b-lysin, transferrin, 7S immunoglobulin, elfin, leukocyte proteinase inhibitor, human b3 defensin and cystatin E, barrier against microbial inoculation which plays an important role in a range of bacteria such as Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa or Enterobacter aerogenes that involved in reducing biofilm formation and infection control.
Other important features of this invention are the simultaneous presence of factors, proteins, and structures that each play an important role in wound healing. These include:
a. Angiogenesis
By preserving the properties of angiogenic factors and stimulating fibroblast cells, the vascular endothelial cells are proliferated, which facilitate the wound healing in chronic and untreatable wounds. Angiogenic factors are selected from angiopoietin-2 (ANG-2), vascular endothelial growth factor (VEGF), IL-8, angiogenin, interferon-?, IL-6, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) and platelet-derived growth factor (PDGF. The extracellular matrix amniotic membrane plays an important role in supporting the migration of endothelial cells due to the presence of RGD receptors.
b. Low Immunogenicity
Reduction or non-expression of molecules such as major histocompatibility complex class I (MHC1)—human leukocyte antigen (HLA), including antigens Ia (HLA-A, B, C) and Ib (HLA-G, E). HLA II class molecules (HLA-DP, -DQ, -DR) and costimulatory molecules (CD80, CD86) lead to low immunogenicity.
c. Anti-inflammatory
Placenta-derived and amniotic membrane cells have anti-inflammatory properties – by inhibiting T-cell proliferation, stimulate T-regulatory lymphocytes, blocking maturation of antigen-presenting cells (APCs), Monocytes inhibit dendritic cells and inhibit the migration of macrophages and natural killer (NK). They secreted many anti-inflammatory agents such as hyaluronic acid, interleukin-10 (IL-10), indoleamine 2,3-dioxygenase (IDO) enzyme, transforming growth factor ß (TGF-ß), hepatocyte growth factor (HGF), and prostaglandin E214,44,47,50,51, and reduce the expression of type 1 helper cells (Th1) inflammatory cytokines which reduces inflammatory cytokines. In addition, the secretory factors in peripheral blood monocytes by altering the differentiation of M1 to M2 macrophages, which enhances the anti-inflammatory profile of M2 regulatory cells such as macrophages. This effect is very useful in remedial medicine because it improves tissue and wound healing.
d. Antifibrotic Properties
The amniotic membrane reduces the risk of scarring and adhesion due to the secretion of TIMP-1, -2, -3 and -4, which reduces the activity of proteases and (suppresses IL-1, IL-6, IL-8 and inflammatory cells) in the wound area.
e. Growth Factors
Growth factors that help stimulate epithelialization and improve wound healing. The growth factors are selected from epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), heparin binding epidermal growth factor (HB-EGF), hepatocyte growth factor (HGF), platelet derived growth factor BB (PDGF-BB), placental growth factor (PlGF), and vascular endothelial growth factor (VEGF), pigment epithelium-derived factor (PEDF), tissue inhibitors of metalloproteinase 1, 2, 3, 4 (TIMP-1, TIMP-2, TIMP-3, TIMP-4), and thrombospondin-1 (VEGF)-A. These exosomes contain a large amount of alpha-2-macroglobulin (a2M), a protein that likely is the main component of exosomes in enhancing wound healing.
f. Mechanical strength:
Creating a bond between the free amide group and the carboxyl proteins of the amniotic membrane and other components, the double cross-linking of the components of the amniotic membrane increases the mechanical strength of the amniotic membrane. This helps to cover the wound surface for longer duration and play a role of the skin.
g. Release of biologic factors affecting wound healing:
Release of growth factors from biological composite sheet is so destruction and diffusion. Due to the reduced rate of degradation compared to fresh amniotic membrane, growth factors are released at the wound surface in several stages and there is a concentration of growth factor at the wound surface over a longer period of time than fresh amniotic membrane.
h. Structural and behavioural similarities to skin:
Biological composite sheet is composed of the basement membrane of the amniotic membrane and the particles or extract adhesive to the basement membrane.
The epithelium and basement membrane of the amniotic membrane coated with Wharton-rich hyaluronic acid gel like act the epidermis and form a suitable substrate for keratinocyte cell growth and particles and extracts effective ingredients play as a dermis-like are effective in the formation of dermal extracellular matrix. Umbilical cord matrix, on the one hand, covers the base of the amniotic membrane, on the other hand, covers the CM / PL particles, and ultimately forms the final structure by treatment and dehydration.
Example 10 - Preclinical efficacy studies
a. Total Protein Assay and Growth Factor Test:
Samples of 5 × 5 cm2 amniotic membrane and enriched amniotic membrane placed in buffer phosphate solution (PBS). It was then incubated at 37 °C on a shaker with gentle stirring for 24 h, 72 h and day 7. 100 µl was removed from the solution and stored at -80 °C. Protein concentration was measured in three replications by BCA (Bicinchoninic Acid) method. Total protein assay in 3 time point is described in table 2 below:
Time point AM (amniotic membrane) (µg/µl) EN-AM (Enriched amniotic membrane) (µg/µl)
24 hours 1/09±0.21 2/93±0.52
72 hours 1/73±0.32 2/37±0.35
7 days 1/91±0.33 3/19±0.71

b. BCA (Bicinchoninic Acid) Protein Assay:
In the microplate tubes, a serial dilution of BSA was prepared. 0.1 mL of each standard and sample into labelled test tubes was pipetted. 2.0 mL of water was added to each tube and mix by inversion. Water is used as blank solutions for standard curve and protein samples. Microplate tubes are incubated for 30 minutes in a 37°C in water bath. All tubes kept at room temperature for 10 min. Measure the absorbance at 562 nm. Fig. 2 describes the result report of the BCA (Bicinchoninic Acid) Protein Assay.
c. Study of proteins with SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis):
To prepare the sample, 27 ml of 5% SDS (weight / volume) was added to the samples. The mixture was homogenized after 2 min by a homogenizer at 11000 rpm. The homogenate mixture was incubated at 85°C for one hour until complete. Proteins soluble. The sample was centrifuged at 8500 g for 5 min at room temperature. The 1: 1 solution was mixed with buffer (including 0.5 M Tris-HCl at pH 8.6, SDS 4% and 20% glycerol) in the presence of 10% beta-mercaptoethanol.
20 µg of protein was loaded in 4% polyacrylamide gel for continuous gel and 10% for batch gel (15 mA flow). After electrophoresis, gel was stained with 0.1% coma R-250, 45% methanol and 10% gel electrophoresis. Detergent solution including water ratio (8): glacial acetic acid (1): methanol (1) (V / V / V), a pure protein ladder as a weight marker. A molecule of 10 to 118 kDa was used.
Electrophoresis results of dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The concentration of protein in group B1 at time 24h is higher than in group A1. The protein concentration in group B2 at time 72h is higher than that of group A2. The concentration of protein in group B3 at time Day 7 is higher than that of group A3. Note: Protein concentration was measured on SDS page and was repeated 3 times for all samples. Fig. 3 shows SDS page staining with Coomassie blue
d. In vitro scratch assay:
In vitro scratch assay is performed for study cell migration in vitro. The major advantages of this method are that it mimics to migration of cells in vivo and patterns of migration either as loosely connected population (e.g., fibroblasts) or as sheets of cells (e.g., epithelial and ECs) also mimic the behaviour of these cells during migration in vivo.
In current study, Human fibroblasts were obtained from dermatology clinic of ROYAN Institute. About 1× 105 fibroblasts were seeded into 60-mm dishes with 10 mg/ml fibronectin ECM substrates, cultured to confluence.
e. Scratch or wound assay
The fibroblast cell monolayer in a straight line scratched with a 0.1–10 µL pipette tip. The debris and smooth edge of the scratch removed by washing with 1 ml of the growth medium (DMEM f12) and then 5 ml of medium with Amniotic membrane and Enriched Amniotic membrane extract add to plate for 36h for the in vitro scratch assay.
The amniotic membrane extract and enriched amniotic membrane extract was provide and after scratching in the cells cultured in plate 6 well, the extract was used for wound healing and cell migration, which each of the extracts had a better effect on the cell, which could more rapidly induce cell migration and complete the space created by the scratch.
Negative control received serum-free medium without AME and positive control well were fed with complete culture medium (DMEM+FBS10%+AB1%).
To obtain the same field during the image acquisition created a marking to be used as reference points close to the scratch. The reference points could be made by etching the dish lightly with a razor blade on the outer bottom of the dish.
The dish is placed in a cell culture incubator at 37 °C for 24 h. At 0, 12, 24 and 36 hours after scratching, digital images of cells were captured by a called Olympus cell Sense software equipped with a digital camera.
Percentage of scratch closure was calculated as follows = [(At0 -At)/At0]*100, in which at is the scratch area at time 0, and at is the correspondent scratch area at 12, 24, and 36 hours. Fig. 4 shows fibroblast cell migration after scratching. Fig. 5 describes the ratio of cell migration in Scratch assay. Table 3 below describes percentage scratch closure.
Time point AM
(% Scratch closure) Control +
(% Scratch closure) Control –
(% Scratch closure) Enriched AM
(% Scratch closure)
0 0 0 0 0
12 hours 31.84±0.9 27.27±1.6 26.33±0.6 34.39±0.8
24 hours 63.33±1.5 61.66±1.8 56.11±0.8 71.47±1.3
36 hours 100±1.1 100±0.9 83.38±1.2 100±0.9

Example 11 - Toxicity and safety testing
a. Cytotoxicity
Cytotoxicity testing is performed by MTT assay in (Mitochondrial activity of cells) accordance with the following steps:
Six samples of the amniotic membrane product of each of them are cut by 1 cm2. Fibroblast cells in the logarithmic phase of the trypsinized flasks and 9 samples each of which 500 thousand cells are inoculated within the wells of the two plates of 6 houses. Plates in the incubator 37 °C with CO2 5% are kept for 2 days. After 2 days of culture Medium, DMEM + 10% FBS is replaced by a fresh medium. The sample of amniotic membrane in the size of 1 × 1 cm2 carefully positioned in the middle of the plate wells and 3 wells remain as a witness.
As a negative control, three pieces of amniotic membrane are incubated in 70% ethanol as described above to completely kill their cells and put in three separate wells. After 24 hours, the specimens are carefully removed from the cells and the culture medium is pulled. All wells are washed with PBS to remove non-adhesive cells. Each well is supplemented with 2970 µl of MTT solution at a concentration of 0.5 mg / ml and the plates are incubated in a 37 °C incubator with 5% CO2 for 4 h. After 4 hours, the sample is extracted and 2970 µl of isopropanol is added. After 3 h, the optical absorption induced by formazan crystals dissolved in isopropanol is calculated at 570 nm. The survival coefficient of the sample cells was calculated according to the following formula:

Fig. 6 describes report of irradiation and skin sensitization study on biological composite sheet
b. Preclinical and laboratory in-vivo evaluation
17 mice were prepared for this test. Animals kept in quarantine for a week, to ensure they are well adapted to new conditions and health, once they are received. The health, respiratory system, skin health and quality of eating of these animals evaluated during this period.
Pre-surgical procedures:
The animals under study are first examined and then weighted. After calculating the dose of anesthetic drugs for each animal (ketamine 80 mg / kg and xylazine 5 mg / kg), the shaving of the desired area is done using a special shaving gel. Asepsis and preparation of the clean room were performed, and the animals were divided into 3 groups of 5 to study. Next, the wound in the back of the animal using a surgical punch with dimensions of 2×2 cm2 for Full thickness dermal defect is created. Then, the prepared Biological Composite Sheet, 2/5 cm in size, are placed on the wound and fixed to the skin by suture.
It is noted that in the control group, the membrane is not transplanted and the wound is only dressed. Three-layer wound dressings in this project include:
1- Covering the wound site or grafting with sterile dressing pads (like as tegadarm)
2- Absorption of potential ulcers by using cotton band (Vibriel)
3- Fixing the dressing using a long-woven band and leukoplast adhesive.
Post-operative measures:
Animal studies after surgeries were performed for 30 to 45 minutes in the warm place at a temperature of 37 degrees for recovery. They also received antibiotics (gentamicin 15mg / kg) and analgesics (tramadol 5mg / kg) for 5 days after complete removal from anesthesia to prevent possible secondary infections and to reduce postoperative pain.
Efficacy In vivo: Examination of the wound healing process:
The wound dressing is changed every 2 days and the wound healing conditions are checked. Wounds are measured using a graded ruler. This review will continue for 21 days.
Irritation and Intra-cutaneous reactivity:
In addition to animal testing, pathological tests are also performed. Histological examination of the location of the grafts for mice used in the animal test is also performed.
At the end of 21 days of study, mice in each group were sacrificed according to the ethics of working with the animal, and the wound sample was removed from the skin behind them and sent to the pathology laboratory for pathological examination.
Cut the samples first to obtain two or three separate sections of each samples (from the margins of the samples and the middle of the samples). The samples were then blocked in paraffin. The cuts were prepared by microtome and fixed on the slide. Hematoxylin-Eosin staining (H&E, basic staining) and Masson's trichrome staining (collagen staining) were performed on the slides of each sample and after preparation, for standard microscopic detection of the samples, the following standard method was used as the evaluation method. And the relevant scoring and indexing. Indicators are used for semi-quantitative evaluation include: a) Epithelization; b) Inflammatory cells (PMNL); c) Fibroblasts; d) New vessels (vascularization); e) Collagen
c. Acute toxicity:
To assess acute toxicity within the first 24 hours after general health transplantation, the appetite and motility and body temperature of the laboratory animal were examined every hour with symptoms such as anorexia, immobility, increase or decrease in body temperature, tremor. There is no need to observe sweating or urination.
Note: Due to the locality of the product used and the likelihood of the product being removed by the laboratory from the liver and kidney, the above case will not be evaluated.
d. Sub Chronic Toxicity Test:
To evaluate the Sub Chronic Toxicity Test within 14 days after a general health transplant, the appetite and motility and body temperature of the animal under study are checked every hour with symptoms such as anorexia, immobility, increase or decrease in body temperature. Do not observe, tremble, sweat, or decrease urine.
Fig. 7 shows wound created in the back of the animal using a surgical punch with dimensions of 2×2 cm2 for Full thickness dermal defect
Fig. 8 shows the H&E stained sections of post wounding
Fig. 9. shows the H&E stained after day 21 and re-epithelialization process was completed