Claims:We claim,
1. A biological composite sheet comprising an amniotic membrane; micronized tissue particles of chorionic membrane, umbilical cord matrix and placenta disc; and extracts of micronized tissue particles;
wherein extracts are prepared by sonication and ultracentrifugation of micronized tissue particles;
wherein micronized tissue particles are applied on one side of the amniotic membrane; and extracts are applied on the micronized tissue particles;
wherein the ratio of chorionic membrane to umbilical cord matrix to placenta disc is 8:4:2;
wherein the amniotic membrane is placed on the surface of epithelium which is placed on the surface of the drying fixture;
wherein the pore size of micronized tissue particles is in the range of 70 to 350 µm; and
wherein the amniotic membrane, micronized tissue particles and extract of micronized tissue particles are treated with crosslinking agent.
2. The biological composite sheet of claim 1 wherein, the crosslinking agent is 1-ethyl-3- (3-dimethylamino propyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) with a concentration of 0.3-1 mM.
3. The biological composite sheet of claim 1 wherein, the extracts of micronized particles are responsible for fast release of growth factors; and the micronized particles are responsible for sustain release of growth factors;
wherein the growth factors are selected from Fibroblast Growth Factor 2 (FGF2), Vascular Endothelial Growth Factor (VEGF), Platelet Derived Growth Factor BB (PDGF-BB) and Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF).
4. The biological composite sheet of claim 1 wherein, the drying fixture is made up of Polytetrafluoroethylene (Teflon), Thermolon ceramic or Polyoxymethylene (Delrin).
5. A method of preparation of biological composite sheet of claim 1 comprising the steps:
a. micronizing the umbilical cord matrix, chorion membrane and placenta disc by mechanical grinding; wherein the pore size of the micronized tissue particles is in the range of 70 to 350 µm and; wherein the ratio of chorionic membrane to umbilical cord matrix to placenta disc is 8:4:2;
b. applying the said the micronized tissue particles on the one side of the amniotic membrane;
c. preparing extract using sonication and ultracentrifugation of micronized particles of step (a);
d. applying the extract obtained in step (c) on the surface of micronized particles which is coated on the one side of the amniotic membrane as in step (b);
e. treating component of step (d) with cryoprotectant and cytoprotectant agent; and
f. dehydrating the component of step (e) by lyophilization to obtain the final product;
wherein the size of amniotic membrane is 3×3 cm2, 2×4 cm2, 5×5 cm2, 6×6 cm2 or 10×10 cm2;
wherein the amniotic membrane is placed on the surface of epithelium which is placed on the surface of the drying fixture; and
wherein the amniotic membrane, micronized tissue particles and extract are treated with crosslinking agent.
6. The method of preparation of biological composite sheet of claim 5 wherein, the crosslinking agent is 1-ethyl-3- (3-dimethylamino propyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) with a concentration of 0.3-1 mM.
7. The method of preparation of biological composite sheet of claim 5 wherein, the extracts of micronized particles are responsible for fast release of growth factors; and the micronized particles are responsible for sustain release of growth factors;
wherein the growth factors are selected from Fibroblast Growth Factor 2 (FGF2), Vascular Endothelial Growth Factor (VEGF), Platelet Derived Growth Factor BB (PDGF-BB) and Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF).
8. The method of preparation of biological composite sheet of claim 5 wherein, the drying fixture is made up of Polytetrafluoroethylene (Teflon), Thermolon ceramic or Polyoxymethylene (Delrin).

, Description:This invention is an improvement or modification of the invention claimed in the complete specification of the main patent application no. 202021017005 / main patent no. 366526, having date of filing April 20, 2020.
Title:
Biological composite sheet comprising of enriched amniotic membrane
Field of the invention:
The present invention is related to biological composite sheet comprising an amniotic membrane, micronized particle of chorionic membrane, umbilical cord matrix and placenta disc and extracts of micronized tissue particles; wherein the one side of the amniotic membrane is coated and enriched with micronized particle of chorionic membrane, umbilical cord matrix and placenta disc, and the extracts are applied on the micronized particle. 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.
The Indian patent IN366526 discloses a biological composite sheet comprising an amniotic membrane and micronized tissue particles of chorionic membrane, umbilical cord matrix and placenta disc; wherein micronized tissue particles are applied on one side of the amniotic membrane.
In the present invention, the biological composite sheet comprising an amniotic membrane, micronized particle of chorionic membrane, umbilical cord matrix and placenta disc and extracts of micronized tissue particles; wherein the one side of the amniotic membrane is coated and enriched with micronized particle of chorionic membrane, umbilical cord matrix and placenta disc, and the extracts are applied on the micronized particle. The extracts are prepared by sonication and ultracentrifugation of micronized tissue particles. The biological composite sheet of the present invention has improved biological and physical properties. The invention further related to the method of preparation of biological composite sheet and its use in would healing.
Summary of the invention:
The present invention is related to the biological composite sheet comprising an amniotic membrane, micronized particle of chorionic membrane, umbilical cord matrix and placenta disc and extracts of micronized tissue particles; wherein the one side of the amniotic membrane is coated and enriched with micronized particle of chorionic membrane, umbilical cord matrix and placenta disc, and the extracts are applied on the micronized particle. The extracts are prepared by sonication and ultracentrifugation of micronized tissue particles. The biological composite sheet of the present invention has improved biological and physical properties.
In one embodiment of the present invention, biological composite sheet comprising an amniotic membrane, micronized particle of chorionic membrane, umbilical cord matrix and placenta disc and extracts of micronized tissue particles; wherein the one side of the amniotic membrane is coated and enriched with micronized particle of chorionic membrane, umbilical cord matrix and placenta disc, and the extracts are applied on the micronized particle. The extracts are prepared by sonication and ultracentrifugation of micronized tissue particles
In one aspect, the ratio of chorionic membrane to umbilical cord matrix to placenta disc used in the biological composite sheet are 8:4:2, 8:6:4, 8:4:4, 8:6:6, 8:5:3, 8:3:3, 4:1:4, 4:2:4, 4:1:3, 3:1:4, 3:2:4, 2:1:4, 2:2:4, 4:1:2, 4:2:3, 4:1:3, 4:1:1 or 4:2:2. The most preferred ratio of chorionic membrane to umbilical cord matrix to placenta disc used in the biological composite sheet is 8:4:2. The pore size of micronized tissue particles is in the range of 70 to 350 µm.
In another aspect, the amniotic membrane is placed on the surface of epithelium which is placed on the surface of the drying fixture. The drying fixture is made of Polytetrafluoroethylene (Teflon), Thermolon ceramic or Polyoxymethylene (Delrin). The amniotic membrane, micronized tissue particles and extract of micronized tissue particles are treated with crosslinking agent.
In another embodiment, the cross-linking agents are used for the adhesion of mixture of chorionic membrane, umbilical cord matrix and placenta disc particles to the surface of the amniotic membrane.
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:
Figure 1 describes biological composite sheet comprising an amniotic membrane; micronized particle of chorionic membrane, umbilical cord matrix and placenta disc; and extracts of micronized tissue particles; wherein the one side of the amniotic membrane is coated and enriched with micronized particle of chorionic membrane, umbilical cord matrix and placenta disc, and the extracts are applied on the micronized particle.
Figure 2 shows a graphical representation of Total protein assay
Figure 3 shows SDS page staining with Coomassie blue
Figure 4 shows fibroblast cell migration after scratching.
Figure 5 shows generation of full-thickness steps and silicon ring fixation.
Figure 6A shows time-point images from in vivo would healing study
Figure 6B shows histological analysis of wound healing
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 amniotic 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.
The amniotic membrane and powdered mixture of chorionic membrane, umbilical cord matrix and placenta disc are used in preparation of biological composite sheet, wherein the membrane or membrane particles are collected from Placenta and Umbilical cord. The biological materials (Placenta and Umbilical cord) were procured form International Bionano Institute (IBI), Section 150, Golsorkh 13, Mahestan Blvd, Shams Abad, Tehran, Iran. Alternatively, no material used to prepare biological composite sheet was procured form India.
After the delivery of the new born, the placenta and umbilical cord are discarded by the hospitals as it is considered as medical waste or biohazard material. Amniotic membrane and chorionic membrane are part of the placenta. The biological composite sheet comprising amniotic membrane coated with particle made up of powdered mixture of chorionic membrane, umbilical cord matrix and placenta disc. The membranes or membrane particles are collected from Placenta and Umbilical cord which are medical waste material for the hospitals. Thus, in preparation of the inventive biological composite sheet, no harm is made to human embryo or any human organ. Also, there is no need to depend on the human donor to obtain the membranes or membrane particles.
In one embodiment of the present invention, biological composite sheet comprising an amniotic membrane; micronized particle of chorionic membrane, umbilical cord matrix and placenta disc; and extracts of micronized tissue particles; wherein the one side of the amniotic membrane is coated and enriched with micronized particle of chorionic membrane, umbilical cord matrix and placenta disc, and the extracts are applied on the micronized particle. The extracts are prepared by sonication and ultracentrifugation of micronized tissue particles. This embodiment is described in Figure 1.
In one aspect, the ratio of chorionic membrane to umbilical cord matrix to placenta disc used in the biological composite sheet are 8:4:2, 8:6:4, 8:4:4, 8:6:6, 8:5:3, 8:3:3, 4:1:4, 4:2:4, 4:1:3, 3:1:4, 3:2:4, 2:1:4, 2:2:4, 4:1:2, 4:2:3, 4:1:3, 4:1:1 or 4:2:2. The most preferred ratio of chorionic membrane to umbilical cord matrix to placenta disc used in the biological composite sheet is 8:4:2. The pore size of micronized tissue particles is in the range of 70 to 350 µm.
In another aspect, the amniotic membrane is placed on the surface of epithelium which is placed on the surface of the drying fixture. The drying fixture is made of Polytetrafluoroethylene (Teflon), Thermolon ceramic or Polyoxymethylene (Delrin). The amniotic membrane, micronized tissue particles and extract of micronized tissue particles are treated with crosslinking agent.
In certain embodiment, the cross-linking agents are used for the adhesion of mixture of chorionic membrane, umbilical cord matrix and placenta disc particles to the surface of the amniotic membrane. The crosslinking is carried out using chemical cross-linking agents, physical cross linking or by biological cross-linking agents. The chemical crosslinking agents are selected from aldehydes such as glutaraldehyde, carbodiimides such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide [EDC], isocyanates such as hexamethylene diisocyanate [HMDI], photo initiator agents such as rose Bengal, riboflavin, carbohydrates such as ribose, glucose, plant extracts such as oleuropein, genipin, and Myrica rubra, polyethylene glycol (PEG) polymers. The physical crosslinking agents are selected from dehydrothermal, UV irradiation. The biological crosslinking agents are selected from transglutaminase.
In yet another embodiment, biological composite sheet comprising an amniotic membrane; micronized particle of chorionic membrane, umbilical cord matrix and placenta disc; and extracts of micronized tissue particles; wherein the extracts of micronized particles are responsible for fast release of growth factors (such as Fibroblast Growth Factor 2 (FGF2), Vascular Endothelial Growth Factor (VEGF), Platelet Derived Growth Factor BB (PDGF-BB) and Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF)), and the micronized particles are responsible for sustain the release of growth factors (such as F Fibroblast Growth Factor 2 (FGF2), Vascular Endothelial Growth Factor (VEGF), Platelet Derived Growth Factor BB (PDGF-BB) and Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF)).
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 non infected wounds.
Different ratio/concentration of coating/enriching material (mixture of chorionic membrane, umbilical cord matrix and placenta disc particles and extracts of the micronized particles) 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 on one side of an amniotic membrane in a scaffold, layer or as a coating, with crosslinking agent.
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. powdering 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. preparing extract using sonication and ultracentrifugation of micronized particles of step (n);
q. coating the surface of the amniotic membrane with particles obtained in step (n);
r. applying the extract on the surface of micronized particle, which is coated on the amniotic membrane as in step (q);
s. treat component of step (r) with cryoprotectant and lyoprotectant agent;
t. dehydration by lyophilization;
u. packaging
In another embodiment of the present invention, the method of preparation of biological composite comprises:
a. powdering the umbilical cord matrix, chorion membrane and placenta disc by cryogenic method or by sonication;
b. incubation of tissues in preservative solution for overnight;
c. preparing extract using sonication and ultracentrifugation of micronized particles of step (a);
d. sizing and cutting the amniotic membrane, wherein the size is selected from 3×3 cm2, 2×4 cm2, 5×5 cm2, 6×6 cm2 and 10×10 cm2;
e. coating the surface of the amniotic membrane with particles obtained in step (a);
f. applying the extract on the surface of micronized particle, which is coated on the amniotic membrane as in step (e);
g. treating component of step (f) with cryoprotectant and cytoprotectant agent; and
h. dehydrating the component of step (g) by lyophilization.
In another embodiment of the present invention, the method of preparation of biological composite comprises:
a. powdering the umbilical cord matrix, chorion membrane and placenta disc by cryogenic method or by sonication; treating the particles of step (a) with crosslinking agent;
b. preparing extract using sonication and ultracentrifugation of micronized particles of step (a);
c. coating the one side of the amniotic membrane with particles of step (a);
d. applying the extract prepared by step (b) on the surface of micronized particle, which is coated on the one side of amniotic membrane as in step (c);
e. treating component of step (d) with cryoprotectant and cytoprotectant agent; and
f. dehydrating the component of step (e) by lyophilization.
In another embodiment of the present invention, the method of preparation of biological composite comprises:
a. micronizing the umbilical cord matrix, chorion membrane and placenta disc by mechanical grinding; wherein the pore size of the micronized tissue particles is in the range of 70 to 350 µm and; wherein the ratio of chorionic membrane to umbilical cord matrix to placenta disc is 8:4:2;
b. applying the said the micronized tissue particles on the one side of the amniotic membrane;
c. preparing extract using sonication and ultracentrifugation of micronized particles of step (a);
d. applying the extract prepared by step (c) on the surface of micronized particles which is coated on the one side of the amniotic membrane as in step (b);
e. treating component of step (d) with cryoprotectant and cytoprotectant agent; and
f. dehydrating the component of step (e) by lyophilization to obtain the final product;
wherein the size of amniotic membrane is 3×3 cm2, 2×4 cm2, 5×5 cm2, 6×6 cm2 or 10×10 cm2;
wherein the amniotic membrane is placed on the surface of epithelium which is placed on the surface of the drying fixture; and
wherein the amniotic membrane, micronized tissue particles and extract are treated with crosslinking agent.
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 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
Micronized particles are combined in Dulbecco's Modified Eagle F-12 medium (DMEM-F12, 1:1) and sonicated (UP200S-Heilescher) 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. The extract obtained is free of cellular debris and contains the growth factors such as Fibroblast Growth Factor 2 (FGF2), Vascular Endothelial Growth Factor (VEGF), Platelet Derived Growth Factor BB (PDGF-BB) and Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF).
Example 5 - Tissues Treatment with a Cross-Linking Agent
The amniotic tissue and each of the micronized tissues are treated with crosslinking agent. The homogenized tissues are treated with the crosslinking agent. The crosslinking is carried out using chemical cross-linking agents, physical cross linking or by biological cross-linking agents. The chemical crosslinking agents are selected from aldehydes such as glutaraldehyde, carbodiimides such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide [EDC], isocyanates such as hexamethylene diisocyanate [HMDI], photo initiator agents such as rose Bengal, riboflavin, carbohydrates such as ribose, glucose, plant extracts such as oleuropein, genipin, and Myrica rubra, polyethylene glycol (PEG) polymers. The physical crosslinking agents are selected from dehydrothermal, UV irradiation. The biological crosslinking agents are selected from transglutaminase.
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 Polytetrafluoroethylene (Teflon), Thermolon ceramic or Polyoxymethylene (Delrin). Its 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 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.
Then, the extract obtained from sonication and ultracentrifugation of micronized particles are applied on the surface of micronized particles which is coated on 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 and 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 – Best method of performing the invention:
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, and methods described and claimed herein are made and evaluated, and are intended to be purely exemplary and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., concentration, ratios of particles used, etc.) but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric. There are numerous variations and combinations of reaction conditions, e.g., powdered mixture, concentrations, solvent mixtures, temperatures, pressures and other reaction ranges and conditions that can be used to optimize the product purity and yield obtained from the describe process. Only reasonable and routine experimentation will be required to optimize such process conditions.
Example 9a -Preparation of micronized particles mixture
The umbilical cord matrix, chorion membrane and placenta disc are obtained from placental tissue originating in a hospital, where it was collected during a Cesarean section birth. The powdering/micronization of umbilical cord matrix, chorionic membrane and placenta disc were performed using Knife mill PULVERISETTE 11 and UP200S-Heilescher sonicator, individually. The particles mixture was obtained by mixing the 8 parts of powdered/micronized chorionic membrane, 4 parts of powdered/micronized umbilical cord matrix and 2 parts of powdered/micronized placenta disc wherein the particle size of placenta disc is 70 to 350 µm having polymorphic shapes, the particle shape of umbilical cord matrix is substrate pattern and the particle shape of chorionic membrane is flat with two side.
Example 9b – Preparation of extract
Micronized particles obtained in example 9a are combined with Dulbecco's Modified Eagle Medium F-12 medium (DMEM-F12, 1:1) and sonicated (UP200S-Heilescher) 3-times in maximum power 25% duty cycle for 20 min at 4 ºC. 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 Extract concentration is 1.5 mg / cm2 of total protein optimized.
Example 9c -Preparation of biological composite sheet/film
The biological composite sheet is prepared as follow: (1) sizing and cutting the amniotic membrane wherein the size is 3×3 cm2, (2) the particles made up of powdered mixture of chorionic membrane, umbilical cord matrix and placenta disc using the ratio of 8:4:2, respectively (as explained in Example 9a) is treated with carbohydrates agent (sucrose) in concentrations (4 -14 mg/ml) for duration time 5-20 hours at 37 °C and humidity at 85-95%, CO2 5% and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) with a concentration of 0.3-1 mM for duration time 5-20 hours at 37 °C and humidity at 85-95%, CO2 5%, (3) the treated particles having the amount of 0.1-14 mg/cm2 is poured on one side (single surface) of amniotic membrane and dispersed uniformly with cell spreaders that can be made from glass, plastic, or metal, in L or T shapes and freeze at -80°c for 24 hours, (4) extract prepared by sonication and ultracentrifugation of micronized tissue particles (as explained in Example 9b) is applied on the surface of micronized particles which is coated on the one side (single surface) of the amniotic membrane (as explained in step 3); wherein the Extract concentration is 1.5 mg/cm2 of total protein, (5) treating the coated amniotic membrane with cryoprotectant wherein, tissues are incubated in PBS, Dextran (3-5 mg/ml); Sucrose (4-5 mg/ml) and glycerol (5-7 mg/ml) for 18 hours at 37 °C and humidity 85-95%, CO2 5%, (6) Biological composite sheet fabricates after amniotic membrane is coated with particles followed by 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 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 9d -Preparation of biological composite sheet/film
In another aspect, the biological composite sheet can be prepared as follows: (1) sizing and cutting the amniotic membrane wherein the size is 3×3 cm2, (2) the particles made up of powdered mixture of chorionic membrane, umbilical cord matrix and placenta disc using the ratio of 8:4:2, respectively (as explained in Example 1) is treated with carbohydrates agent (sucrose) in concentrations (4 -14 mg/ml) for duration time 5-20 hours at 37 °C and humidity at 85-95%, CO2 5% and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) with a concentration of 0.3-1 mM for duration time 5-20 hours at 37 ° C and humidity at 85-95%, CO2 5%, (3) the micronized particles and the extract of of micronized tissue particles (as explained in Example 9b) applied on one side (single side) of amniotic membrane dispersed uniformly with cell spreaders that can be made from glass, plastic, or metal, in L or T shapes and freeze at -80°c for 24 hours; (5) treating the coated amniotic membrane with cryoprotectant wherein, tissues are incubated in PBS, Dextran (3-5 mg/ml); Sucrose (4-5 mg/ml) and glycerol (5-7 mg/ml) for 18 hours at 37 °C and humidity 85-95%, CO2 5%, (6) Biological composite sheet fabricates after amniotic membrane is coated with particles followed by 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 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

Various modifications and variations can be made to the compounds, compositions and methods described herein. Other aspects of the compounds, compositions and methods described herein will be apparent from consideration of the specification and practice of the compounds, compositions and methods disclosed herein. It is intended that the specification and examples be considered as exemplary.
Example 10 – 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, 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 11 – Experimental studies
a. Total Protein Assay and Growth Factor Test:
Samples of 5 × 5 cm2 amniotic membrane, amniotic membrane coated with micronized particles, amniotic membrane coated with extract and amniotic membrane coated with micronized particles followed by extract 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 is explained by figure 2. The A1 represents amniotic membrane extract; B1 represents amniotic membrane coated with micronized particles; B2 represents amniotic membrane coated with extract; and B3 represents amniotic membrane coated with micronized particles followed by extract (the product of present invention).
b. 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.
Figure 3 shows SDS page staining with Coomassie blue. Protein concentration was measured on SDS page and was repeated 3 times for each Group A and B. Group A1, A2 and A3 are related to amniotic membrane, Group B1 represents amniotic membrane coated with micronized particles; Group B2 represents amniotic membrane coated with extract; and Group B3 represents amniotic membrane coated with micronized particles followed by extract (the product of present invention). 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.
c. In vitro scratch wound model assay:
The in vitro scratch assay is an easy, low-cost and well-developed method to measure cell migration in vitro. The basic steps involve creating a “scratch” in a cell monolayer, capturing the images at the beginning and at regular intervals during cell migration to close the scratch, and comparing the images to quantify the migration rate of the cells. Compared to other methods, the in vitro scratch assay is particularly suitable for studies on the effects of cell–matrix and cell–cell interactions on cell migration, mimic cell migration during wound healing in vivo and are compatible with imaging of live cells during migration to monitor intracellular events if desired. Besides monitoring migration of homogenous cell populations, this method has also been adopted to measure migration of individual cells in the leading edge of the scratch. Not taking into account the time for transfection of cells, in vitro scratch assay per se usually takes from several hours to overnight.
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.
Test protocol:
Human Fibroblast cells about 250,000 were seed into each of the plate (6 well) and incubated for 36 hours in 37 C incubator with 5% CO2 to 80% confluency.
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 phosphate buffer saline.
Samples of 1 x 1 cm2 amniotic membrane extract (A), amniotic membrane coated with micronized particles (B1); amniotic membrane coated with extract (B2); and amniotic membrane coated with micronized particles followed by extract (B3) (the product of present invention) placed in 2500 µL free serum. It was then incubated at 37 C on a shaker with gentle stirring for 36 h. Each time point (1h, 12 h, 24 h and 36 h) 2500 µL was removed and add new free serum. The extracts were store in -80 C freezer.
The extracts were used for wound healing and cell migration, which each of the extracts has a better effect on the cell migration, which could more rapidly induce cell migration and complete the area created by the scratch.
Negative control received serum-free medium.
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 plate is placed in a cell culture incubator at 37 C for 36 h. At 0, 12, 24, and 36 hours after scratching, digital image of cells were record by an 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.
Test result:
Scratch area reduction done by fibroblast migration and proliferation.
After 12 h more than 60% of scratch area was reduced. The complete closure of scratch area in amniotic membrane coated with micronized particles followed by extract (B3) was after 24 h, while the complete closure of scratch area in amniotic membrane coated with micronized particles (B1) and amniotic membrane coated with extract (B2) was after 36 hours.
Figure 4 shows fibroblast cell migration after scratching. Control is DEME without FBS; A represents amniotic membrane extract; B1 represents amniotic membrane coated with micronized particles; B2 represents amniotic membrane coated with extract; and B3 represents amniotic membrane coated with micronized particles followed by extract (the product of present invention). The product of Group B3 has faster scratch closure compare to the product of Group B1 and B2.
d. In Vivo Efficacy Assay (in animal):
Test Protocol:
Introduction:
According to the World Health Organisation, about 164 million people suffer from skin wounds in the world and the distribution of various types of wounds in many regions of the world are similar. Wound type, anatomical depth and underlying diseases are important variable in wound healing. Full-thickness wound healing, process is the complexity of the activity and coordination of intracellular, intercellular and extracellular. Standard treatment for full-thickness wounds diameter greater than 1 cm2, the use of skin autograft is this method with problems such as new wounds at the site of transplantation, pain and lack the source is in patients with high burn rate and scars for replacement autograft uses allografts or dermis products. One of the products of allografts for the treatment of wounds is the use of amniotic membranes.
In order to evaluate the biocompatibility and effectiveness of our product, tests were performed in accordance with the relevant standard and we concluded that this product is biocompatible and effective.
Generation of full-thickness skin models:
After the approval of the animals, the ethics committee will conduct all the tests. 17 wistar rats (male) weight 260 g were preparing from the Animal Care Center of Royan Research Institute. The animals were kept under standard conditions of 21 C and 12 hours cycle of dark light with free access to water and food. The rats were then randomly divided into four groups.
For diabetic rats streptozotocin (STZ) injected intraperitoneally for duration of 5 minutes with at a dose of 150 mg/kg after 14 hours of fasting.
Blood sugar was checked after 72 hours STZ injection and rats with a blood sugar higher than 17 mmol/l were considered diabetic.
Rats were anesthetized by intraperitoneal injection of xylosine and vectamine as prescribed (Ketamine 80 mg/kg and Xylazine 5 mg/kg). Asepsis procedures and preparation of a clean room and surgery site were performed. The hair on the back of the neck and between the two shoulder was shaved with shaving gel and shaver, and then the skin of this area was surgically removed to a full thickness of 2 cm diameter and used silicone ring for prevention of contraction.
Silicone ring thickness were 1 mm, inner diameter of 2 cm and outer diameter of 3 cm and that’s prepared from the engineering department of Royan Research Institute and its sutured with 3-0 nylon thread at the wound site. Figure 5 shows a generation of full-thickness steps and silicon ring fixation
It is noted that in the control group, the membrane is not transplanted and the wound is only dressed. Study group include:
1. Group 1: Control
2. Group 2: amniotic membrane coated with micronized particles (EHAM 1)
3. Group 3: amniotic membrane coated with extract (EHAM 2)
3. Group 4: amniotic membrane coated with micronized particles followed by extract (the product of present invention) (EHAM 3)
Post-operative measures:
Animals after surgeries were recovery for 30 to 45 minutes in the warm place at a temperature of 37 C. They also received antibiotics (Gentamicin 15 mg/kg) and analgesics (Tramadol 5 mg/kg) for 5 days after complete removal from anaesthesia to prevent possible secondary infections and to reduce postoperative pain.
Efficacy In vivo: Examination of the wound healing process:
Secondary 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, rats in each group were sacrificed according to the ethics of working with the animal, and the sample was removed from the skin 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
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 1 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.
Sub Chromic Toxicity Test
To evaluate the Sub Chromic 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.
Clinical evaluation/Conclusion
Images shows that at difference time point after treatment with Group 4: amniotic membrane coated with micronized particles followed by extract (the product of present invention) (EHAM 3), there is no sign of inflammation or infection formation in the wounds site. It was observed that new epithelialization formed in the treated group with EHAM 3 and as a result the wound surface decreased. The duration of wound closure in EHAM 3 was 3 weeks. The duration of would closure in group EHAM 3 was longer than the other groups. The rate of angiogenesis in group EHAM 3 was significantly higher than the other groups. In group EHAM 2, would healing and angiogenesis were higher than group EHAM 1. Figure 6A shows time-point images from in vivo would healing study.
Figure 6B shows histological analysis of wound healing. Total skin thickness measurements showed an increased thickness of EHAM 2 and EHAM 3 treated skin compared with EHAM 1 and untreated wounds (Control). Also, there are significantly more blood vessels in EHAM 3 treated animal compared to EHAM 1, EHAM 2 and untreated groups (Control).