EP4048298A1 - Herstellung und verwendung von therapeutischen hydrogelen - Google Patents

Herstellung und verwendung von therapeutischen hydrogelen

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Publication number
EP4048298A1
EP4048298A1 EP20879268.9A EP20879268A EP4048298A1 EP 4048298 A1 EP4048298 A1 EP 4048298A1 EP 20879268 A EP20879268 A EP 20879268A EP 4048298 A1 EP4048298 A1 EP 4048298A1
Authority
EP
European Patent Office
Prior art keywords
tissue
hydrogel
powder
grinding
amniotic
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP20879268.9A
Other languages
English (en)
French (fr)
Other versions
EP4048298A4 (de
Inventor
Di JING
Jian Ma
Yonghui Wang
Xiuyu Wang
Zhijun Huang
Mingxiang WANG
Volodymyr RYZHUK
Wenting GAO
Peilin Yu
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Biopryme Biologics Inc
Original Assignee
Biopryme Biologics Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from CN201911118465.6A external-priority patent/CN110787117A/zh
Priority claimed from CN201911118116.4A external-priority patent/CN111054486B/zh
Priority claimed from CN202010056374.0A external-priority patent/CN111000871A/zh
Priority claimed from CN202010056372.1A external-priority patent/CN110946814A/zh
Priority claimed from CN202010975599.6A external-priority patent/CN112022880A/zh
Priority claimed from CN202010975598.1A external-priority patent/CN112022879A/zh
Application filed by Biopryme Biologics Inc filed Critical Biopryme Biologics Inc
Publication of EP4048298A1 publication Critical patent/EP4048298A1/de
Publication of EP4048298A4 publication Critical patent/EP4048298A4/de
Pending legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
    • A61L26/0057—Ingredients of undetermined constitution or reaction products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/36—Skin; Hair; Nails; Sebaceous glands; Cerumen; Epidermis; Epithelial cells; Keratinocytes; Langerhans cells; Ectodermal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/50—Placenta; Placental stem cells; Amniotic fluid; Amnion; Amniotic stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K35/56—Materials from animals other than mammals
    • A61K35/63—Arthropods
    • A61K35/64—Insects, e.g. bees, wasps or fleas
    • A61K35/644—Beeswax; Propolis; Royal jelly; Honey
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/4886—Metalloendopeptidases (3.4.24), e.g. collagenase
    • A61K38/4893—Botulinum neurotoxin (3.4.24.69)
    • A—HUMAN NECESSITIES
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    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A61L15/40—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing ingredients of undetermined constitution or reaction products thereof, e.g. plant or animal extracts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
    • A61L26/0061—Use of materials characterised by their function or physical properties
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    • A—HUMAN NECESSITIES
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    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A—HUMAN NECESSITIES
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    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A61L27/3633—Extracellular matrix [ECM]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
    • A61L27/3691—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by physical conditions of the treatment, e.g. applying a compressive force to the composition, pressure cycles, ultrasonic/sonication or microwave treatment, lyophilisation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/52—Hydrogels or hydrocolloids
    • A—HUMAN NECESSITIES
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    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L29/00—Materials for catheters, medical tubing, cannulae, or endoscopes or for coating catheters
    • A61L29/005—Ingredients of undetermined constitution or reaction products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L29/00—Materials for catheters, medical tubing, cannulae, or endoscopes or for coating catheters
    • A61L29/14—Materials characterised by their function or physical properties, e.g. lubricating compositions
    • A61L29/145—Hydrogels or hydrocolloids
    • A—HUMAN NECESSITIES
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    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/005—Ingredients of undetermined constitution or reaction products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L31/145—Hydrogels or hydrocolloids
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
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    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
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    • A61P41/00—Drugs used in surgical methods, e.g. surgery adjuvants for preventing adhesion or for vitreum substitution
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
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    • C12Y304/24—Metalloendopeptidases (3.4.24)
    • C12Y304/24069—Bontoxilysin (3.4.24.69), i.e. botulinum neurotoxin

Definitions

  • the application is in the field of preparation and development of hydrogels, which are optionally optimized and used for therapeutic purposes.
  • Therapeutic materials have been made from human and animal tissues. These materials may be beneficial because they can have natural biological constituents that reduce inflammation, promote healing and/or provide other benefits. See, for example, U.S. Application Pat. Pub. US 2018-0100139 Al. However, for practical use, traditionally prepared compositions lack many of the properties desirable for many therapeutic uses. Such compositions are, therefore, impractical for many applications.
  • preparation steps may be configured to generate hydrogels having characteristics favorable for specific therapeutic uses.
  • the preparation involves cryogrinding of tissue, e.g., amniotic tissue, to produce a powder having a desirable size distribution, addition of a liquid to the powder resulting in a therapeutic hydrogel. Grinding a low temperatures prevents destruction of desirable bioactive components of the tissue.
  • the size distribution of particles within the powder can be used to control the rate at which the hydrogel thickens during use, allowing for selection of thickening and/or solidification times desirable for specific therapeutic procedures.
  • the size distribution also enables the use of the rehydrated powder in a spray system.
  • the preparation involves sterilization of the powder at low temperatures. Such temperature-controlled sterilization allows for the destruction of microorganisms while maintaining desirable bioactive compounds within the tissue derived powder.
  • Hydrogels of the invention may be used in a wide range of therapeutic applications. These include dermal and subdermal applications, oral and esophageal applications, ophthalmic applications, vaginal and urinary tract applications, surgical applications, and the like. Further examples of hydrogel applications are discussed elsewhere herein.
  • Various embodiments of the invention include methods of producing a tissue derived powder.
  • the powder may be configured to produce a paste or a hydrogel upon addition of a liquid such as water, saline, or other bio-compatible liquid.
  • Various embodiments include the methods of manufacture, the powder, the hydrogel, various delivery systems, and/or various methods of treatment.
  • the methods can include, for example, decellularization, chemical alteration, physical alteration, sterilization, and/or addition of therapeutic compounds to produce a therapeutic material (composition).
  • the therapeutic material can be included in a solid (e.g., freeze dried powder), a gel, a paste, a liquid, and/or the like.
  • the therapeutic material may be configured for use in a wide variety of medical applications.
  • the treatment composition may be used to reduce the growth of undesirable tissues (e.g., fibrosis or angiogenesis), to reduce inflammation, to deliver therapeutic compounds (e.g., antibiotics), to treat ocular injuries, to treat burns, and/or the like.
  • tissue derived hydrogels have been suggested as a therapeutic means in the past, it has been difficult to produce a suitable hydrogel while maintaining the desired biological activity in many applications.
  • Various embodiments of the invention solve these technical hurdles.
  • Various embodiments of the invention include a treatment composition comprising extracellular matrix derived from decellularized human or animal tissue, and physically, chemically and biologically processed to alter immune responses or other biological processes; and a carrier matrix is added to the extracellular matrix and is configured to control a viscosity, adhesiveness, solidification and biodegradation rate of the treatment composition.
  • the extracellular matrix is derived from human tissue or animal tissue.
  • Various embodiments of the invention include a method of preparing a bioactive hydrogel, the method comprising: receiving amniotic tissue; freeze-drying the amniotic tissue; grinding the amniotic tissue at a temperature less than -10 degrees Celsius to produce a powder of the amniotic tissue particles, at least 50% of the particles having a diameter less than 200 micrometers; and adding a rehydration fluid to the freeze-dried powder to produce a hydrogel.
  • Various embodiments of the invention include a bioactive hydrogel system comprising: a rehydrated freeze-dried powder of amniotic tissue, the freeze-dried powder having particles of which at least 50% of the particles have a diameter less than 200 micrometers, the freeze-dried powder having bioactive materials whose activity is preserved by grinding the amniotic tissue at a temperature of less than minus 10 degrees Celsius.
  • Various embodiments of the invention include a treatment system comprising: extracellular matrix derived from decellularized human or animal tissue and chemically processed to alter immune responses; and a carrier matrix added to extracellular matrix and is configured to control a viscosity, adhesiveness, solidification and biodegradation rate of the treatment composition.
  • Various embodiments of the invention include a treatment system including a composition comprising: extracellular matrix derived from decellularized human or animal tissue and chemically processed to alter immune responses; and a carrier matrix is added to extracellular matrix and is configured to control a viscosity, adhesiveness, solidification and biodegradation rate of the treatment composition.
  • Various embodiments of the invention include a treatment system including a composition comprising: a powder of ground amniotic tissue having a particle size of D50 (pm) or D50 (pm) less than 200 pm, wherein the amniotic tissue is ground at a temperature less than 0, -10, -20 or -30 degrees C to retain bioactivity of the amniotic tissue.
  • Various embodiments of the invention include a method of making a therapeutic composition, the method comprising: obtaining amniotic tissue; mixing the amniotic tissue, the mixing optionally including homogenization and/or addition of additional therapeutic components; cryo-grinding the amniotic tissue, the grinding optionally including and/or freeze-drying, the grinding resulting in a powder have a particle diameter (D50) less than 200, 100, or 50 pm.
  • D50 particle diameter
  • Various embodiments of the invention include a method of treatment of any one of the ailments discussed herein using the powdered or rehydrated powder discussed herein, wherein the ailment is Joint Injury or Joint Degradation, In-stent Restenosis , striae gravidarum, striae distensae, Retinal Tears, Retinal Detachments, Macular Holes, ocular surface wounds, dry eye, Intraocular injury, Age-Related Macular Degeneration (AMD), regeneration of retinal pigment epithelium (RPE) cells, vaginal or anal trauma, oral disorders, radiation therapy damage, acne, wrinkle elimination, hydration, skin toning (brown spots, hyperpigmentation and skin redness minimization), gastric reflux injury, radiation burns, skin filling, and skin volumizing.
  • the ailment is Joint Injury or Joint Degradation, In-stent Restenosis , striae gravidarum, striae distensae, Retinal Tears
  • FIG. 1 illustrates methods of producing therapeutic hydrogels, according to various embodiments of the invention.
  • FIG. 2 illustrates Methods 200 of grinding, according to various embodiments of the invention. These methods are optionally performed as part of Grind Step 130 illustrated in FIG. 1.
  • FIG. 3 illustrates an exemplary batch grinding system, according to various embodiments of the invention.
  • FIG. 4 illustrates measurements of biological activity, with and without grinding, according to various embodiments of the invention.
  • FIG. 5 illustrates an amount of recombinant human epidermal growth factor (hEGF) released in the hydrogel as a carrier measured by ELISA analysis, according to various embodiments of the invention.
  • FIG. 6 illustrates an effect of the hydrogel on preventing and managing intrauterine adhesions following instillation of chemical agents, according to various embodiments of the invention.
  • FIG. 7 illustrates an effect of the hydrogel on ocular surface wounds and dry eye through the corneal chemical (alkali) burns animal study, according to various embodiments of the invention.
  • FIG. 8 illustrates an effect of the hydrogel on ocular surface wounds through the corneal mechanical injuries animal study, according to various embodiments of the invention.
  • FIGs. 9A 7 9B Illustrate human retinal pigment epithelial (RPE) cells cultivated in vitro using the highly diluted hydrogel, according to various embodiments of the invention.
  • FIG. 10 illustrates case studies of real patients using the hydrogel in managing radiation dermatitis and managing pain, according to various embodiments of the invention.
  • FIG. 11 illustrates case studies of real patients using the hydrogel (spray form) in managing oral mucositis and managing pain, according to various embodiments of the invention.
  • FIG. 12 illustrates a case studies self-reported surveys of real patients using the hydrogel in managing chemoradiotherapy induced radiation dermatitis, according to various embodiments of the invention.
  • FIG. 13 illustrates a case studies self-reported surveys of real patients using the hydrogel in managing chemoradiotherapy induced oral mucositis, according to various embodiments of the invention.
  • FIG. 14 Case studies self-reported surveys of real patients using the hydrogel in managing radiation vaginitis, according to various embodiments of the invention.
  • FIG. 15. Case studies self-reported surveys of radiation proctitis, according to various embodiments of the invention.
  • FIG. 16 illustrates a case studies of real patients using the hydrogel in managing acne vulgaris, according to various embodiments of the invention.
  • FIG. 17 illustrates case studies of real patients using the hydrogel in managing striae gravidarum in combination with laser treatment, according to various embodiments of the invention.
  • FIGs. 18A & 18B illustrate case studies of real patients' satisfaction rates regarding improvements achieved using the hydrogel in mesotherapy micro-injections for skin regeneration, hydration, filling and volumizing, according to various embodiments of the invention.
  • the powder of various embodiments of the invention, is typically configured to be rehydrated to form a paste, hydrogel or solution.
  • the paste, hydrogel or solution may then be applied in therapeutic methods as described elsewhere herein.
  • the powder and/or hydrogel includes materials derived from a living organism and optionally one or more additional components.
  • the materials may comprise any combination of human or animal tissue, cells, proteins, collagen, mucopolysaccharide, amniotic tissue, placenta tissue, amniotic sac tissue, umbilical cord tissue, cartilage, stem cells, enzymes, proteins, hormones, bacteria, yeasts, algae, and/or the like.
  • the one or more optional components may include, for example, trace elements, an antimetabolite agent, an antifungal agent, a pain reliever, an muscle cells, differentiated stem cells, skin cells, nerve cells, immunological cells, a vitamin, viruses, biological compositions, cells, cross-linking materials, hydrogel matrix structure, viscosity control compounds, cross-linking compounds, pharmaceuticals, anti inflammatory agents, antibodies, T-cells, vaccines, immune system repressors or promotors, antibiotics, anti-viral agents, enzymes, peptides bacteriophage, thickener, buffer, salt, fat (e.g., omega-3s), natural oils, aloe, mineral oil, antioxidants, coloring agents, cosmetics, fibrin, stem cell scaffolding, moisturizers, sun screen, tea extracts, vitamin C, hyaluronic acid, lactic acid, alpha- or beta-hydroxy acids, collagen, a colloid, hormones, preservatives, sweetener, superoxide dismutase (SOD), gluta
  • the carrier Matrix As they carry the ground tissue and added components. Various combinations of these components may be added/combined before and/or after a grinding process in which the powder is produced.
  • the powder in a dried form, as a shelf life of 3-5 years or greater than 5 years.
  • the therapeutic hydrogel includes human or animal amniotic tissue.
  • Specific hormones that may be included in the powder or rehydrated powder include growth hormones, interferon, adrenocorticotropic hormone (ACTH), cortisol, estrogen, kisspeptin, leptin, melanocyte-stimulating hormone (MSH), melatonin, norepinephrine, oxytocin, Peptide YY, progesterone, prolactin, prostaglandins, relaxin, serotonin, somatostatin, thyroid hormones, vitamin D, and/or the like.
  • ACTH adrenocorticotropic hormone
  • cortisol cortisol
  • estrogen kisspeptin
  • leptin leptin
  • MSH melanocyte-stimulating hormone
  • melatonin norepinephrine
  • oxytocin Peptide YY
  • progesterone prolactin
  • prostaglandins relaxin
  • serotonin serotonin
  • somatostatin
  • mammalian amniotic tissue is combined with a collagen thickener and homogenized prior to grinding to the powder.
  • an antibiotic is added to the resulting powder.
  • Thickening may be initiated by rehydration, light, exposure to air, or mixing with an activation agent.
  • some embodiments of the powder or rehydration liquid include a light activated cross-linking agent.
  • human and/or animal tissue is homogenized and combined with stem cells prior to grinding and following grinding materials configured to function as a stem cell growth scaffolding are added to the resulting powder.
  • the human or animal tissue is optionally sterilized prior to being combined with the stem cells.
  • porcine amniotic tissue is combined with a hormone and homogenized. Following homogenization, the mixture is freeze-dried (e.g., lyophilized) and ground to produce the powder.
  • the hormone may be selected to promote growth, to reduce inflammation, to increase or decrease vascularization, to stimulate stem cell growth, to differentiate stem cell growth to a specific cell type (e.g., differentiate), to promote nerve growth or blood vessel growth.
  • amniotic tissue is combined with a thickener, homogenized and freeze dried prior to grinding.
  • the thickener may be selected to control thickening of a hydrogel or paste after the powder resulting from the grinding is rehydrated.
  • the powder includes ground tissue and is added to a liquid to produce a hydrogel or paste, wherein the liquid includes any combination of a buffer, salts, a thickening agent, an antibiotic, and/or any other material disclosed herein as being a component of the powder in the various embodiments disclosed herein.
  • Ophthalmic products are provided as powder and can be mixed with purified water on site.
  • the powder can include decellularized porcine placenta tissue, carboxymethyl, cellulose sodium, sodium chloride, potassium chloride, and optionally Polyquaternium-1 as a preservative.
  • the components of the powder, hydrogel, paste or solution may be mixed in any order before, during and/or after steps of grinding, homogenization, cooling, sterilization, drying, and/or rehydration.
  • FIG. 1 illustrates methods of producing therapeutic hydrogels, according to various embodiments of the invention.
  • the illustrated methods are optionally adapted to generation of therapeutic pastes or solutions, by varying an amount of hydrating liquid.
  • the methods include producing a powder ground to achieve desirable characteristics, optionally sterilizing the powder, and rehydrating the power for a variety of therapeutic uses, some of which are described further elsewhere herein.
  • the steps illustrated in FIG. 1 are optionally performed in alternative orders.
  • tissue having desirable properties is obtained.
  • This tissue can include human or animal tissues.
  • the tissue may include amniotic tissue obtained from humans, swine, fowl, sheep, suidae, porcine, equine, bovine, ovine, murine, molluscs, amphibians, rabbit or other mammals or fish, animal embryotic tissue, and/or other suitable sources.
  • the matrix is derived from a combination of amniotic tissues (e.g., placenta, sac or umbilical cord) and fish skin.
  • the tissue includes placenta tissue obtained from domestic rabbit, pigs, sheep or cattle.
  • the tissue is selected to include components having a variety of therapeutic properties such as anti-inflammatory compounds, growth stimulators, anti-bacterial properties, anti-scarring properties, and/or the like.
  • Amniotic tissue is optionally obtained as afterbirth or during slaughtering on animals.
  • Obtain Tissue Sep 110 includes obtaining cultured cells, e.g., bacteria, yeasts, algae etc., rather than or in addition to obtaining tissues of higher-level organization.
  • the tissue optionally includes one or more of amniotic sac tissue, amniotic fluid, ocular tissue, lymph node tissue, neural tissue, umbilical cord tissue, and/or the like.
  • extracellular matrix is derived from varied tissue, such as skin, dermis, urinary bladder, small intestine, mesothelium, pericardium, heart valve, fascia lata, liver, lung, heart, adipose, skeletal, blood vessel, nerve conduits, cartilage, cornea, breast, colon, placenta, amnion or other tissues and organs.
  • tissue such as skin, dermis, urinary bladder, small intestine, mesothelium, pericardium, heart valve, fascia lata, liver, lung, heart, adipose, skeletal, blood vessel, nerve conduits, cartilage, cornea, breast, colon, placenta, amnion or other tissues and organs.
  • a Mix Step 120 the obtained tissue is mixed with additional components, such as one or more of those discussed elsewhere herein.
  • Mix Step 120 may be performed in several stages before or after any of the various steps illustrated in FIGs. 1 and 2.
  • tissue may be mixed with a thickener, a growth hormone, an antibiotic, and/or anti-inflammatory agent prior to grinding, and then the resulting powder may be mixed with stem cells following sterilization.
  • the tissue optionally includes an extracellular matrix and the added materials represent a carrier matrix.
  • Mix Step 120 optionally includes homogenization of tissue or additional components.
  • tissue may be homogenized prior to freeze-drying, prior to grinding, and/or prior to rehydration.
  • Mix Step 120 optionally occurs, at least in part, during grinding.
  • Mix Step 120 optionally further includes decellularization of the tissue.
  • Decellularization can be accomplished by physical methods such as: 1) Multiple freeze-thaw cycles in which intracellular ice crystals disrupt cell membrane; 2) Force and hydrostatic pressure which can burst cells; and 3) Non- thermal irreversible electroporation in which pulsed electrical fields disrupt cell membranes.
  • Decellularization can be accomplished by chemical methods such as 1) alkaline treatment in acid is used to solubilize cytoplasmic components of cells and disrupts nucleic acids; 2) Flypotonic and hypertonic solutions in which lyse cells by osmotic shock and the shock disrupts DNA-protein interactions; 3) use of non-ionic detergents (Triton-x 100) which disrupt DNA-protein interactions, disrupt lipid-lipid and lipid- protein interactions and to a lesser degree disrupt protein-protein interaction; 4) use of Ionic detergents (e.g., Sodium dodecyl sulfate, Sodium deoxycholate, Triton X-200) which solubilize cell and nucleic membranes, and tend to denature proteins; 5) use of zwitterionic detergents (e.g., CHAPS, Sulfobetaine- 10 and -16 (SB-10, SB-16)) which exhibit properties of non-ionic and ionic detergents; 6) use of Alcohols which cause cell lysis by dehydration and
  • Decellularization can be accomplished by biologic means such as: 1) the use of enzymes (e.g., Nucleases which Catalyze the hydrolysis of ribonucleotide and deoxyribonucleotide chains; Trypsin which Cleaves peptide bonds on the C-side of Arg and Lys; and Dispase: Cleaves specific peptides, mainly fibronectin and collagen IV); or 2) the use of Non-enzymatic agents such as: a) Chelating agents (EDTA, EGTA) which bind metallic ions, thereby disrupting cell adhesion to ECM (extracellular matrix), or b) Protease inhibitors (phenylmethylsulfonylfluoride, aprotonin, leupeptin) which inhibit many proteases needed to maintain the native ECM ultrastructure.
  • enzymes e.g., Nucleases which Catalyze the hydrolysis of ribonucleotide and deoxyribonucleotide chains; Tryp
  • a Grind Step 130 the tissue and some or all of any added components are ground to produce a powder.
  • tissue grinding systems and methods have been developed. In various embodiments, these systems and methods result in a powder having a desirable size distribution, e.g. a small size and small variation in size.
  • the resulting powder is typically highly dissolvable in water or other biocompatible fluids.
  • the powder may be stored in a dried form and later mixed with a liquid to form a solution, paste or hydrogel.
  • An aspect of the powder found in various embodiments is that it is produced using a manufacturing process configured to maintain desirable chemical and biological characteristics of biological materials ground to make the powder, while also achieving the desirable size distribution.
  • Grind Step 130 is optionally repeated multiple times under different conditions, e.g., using different grinding mechanisms to achieve a desired powder.
  • the grinding is optionally performed at low temperature and/or the materials being ground can include freeze-dried components.
  • the grinding may be performed in a temperature-controlled grinder configured to maintain the ground materials in various temperature ranges (optionally below room temperature) during the grinding process.
  • the temperature is optionally selected to preserve biological and/or therapeutic activity of the components being ground.
  • Grind Step 130 Further details of Grind Step 130 are discussed elsewhere herein, for example with respect to FIG. 2.
  • additional components are added to the resulting powder resulting from grinding, as a stage of Mix Step 120.
  • a Sterilize Step 140 the powder produce in Grind Step 130 is sterilized. While any of the well- know methods of sterilization may be used in Sterilize Step 140, various embodiments of the invention include specialized approaches to sterilization which are configured to kill undesirable contaminants/constituents (e.g., undesirable virus and/or bacteria) while at the same time preserving the desirable bioactivity and/or therapeutic aspects of the powder.
  • undesirable contaminants/constituents e.g., undesirable virus and/or bacteria
  • Sterilize Step 140 is performed prior to grinding.
  • the tissue can be soaked in 0.15-0.18% peracetic acid/4-4.8% ethanol solution (or alternatively Triton X-100) on shaker (room temperature, 140-160 rpm) for 2 hours. Then Rinsed with Dl water on shaker (room temperature, 140-160 rpm) for three times, 10 min/time. Sterilization is optionally performed on both the original tissue and after grinding.
  • Sterilization Step 140 includes cooling the powder while providing a dose of sterilizing radiation.
  • This radiation can include, for example, alpha particles, high energy electrons, high energy ions, neutrons, protons, gamma rays, x-rays, and/or ultraviolet light.
  • Cooling the powder during sterilization can include use of a refrigerant, thermoelectric cooling, evaporative cooling, and/or the like.
  • Sterilization Step 140 includes moving the powder relative to a source of sterilizing radiation during the sterilization process such that localized heating caused by the sterilizing radiation is minimized.
  • Relative movement can be achieved by, for example, directing a high energy electron beam (e-beam) over a thin layer of the powder in a raster pattern. Or, by placing the powder in a sealed vial and rotating the vial while exposing it to an e-beam or other sterilizing radiation.
  • a typical e-beam dosage may be 15KgY. Both movement and cooling are optionally used together during Sterilization Step 140, to minimize damage to desirable characteristics of the powder while achieving a needed level of sterilization.
  • a regulatory standard of sterilization may be required.
  • the combination of both cooling and relative movement can be used to achieve this standard while optimizing preservation of desirable bio-functionality.
  • Advantages of relative movement during sterilization are optionally achieved by pulsing the radiation source.
  • the powder generated in Grind Step 130 is stored for future use. Shelf life of the powder may be extended by storage at a controlled room temperature, storage below 10, 5 or 0 degrees Celsius storage as a dried powder, and/or addition of preservatives, such as anti oxidants. In some embodiments, storage takes place in a sealed vial and/or a delivery device. (See, for example, Apply Step 170).
  • the powder may be stored under vacuum or under an inert gas.
  • the powder produced in Grind Step 130 is rehydrated.
  • the amount of liquid used in to rehydrate the powder can be selected to determine whether the resulting product is a paste, hydrogel or solution.
  • the amount of liquid can also be used to control viscosity of a hydrogel.
  • liquids used to rehydrate the powder include, water, saline, normal saline, hypertonic saline, buffered saline, buffered liquids, eatable oils, juices, dairy products, and/or the like.
  • the fluid used is optionally adapted to the intended use.
  • the salt concentration and/or pH of the hydration liquid may be adjusted to match a normal pH of a part of the body in which treatment is intended to occur. (pH and salt concentration being different in the eye relative to the heart or urethra, etc.).
  • the fluid used for rehydration of the powder optionally includes one or more of the additional hydrogel components discussed herein.
  • Rehydrate Step 160 is performed immediately prior to use.
  • a hydrogel produced by rehydration my thicken (an increase in viscosity) in the few hours after hydration.
  • a smaller particle size of the particle may result in quicker thickening times. This provides an advantage in some examples, for example, a thickening time of less than 30, 20 or 15 minutes may be desirable in ophthalmic repairs so that a surgeon doesn't have to wait for a suitable viscosity to be reached for too long during surgery.
  • Rehydrate Step 160 is optional in embodiments in which the powder is used therapeutically in a dried form. For example, applied to a wound as part of a dry wound dressing or attached as a coating to a canula, stint or incubation tube. In some embodiments, rehydration occurs when the powder comes in contact with body fluids. For example, in an application in which a suture is coated with the powder, rehydration occurs when the suture penetrates the body during application of stitches to a wound.
  • the powder including ground tissue is added to a mechanical delivery device, optionally after rehydration, to create a therapeutic delivery system.
  • the powder is stored in a sterilized and sealed vial.
  • the power is included in a cosmetic, a dermal cream, a sunscreen, a wound dressing, a bandage (e.g, a hydrocolloid gel bandage or a Band-AidTM), an eye drop, an anti-inflammatory cream, an antipruritic cream, a wound closure strip, surgical sponge, tampon, suppository (anal or vaginal), an injection device, an intrauterine device, a stent, a catheter, an transdermal cannula, a nasal or oral cannula, a transdermal patch, a device configured to be implanted in a living person or animal, and/or the like.
  • a cosmetic e.g, a dermal cream, a sunscreen, a wound dressing, a bandage (e.g, a hydrocolloid gel bandage or a Band-AidTM), an eye drop, an anti-inflammatory cream, an antipruritic cream, a wound closure strip, surgical sponge, tampon, suppository (anal or
  • the powder (wet or dry) may be delivered using an injection device, cosmetic dispenser, a micro-needle or array thereof, spray device, a nebulizer, a canula, a catheter, a stent, added to a surgical stable or suture material (absorbable or non-absorbable), a drinkable solution, and/or the like.
  • the powder may be included dry or rehydrated to a paste, hydrogel, spray, gel, and/or liquid solution, as appropriate for the particular system.
  • the powder may be placed in a spray bottle, sprayed on wound or in mouth, placed in a wound dressing, placed in a drinkable liquid, injected, added with micro-needles,
  • an end user can select between hydrogel or paste or solution (in which the powder is fully dissolved) by selecting a rehydration volume and/or fluid.
  • the treatment composition may be used to reduce the growth of undesirable tissues (e.g., fibrosis or angiogenesis), to reduce inflammation, to deliver therapeutic compounds (e.g., antibiotics), to treat ocular injuries, to treat burns, and/or the like.
  • the powder may be attached to a device configured to be inserted into a body, such as a suture, surgical staple, sponge, canula, catheter, replacement joint part, surgical rod, brace, screw or pin, stent, electrode, sensor, drug delivery device (e.g., insulin pump), piercing, breast implant, and/or the like.
  • a device configured to be inserted into a body
  • the powder or hydrogel is placed in a pressurized or non- pressurized squirt bottle.
  • the powder is placed in an aerosol dispenser. Spray applications are possible because of the relatively small particle size.
  • the powder and/or hydrogel can be sprayed on a wound, injury, or other oral or dermal trauma.
  • the tissue derived powder, paste and/or hydrogel may be used to reduce inflammation and promote healing associated with body piercings and/or tattoos. For example, to promote healing of infected oral or vaginal piercings.
  • the grinding systems (as used in Grind Step 130) of various embodiments of the invention can be divided into “batch” and “continuous” systems.
  • batch grinding systems a quantity of material is ground together, for the entire quantity the grinding starts at the same time and ends at the same time.
  • Batch grinding may occur in a container, such as a bowl-shaped vessel.
  • grinding balls may be added to the container. These grinding balls are optionally moved using stirring rotors, e.g. 01O*3Omm.
  • material to be ground is provided at an input and ground product exits an output. Examples of continuous grinding systems include screw grinders. Such grinders can include regions having different grinding implements.
  • These implements can include, for example, feeder structures (e.g., one, two or more screws) configured to drive material forward, region including burrs, regions including blades, regions include augers, regions including gears or other meshing parts, tapered regions, regions with rollers, regions including mincer augers, and/or regions including grinding balls. Different regions may rotate at different speeds.
  • a continuous grounder may include feeder regions separated by roller and/or grinding ball regions. While batch grinding systems are discussed herein for the purpose of example, the teachings and examples provided are readily applied to continuous grinding systems. For example, the cooling systems described herein may be used for batch and continuous grinding systems.
  • Characteristics of the grinding process that can be controlled to optimize the resulting product include grinding speed, grinding ball size(s), grinding region structure & dimensions, grinding taper, vessel temperature, grinding fluid formula & temperature, grinding time, and/or the like. In various embodiments, these characteristics are chosen to achieve a desired particle size and distribution while also preserving the bioactivity of compounds within the resulting powder. Specifically, a powder size small enough to have good solubility and/or hydrogel thickening times is achieved while maintaining the grinding process at a temperature at which bioactivity is maintained.
  • a temperature-controlled grinding tank has a double wall and includes connections configured for flow of a refrigerant between the walls.
  • the inner and outer walls may include different materials, e.g., nylon and stainless-steel, respectively.
  • Grinding balls of one or more sized are disposed in the tank during grinding.
  • the grinding ball(s) tank and/or other grinding parts comprise a hard material such as zirconia, nickel, titanium, carbide and/or stainless-steel (e.g., 316L SS).
  • 316L SS stainless-steel
  • Several (1, 2, 3, 4 or more) different sizes of grinding balls may be used in the same operation.
  • 1, 2, 3, 5, 8 and 10 millimeter (diameter) grinding balls may be used together, in various combinations, in a cryogenic grinding process.
  • the number and sizes of the grinding balls may be selected to generate a desired medium/average particle size and desired size distribution.
  • a refrigerant such as an ethanol solution, e.g., 99.7% EtOH, is used as a refrigerant to keep the grinding tank at a temperature between -30 and -50 degrees Centigrade.
  • Alternative refrigerants may include, for example, dry ice, ethylene glycol, acetone, water, ethanol, o-Xylene, m-Toluidine, Acetonitrile, Pyridine and methanol.
  • the grinding balls have three different diameters of 10 mm, 5 mm and 2 mm, respectively, and the quantity of balls used is in the respective ratio 1:2:3.
  • One, two, three or more sizes of grinding balls may be used.
  • the grinding balls may have different densities, e.g., smaller balls having higher densities.
  • the size ratio was tested among the three different sizes 1:2:3. Of course, it can be 1:5:10, 1:3:6, 1:4:20, or other ratio combination.
  • Thee size grinding balls can maximize the efficiency and effectiveness of grinding process.
  • Various embodiments include at least two or at least three sizes of grinding balls. The largest to smallest having ratios of at least 1:3, 1:4, 1:5 or 1:10 in diameter.
  • the diameters of the grinding balls and the ratios among the grinding balls are selected based on the desired powder particle size.
  • FIG. 2 illustrates Methods 200 of grinding, according to various embodiments of the invention. These methods are optionally performed as part of Grind Step 130 illustrated in FIG. 1.
  • a mixture of tissue and any of the other optional components discussed herein are ground to a powder of desired characteristics.
  • human or animal amniotic tissue may be ground to a fine powder, which when rehydrated forms a therapeutic solution, paste or hydrogel.
  • the powder is ground to a size or distribution as discussed elsewhere herein (e.g., see examples below). Grinding can be accomplished in either a patch process or a continuous process.
  • grinding is performed at a reduced temperature.
  • a cooling system is used to maintain the ground mixture at temperature below 0 degrees C during grinding.
  • the tissue and/or other materials ground are optionally freeze-dried prior to grinding. Grinding may be performed using wet or try methods. In wet methods a grinding liquid is added to the mixture to be ground. Optionally, this liquid is one whose freezing point is below the grinding temperature.
  • the steps illustrated in FIG. 2 are optionally performed in alternative orders. For example, selection of grinding implements may be performed before adding materials to be ground.
  • Method 200 illustrated in FIG. 2 is used for batch grinding of tissue.
  • batch grinding one batch of material is ground at a time in a particular grinding device.
  • batch grinding is performed in a large container, alternatively referred to herein as a tank, vessel, or vat. This container may be open, closed or sealed during grinding.
  • a desired particle size and size distribution for the resulting powder is selected.
  • the selection is optionally based on desired properties of the final product. For example, the rate at which a hydrogel or paste of powdered tissue thickens has been found to be controllable by selecting a particle size, hydrogels including smaller particles increase in viscosity at a greater rate relative to hydrogels including larger particles. Solubility has been found to be controllable by selecting both particle size and size distribution, smaller particles being easier to solvate.
  • the generation of smaller particles is limited by the goal of preserving the bioactivity of the material (including tissue) ground. Too much grinding can substantially reduce bioactivity by destroying biological structures. Too much grinding and/or poor grinding conditions can also reduce by bioactivity by causing chemical degradation of bioactive materials. For example, localized heat of grinding can cause oxidation, denaturation, or other reactions of proteins.
  • an optional Freeze Step 220 a mixture of materials to be ground is frozen. This freezing may include freeze-drying the mixture produced and optionally homogenized in Mix Step 120. Freezing and drying may occur together by sublimation, or freeze-drying.
  • Add Mixture Step 225 the mixture to be ground is added to a grinding tank.
  • Add Mixture Step 225 is optionally performed prior to Freeze Step 220 or prior to all or part of Mix Step 120. For example, some components may be separately added to the grinding tank wherein they are mixed, and/or freezing may occur in the grinding tank.
  • a Select Balls Step 230 selecting one or more grinding balls and adding the selected grinding balls to the grinding tank.
  • the grinding balls may be selected based on a desired particle size and distribution in the powder that results form grinding.
  • a Cool Step 235 the grinding tank is cooled. This may be accomplished, for example, by circulating a refrigerant within a double wall of the grinding tank.
  • the refrigerant may be any of those known in the art of refrigerant systems.
  • the interior of the grinding tank is maintained during grinding at temperatures less than 10, 0, -15, -25, -30, -40 or -50 degrees C, or any range therebetween.
  • a grinding fluid is added to the grinding tank. This grinding fluid comes in contact with the material being ground.
  • the grinding fluid may server as a lubricant during the grinding process, resulting in a narrower resulting particle size distribution and/or reducing grinding times needed to reach a desired medium or average particle size.
  • the grinding fluid may also serve to maintain thermal transport and equilibrium within the material being ground.
  • the grinding fluid is optionally also a cooling fluid.
  • the cooling fluid may be pre-cooled to a desired grinding temperature and/or may include an ice bath configured to maintain the desired temperature.
  • the grinding fluid is preferably a liquid at the desired grinding temperature when combined with the material to be ground.
  • the grinding fluid optionally includes saline, normal saline, a salt solution, and/or one or more alcohols and/or any of the suitable cooling fluids or refrigerants discussed herein.
  • a Grind Step 245 stirring rotors are used to move the grinding balls within the grinding tank to grind the material into a powder of the desired particle size and size distribution.
  • grinding speeds between 500-3000 rpm, 1000-3000 rpm, 1800-2000 rpm or 1500-2500 rpm are used. Although other speeds may be used depending on the size and other characteristics of the grinder. Grinding may continue until the desired particle size is achieved, for example using various equipment the grinding may take between 5-30, or 10-15 minutes. Although other grinding times are possible.
  • the resulting particle size is directly related to the cryogenic grinding time.
  • the ground material (now a powder) is dried to remove (e.g., evaporate or sublimate) the grinding fluid and/or liquids found in the original material to be ground.
  • Dry Step 250 may be performed within part of the grinding system or within a separate device. Drying is optionally performed under negative pressure, e.g., vacuum.
  • the powder is added to a suitable container.
  • the tried powder may be added to a sealed vial or other sealable container prior to Store Step 150.
  • the Method 200 illustrated in FIG. 2 is used for continuous grinding using a continuous grinding system.
  • material is continuously provided to an input of the grinding system and a ground powder is continuously expelled at an output of the grinding system.
  • Ground powder may exit the output at the same time that additional material is provided at the input.
  • Selection Step 215, is performed as described elsewhere herein.
  • Freeze Step 220 the mixture of materials to be ground are frozen as described elsewhere herein.
  • Freeze Step 220 is optionally performed using a continuous freeze-trying system in which a continuous quantity of freeze-dried mixture (including tissue) is produced and provided to the grinding system.
  • the material (e.g., tissue) to be ground is added to the continuous griding system at an input.
  • the output of a freeze-trying system is feed directly to this input.
  • the continuous grinding system includes multiple inputs at which different components to be ground can be added. For example, freeze-dried amniotic tissue may be added at a first input port and a preservative, an antibiotic and stem cells may be provided at one or more different inputs to the continuous grinding system. As such, different components of the mixture may be subject to different types and amounts of grinding.
  • one or more grinding implements are selected for use in the continuous grinding system. These grinding implements may include any combination of the grinding implements discussed elsewhere herein.
  • Cool Step 235 at least part of the continuous grinding system is cooled such that the material being ground is kept at a controlled temperature, e.g. at any of the grinding temperatures or temperature ranges discussed elsewhere herein.
  • This cooling may be performed, for example, by passing a refrigerant through a cooling jacket of the continuous grinding system and/or by adding a cooling liquid to the material being ground.
  • a grinding fluid is added to the continuous grinding system.
  • a grinding fluid is optionally also a cooling fluid.
  • the grinding fluid is optionally added at a different input to the grinding system relative to the tissue to be ground.
  • a Grind Step 245 the continuous grinding system is used to grind the material to be ground.
  • Grind Step 245 optionally includes turning of one or more grinding spindle to both grind the material and drive the material through the grinding system. The result of Grind Step 245 a powder of the desired particle size and size distribution is produced.
  • optional Dry Step 250 the ground material (now a powder) is dried as described elsewhere herein. Where a continuous grinding system is used in Grind Step 245, the drying is optionally also performed using continuous trying system. In an optional Package Step 255, the powder is added to a suitable container as discussed elsewhere herein.
  • FIG. 3 illustrates an exemplary Batch Grinding System 300 including Grinding Balls 310, a Nylon Grinding Tank 320 and a Refrigerant Cycling System 330, according to various embodiments of the invention.
  • the refrigerant is optionally supplied using a refrigerant compressor, silicon transfer tubing and suitable temperature control electronics.
  • Various embodiments of the invention include the use of dry batch grinding to produce therapeutic powder and/or hydrogel. It was demonstrated that particle size could be controlled while maintaining desirable therapeutic properties at levels not previously demonstrated.
  • Dgo the maximum particle diameter below which 90% of the sample volume exists.
  • D50 the maximum particle diameter below which 50% of the sample volume exists.
  • Dio the maximum particle diameter below which 10% of the sample volume exists.
  • Various embodiments of the invention include the use of wet batch grinding to produce therapeutic powder and/or hydrogel. It was demonstrated that particle size could be controlled while maintaining desirable therapeutic properties at levels not previously demonstrated. In some embodiments, wet grinding produced more favorable results relative to dry grinding results.
  • GaGs GaGs (Glycosaminoglycan Assay BlyscanTM, B1000, Biocolor, UK) are illustrated in FIG. 4. (***p ⁇ 0.001 compared with No.0 sample).
  • the results indicate that the grinding methods produce a desirable powder while protecting proteins and other therapeutic components from being degraded or destroyed.
  • the grinding performed with different ratios of four sizes of grinding balls has no significant effect on the collagen and elastin.
  • GaGs decreased as shown after grinding, which demonstrated that GaGs are still present but might be damaged somewhat during the cryogenic grinding process. This illustrates a fine balance between optimizing particle size and maintaining biological activity, as achieved by various embodiments of the invention.
  • the powder (retaining biological activity) has a Dso (pm) of less than 250, 200, 150, 125, 100, 75, 50 or 39.3, or any range therebetween.
  • the powder (retaining biological activity) has a Dio (pm) of less than 100, 50, 40, 24 or 10, or any range therebetween.
  • the powder (retaining biological activity) has a D 90 (pm) of less than 650, 500, 450, 400, 350, 250, 200, 150, or 136, or any range therebetween.
  • smaller D 90 , D 50 and Di 0 may be achieved using different grinding implements and grinding times. A lower limit on the sizes results from the reduction in biological activity that would result from further grinding. It is anticipated that sufficient biological activity may remain where Dso is less than 10 in some embodiments.
  • Sterilization Irradiation such as E Beam, or other radiation.
  • Hypertonic and hypotonic solutions Cell lysis by osmotic shock, disrupt DNA-protein interactions.
  • Triton X-100 Disrupt DNA-protein interactions, disrupt lipid- lipid and lipid-protein interactions and to a lesser degree protein-protein interaction.
  • Sodium deoxycholic acid Solubilize cell and nucleic membranes, denatures proteins.
  • DNase I Digesting single or double stranded DNA to produce single or double stranded oligodeoxynucleotides.
  • MgCh As a cofactor, magnesium can activate DNA enzyme activity.
  • PMSF Inhibiting many proteases to maintain the native ECM ultrastructure.
  • EDTA Chelating divalent metal ions, which helps cells separate from ECM proteins by isolating metal ions and inactivates the remaining DNA enzymes.
  • Porcine pepsin and hydrochloric acid Mixing the decellularized ECM and pepsin in acidic environment breaks down proteins into smaller peptides creating a uniform hydrogel.
  • Optional Defoamer Avoid adverse effects caused by non-sterile air bubbles.
  • Decellularization characterization Xenogeneic and allogeneic cellular antigens are, by definition recognized as foreign by the host and therefore induce an inflammatory response or an immune- mediated rejection of the tissue. However, components of the ECM (extracellular matrix) are generally conserved among species and are tolerated well even by xenogeneic recipients The criteria for evaluating decellularization is as follows: Less than 50 ng dsDNA per mg ECM dry weight should be present; DNA fragment length should be less than 200 bp; no visible nuclear material in stained tissue sections.
  • Various embodiments of the invention include a lyophilized decellularized ECM (Extracellular Matrix) powder (also referred to herein as a "tissue derived powder”), which may include additional components and may be hydrated to produce a hydrogel. .
  • ECM Extracellular Matrix
  • tissue derived powder also referred to herein as a "tissue derived powder”
  • Alternative methods of producing this tissue derived powder include, for example, (1) Sterilization before decellularization to inactivate virus; (2) Cryogenic grinding after the digestion/solubilization process to increase tissue specific surface area and accelerate digestion of ECM; (3) Shaking the solution with electric mixer rather than thermal shaker oscillator to digest tissues more thoroughly; (4) Adding freeze-drying and cryo-grinding after neutralization to make lyophilized powder of the hydrogel that is easier to dissolve; (5) Optionally adding defoamer into the hydrogel to avoid adverse effects caused by bubbles; (6) Sterilization by irradiation such as e-beam.
  • the hydrogel is configured to increase solubility following mixing with polyethylene glycol.
  • the hydrogel is configured to increase viscosity, adhesiveness, and/or solidification time of the hydrogel following mixing with additives, exposure to air, being applied to a patient, or mixing with an activation fluid; relative to ECM tissue without the carrier matrix.
  • the hydrogel is configured to increase density following mixing with additives, for example, sodium hyaluronate, carbomer, being applied as an intra-ocular injection to a patient.
  • a novel multi-use wound care product can be configured by adding with preservatives to the hydrogel, unlike existing collagen or acellular tissue powder wound care products in the market which are only for single use and only used as a powder/paste.
  • preservatives for example, potassium sorbate is added to the hydrogel for skin wound care, oral wound care and vaginal/anal wound care to serve a single patient multi-use purpose; and polyquaritum-1 is added to the hydrogel for ocular surface wound care to serve a single patient multi-use purpose.
  • This novel multi-use wound care product can be applied as powder/paste to wound site and the reminder powder/paste can be mixed with saline, isotonic solution or water to form a multi-use hydrogel or spray to provide continuous wound care management to wound site.
  • this novel multi-use wound care product can be directly mixed with saline, isotonic solution or water to form a multi-use hydrogel or spray.
  • a Rough Washing Step the tissue is washed to remove unwanted matter.
  • the materials removed can include umbilical cord and impurities, some large blood vessels and blood fluid.
  • Test standard visually no foreign matter. This step may include: Wash 3 times with PBS. Test standard: and visually checked for the absence of blood. Repeated freezing and thawing 3-5 times at -80 -25°C, to decellularize. In the flow of Dl water, rub the tissue repeatedly with a mesh for 5 min, carefully remove blood vessels and dirty tissues. Rinse the tissue with Dl water on shaker (room temperature, 140-160 rpm) for 30 min.
  • a Decellularization Step In a flow of Dl water, rub the tissue repeatedly with a mesh for 5 min, carefully remove blood vessels and dirty or sullied tissues. Rinse the tissue with 0.05% Trypsin / 0.02 EDTA diluted with Dl water on shaker (room temperature, 140-160 rpm) for 60 min. In the flow of Dl water, rub the tissue repeatedly with a mesh for 5 min, carefully remove blood vessels and dirty or sullied tissues. Rinse the tissue with 3% Triton diluted with Dl water on shaker (room temperature, 140- 160 rpm) for 60 min.
  • decellularization can include steps of: Rinse the tissue with hypertonic solution on shaker (room temperature, 140-160 rpm) for 30 min. Then tissue was rinsed with hypotonic solution on shaker (140-160 rpm; 30 min/time). Repeat three times.
  • Making lyophilized hydrogel powder can include: Optionally freezing the tissue for 4-8 hours at -80°C. Lyophilization for 48 to 72 h.
  • a Digestion Step Cutting the lyophilized ECM in to 1-3 cm 2 pieces. Cryogenic grinding the lyophilized decellularized ECM into powder. Dilute decellularized lyophilized ECM with (optionally porcine) pepsin (lmg/mL)/ HCL (0.01 M) diluted with Dl water until desired concentration. Shake solution at room temperature with 180-200 rpm for 24-48 h with electric mixer. Optionally, assuring that the solution does not rise above 35 degree Centigrade.
  • hydrogel has to be neutralized to induce polymerization: This is optionally done by adding 1/10 of total volume 0.1M NaOH and add PBS to obtain desired concentration.
  • Sterilization may then be accomplished by irradiation such as e-Beam radiation.
  • Packaging may occur in sealed evacuated vials.
  • various embodiments of the invention include making a tissue derived powder which can be rehydrated to a lyophilized extracellular matrix hydrogel, and other freeze-dried materials, such as protein, collagen, mucopolysaccharides and lyophilized tissue into uniform and fine lyophilized powder and maintain the biological activity of the extracellular matrix found within these tissue.
  • the resulting materials can be used in various therapeutic and non-therapeutic applications, such as treatment of various types of wounds, including: superficial wounds and complicated wound (pressure ulcers, venous ulcers, diabetic ulcers, chronic vascular ulcers, tunneled/undermined wounds, surgical wounds, and draining wounds.), which are difficult to treat using traditional dressings.
  • the resulting materials may also provide regeneration ability and play a key role of providing growing cells with a microenvironment that resembles their natural niche.
  • the carrier matrix (the combination of tissue derived powder (optionally including added components) and liquid used to rehydrate, which conveys biological active components) is configured to increase viscosity, adhesiveness and solidification time after being placed in contact with a patient for treatment.
  • a hydrogel may be thus configured by control of particle size and distribution, and/or viscosity can be controlled by the addition of additional components such as thickening agents.
  • the hydrogel viscosity and adhesiveness increases, and solidification times shorten in the right powder size and concentration and at a right temperature (e.g., body temperatures).
  • the Carrier Matrix is configured to increase viscosity adhesiveness, and/or solidification time of the hydrogel following mixing with additives, exposure to air, being applied to a patient, or mixing with an activation fluid; relative to ECM tissue without the Carrier Matrix.
  • the thickening process of the hydrogel includes collagen polymerization that occurs at 37 °C and neutral pH ( ⁇ 7.4) due to collagen's molecular structure, which is the unique supercoiled triple helix. This process relies on the unique properties of collagen.
  • collagen may be an added component to the powder of various embodiments of the invention, or may be intrinsic to the tissue used. Small particle size appears to accelerate this reaction.
  • tissue derived powder or rehydrated powder further comprises a therapeutic agent, the Carrier Matrix (e.g., hydrogel) being configured to release the therapeutic agent from the composition into a patient.
  • Carrier Matrix e.g., hydrogel
  • the powder can include additional components (other than tissue) including therapeutic agents. Some of these agents, e.g. stem cells, are optionally added after sterilization to preserve the function of biological species.
  • the hydrogel, of various embodiments of the invention can carry a variety of the taught components, such as pharmaceutical agents, exosomes, anti inflammatory drugs, antioxidants, etc. These components can be used alone or in combination with each other. Additional examples of materials that may be included in the hydrogel are listed in Table 4. Table 4.
  • human and/or animal tissue used to produce the powder and hydrogel are selected for inclusion in the composition based on anti-inflammatory compounds and/or effects found within the tissue.
  • the hydrogel includes a combination of compounds that reduced immune response (e.g., have an anti-inflammatory effect) and antibiotics or fungicides that reduce bacterial or fungal infection.
  • the hydrogel can result in a decrease in T-cell response while an antibiotic reduces likelihood of infection.
  • the hydrogel provides less inflammation and decreased chance of infection at the same time.
  • Inflammation has its positive side, but excessive inflammation can slow wound healing and induce scar formation, so the hydrogel containing possible pharmaceutical agents is optionally configured to target excessive inflammation.
  • the hydrogel can help with inflammation during the wound healing, which leads to faster healing with less scar tissues.
  • These properties in hydrogels derived from amniotic tissues may be related to the immune privileged characteristics of the placenta.
  • the tissue-derived hydrogel can simulate an improved environment for promotion and the growth of stem cells and epithelial cells and accelerate wound healing. Fibrosis is mainly associated with a significant increase in the expression of profibrotic cytokines such as TGF-b. In some studies, the hydrogel was shown to suppress of expression and release levels of cytokines such as local TGF-b in hydrogel-treated wounds compared with untreated wounds.
  • One exemplary method begins with an optional Solvate Step 2.1 in which a sterile liquid, e.g., normal saline, is added to the dried tissue derived powder to form a hydrogel.
  • a sterile liquid e.g., normal saline
  • the powder is optionally prepared using the methods illustrated by FIG. 1.
  • any of the therapeutic agents discussed herein may be added to the dry powder in a Solvate Step 2.1.
  • any of these therapeutic agents or additional components may be added as part of the preparation of the dried powder.
  • the formed hydrogel is applied to a wound or injury.
  • the mode of application can vary significantly depending on the nature of the wound or injury, and the desired therapeutic effect.
  • the hydrogel may be injected into an internal body cavity, applied to the skin and then covered with a dressing, applied as part of a wound dressing, applied as a coating on sutures, applied on a surface of an object to be placed in the body (e.g., a replacement hip joint or bone pin.
  • the hydrogel can be applied in vitro and/or in vivo.
  • the hydrogel is reapplied to the wound or injury.
  • a dressing including the hydrogel may be replaced with a fresh dressing including the hydrogel.
  • the hydrogel can be used as a carrier containing one or more pharmaceutical agents such as exosomes.
  • Exosomes carry a variety of proteins, mRNAs, miRNAs, growth factors, and/or lipids as important signaling molecules, which form a new cell-to-cell signaling system that participate in cell communication, cell migration, angiogenesis and so on. It has been reported that the powerful wound repair function of mesenchymal stem cells is related to the secreted exosomes of cells. Therefore, the stabilization and slow release of exosomes in vitro and in vivo is extremely important.
  • the hydrogel can be used as a carrier for mesenchymal stem cell exosomes.
  • FIG. 4 illustrates an amount of recombinant human epidermal growth factor (hEGF) released in the hydrogel as a carrier measured by ELISA analysis, according to various embodiments.
  • hEGF human epidermal growth factor
  • FIG. 5 illustrates an amount of recombinant human epidermal growth factor (hEGF) released in the hydrogel as a carrier measured by ELISA analysis, according to various embodiments of the invention.
  • hEGF human epidermal growth factor
  • Hydrogel acts as a lubricant in the joint cavity, reduces the friction between the tissues, possibly reduces inflammation, and/or at the same time exerts an elastic effect, which cushions the damage of the articular cartilage.
  • Injecting high concentration of the hydrogel into the joint cavity can significantly improve the inflammatory response of synovial tissue, enhance the viscosity and lubrication of joint fluid, protect articular cartilage, promote healing and regeneration of articular cartilage, relieve pain and increase joint mobility.
  • the barrier function of the hydrogel can effectively prevent the diffusion of inflammatory mediators and reduce the stimulation of pain receptors by chemical substances. Achieve the reduction of joint pain.
  • therapeutic agents which may be included in the hydrogel for these applications include: steroids, anti-inflammatory agents, pain relievers (e.g., opioids), collagen, hyaluronic acid, anti-oxidants, and/or the like. Any of the other therapeutic agents discussed herein can be included in the hydrogel for these applications.
  • Interventional therapy is a kind of minimally invasive treatment using modern high-tech means. For example, under the guidance of medical imaging equipment, special instruments such as orthoscopic/endoscopic tools, catheters and guide wires are introduced into the human body to diagnose and treat the diseased state. Interventional therapy uses digital technology to expand the doctor's field of vision. With the help of a catheter, the guide wire extends the doctor's hands. Many treatments can be provided with minimal damage to the tissue. Interventional therapy has the characteristics of no surgery, small trauma, quick recovery and good effect. It is the development trend of future medicine.
  • the hydrogel can be used as a coating after the solidification within 3-5 min to treat medical devices. Since the composition of the hydrogel is similar to that of natural extracellular matrix, it will greatly improve the tissue affinity of medical devices, avoid the formation of scar tissue and reduce the adverse effects.
  • the hydrogel is delivered to a patient as a coating on a catheter or shunt.
  • the hydrogel may be used as a coating to reduce inflammation otherwise induced by a catheter or drainage tube.
  • the hydrogel is used as a coating on a central venous catheter, indwelling foley catheter, peripheral IV lines, arterial lines. Any of the other therapeutic agents discussed herein can be included in the hydrogel for these applications.
  • ISR In-stent Restenosis
  • therapeutic agents can be added to the hydrogel to cover the stents, including anti-inflammatory agents, antioxidants, anticoagulant and/or the like. Any of the other therapeutic agents discussed herein can be included in the hydrogel for these applications.
  • the hydrogel or powder is optionally attached to or applied with a stent. Inflamed tissue resulting from stents can be treated using the hydrogel or powder, for example in the urethra or other vessels/ducts with the body.
  • Intrauterine adhesions occur to a high percentage of women who have had multiple curettage (D&C) surgeries. Scar tissue from uterine surgeries like dilation and curettage (D&C) cause more than 90% of IUA. Also, scar tissue from a Cesarean section or from sutures used to stop hemorrhage, endometriosis, infections of the reproductive organs and radiation therapy treatment can cause IUA.
  • D&C curettage
  • the hydrogel optionally solidifies within 3- 5 min and provides a protective layer on the uterine wall, prevents scar tissue formation and intrauterine adhesions, and provides a moist environment for intrauterine wound healing.
  • the hydrogel also provides anti-inflammatory effect and enhances the endometrial regeneration and thus improves fertility and pregnancy outcomes in clinical practice.
  • FIG. 6 illustrates an effect of the hydrogel on preventing and managing intrauterine adhesions following instillation of chemical agents, according to various embodiments of the invention.
  • Vitrectomy is a one of the standard and common surgeries to treat retinal tears, retinal detachments and macular holes. Often, Vitrectomy with air-fluid exchange requires patients to remain in a face-down position all day and night for a period of time varying from days to weeks. Sealing retinal tears/holes and macular holes during Vitrectomy and Pneumatic Retinopexy by applying a material such as glue or film can be an excellent and novel approach for preventing vitreous fluid from flowing through the open retinal tears into the subretinal space and can also obviate the time needed for postoperative face-down position.
  • a material such as glue or film
  • a cross-linking function of the hydrogel results from the hydrogel's optional collagen molecular structure.
  • This structure includes a unique supercoiled triple helix, which does not depend on an exogenous cross-linking agent.
  • the hydrogel with a concentration of extracellular matrix between 8-10 mg/ml can rapidly solidify in 3-5 min at 37 °C to quickly seal and adhere the retinal tears/holes and macular holes to prevent expansion of the tears and holes.
  • the Carrier Matrix, extracellular matrix, and/or processing thereof is configured to result in temperature triggered thickening or solidification at body temperature (e.g. 37C). This solidification rate is highly advantageous during surgery and is the result of methods used to prepare the hydrogel.
  • Bonding occurs both within the hydrogel and also with surrounding ocular tissue, resulting in adhesion to the surrounding tissue.
  • vitreous humor is removed by vitrectomy, and in some cases Laser surgery, (photocoagulation) or Freezing (cryopexy) is required in order to secure the retina to the eye wall.
  • the solidification of the hydrogel is irreversible and sufficient to repair retinal tears and holes.
  • the application of the hydrogel avoids the uncomfortable and challenging postoperative face-down position.
  • the hydrogel is 100% natural, has great biocompatibility and is biodegradable within 30 days.
  • the native extracellular matrix (ECM) of the hydrogel is a rich reservoir for hundreds of proteins, and a variety of growth factors and cytokines such as epidermal growth factor (EGF), transforming growth factor-b (TGF- b), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), and vascular endothelial growth factor (VEGF), etc.
  • EGF epidermal growth factor
  • TGF- b transforming growth factor-b
  • FGF fibroblast growth factor
  • PDGF platelet-derived growth factor
  • VEGF vascular endothelial growth factor
  • hyaluronic acid one of key components of extracellular matrix (ECM) of the hydrogel, is a primary constituent present in the subretinal locations and can serve as the optimal natural extracellular matrix (ECM) components of retina.
  • the hydrogel provides outstanding benefits in modern retinal treatment: 1. serves as an adhesive to quickly seal/adhere the retina tears/holes and macular holes (including those produced by surgery) and provides extracellular matrix (ECM) microenvironment of retina for tissue repair and regeneration; 2. shortens the postoperative recovery time dramatically since the hydrogel can solidify within 3-5 min thus a postoperative face down position is no longer required or required for a shorter time; 3. better supports the survival, proliferation and directed migration of local/adjacent retinal pigment epithelium (RPE) cells and retinal progenitor cells; 4.
  • RPE retinal pigment epithelium
  • the hydrogel with in-vitro cultured stem cells can be directly applied to the retina tears/holes and macular holes.
  • antifoaming agent is added to the hydrogel to avoid adverse effects causing by non-sterile air.
  • Any of the other therapeutic agents discussed herein can be included in the hydrogel for these applications.
  • Ocular surface wounds such as corneal chemical burns and mechanical injuries are common and serious ocular trauma.
  • corneal neovascularization induced by corneal chemical burns can cause serious visual impairment and even blindness.
  • blood vessels once blood vessels are formed, they are hard to be removed. Therefore, inhibition of neovascularization and regeneration of corneal epithelial cells is very important for the treatment of ocular surface injury.
  • the hydrogel can serve as an excellent substitute ocular surface fluid.
  • the hydrogel can well manage ocular surface wounds, quickly heal ocular wounds, prevents and minimize corneal neovascularization, anti-inflammatory, anti-scarring and anti-angiogenesis.
  • it can be used as eye drops, which makes it suitable for both clinical in office and in home use.
  • the hydrogel Given its unique function of self-crosslinking, the hydrogel can quickly form a protective film within 3 min to protect the cornea from external stimulus and provide a biological environment to manage ocular surface injuries.
  • the hydrogel also demonstrates strong efficacy in managing dry eye.
  • the animal model of corneal chemical (alkali) burns is often used to evaluate the effectiveness in dry eye.
  • therapeutic agents which may be included in the hydrogel for these applications include anti-inflammatory agents, anti-oxidants, anti-angiogenesis agents and/orthe like. Any of the other therapeutic agents discussed herein can be included in the hydrogel for these applications.
  • components are added to the hydrogel (or tissue/powder precursors) to increase density of the hydrogels.
  • the hydrogel can have a higher density than the vitreous humor of the eye in order to sink to and make contact on the retina in the proper patient position.
  • Fig. 7. Illustrates an effect of the hydrogel on ocular surface wounds and dry eye through the corneal chemical (alkali) burns animal study.
  • [0150]A Images of Slit lamp observation.
  • the cornea became turbid, and the transparency decreased significantly, some lesions were visible, and there was no significant difference in angiogenesis between any two groups.
  • corneal turbidity was reduced in each group, and corneal transparency in each hydrogel treatment group was better than that in the model group, but there was no significant difference between the hydrogel treatment groups.
  • the corneal became transparent and no angiogenesis were observed in each hydrogel treatment group. There was a small amount of angiogenesis in the model group.
  • FIG. 8 illustrates an effect of the hydrogel on ocular surface wounds through the corneal mechanical injuries animal study.
  • the hydrogel can serve as an excellent intraocular substitute fluid and intraocular anti-adhesion, anti-scarring and anti-inflammatory for various ophthalmic surgeries including cataract extraction, intraocular lens implantation, corneal transplant surgery, retina attachment, glaucoma laser treatment, and glaucoma incisional surgeries including trabeculectomy, filtering surgery, drainage implant surgery and electrocautery.
  • Glaucoma surgery can make patients more likely to get cataracts later.
  • Other possible risks of glaucoma surgery include: eye pain or redness, eye pressure that's still too high or even too low, loss of vision, infection, inflammation, bleeding in the eye.
  • the postoperative complications of cataract and glaucoma surgery can be reduced through the intraocular injection of the hydrogel.
  • the hydrogel can be used as a carrier containing one or more antibacterial agents such as cefuroxime and delivered as an ocular surface hydrogel and/or intraocular injection.
  • cefuroxime is a second-generation cephalosporin with bactericidal action against many Gram-positive organisms and some Gram-negative organisms.
  • the hydrogel can also be used as a carrier containing anti-metabolite drug such as 5-Fluorouracil (5-FU) or Mitomycin C (MMC) to form a sustained-release drug delivery system, which delays the scar formation after glaucoma surgery. Buffers may be used to adjust the hydrogel for an appropriate physiological pH for various parts of the eye.
  • anti-metabolite drug such as 5-Fluorouracil (5-FU) or Mitomycin C (MMC)
  • Buffers may be used to adjust the hydrogel for an appropriate physiological pH for various parts of the eye.
  • biocompatibility is an important feature of intraocular lenses which may influence their clinical performance in the short and long term.
  • a fully biocompatible artificial intraocular lens is expected to exhibit the following features: elicits no foreign-body reaction, is accepted by the surrounding tissues, has good compatibility with the capsular sac, and provides satisfactory vision over the lifetime of the patient without any further intervention.
  • Uveal biocompatibility is determined by the inflammatory reaction to the artificial intraocular lens formed in the eye.
  • Intraocular lens made from silica gel, polymethyl methacrylate and the hydrogel containing antibiotics by 3D-printing or intraocular lens coated with the hydrogel not only can effectively reduce the incidence of local bacterial infections, but also further increase the biocompatibility of intraocular lenses, reduce local inflammation and scar formation. Thereby, these advantages prolong the service life of intraocular lens and improve postoperative quality of life in cataract patients.
  • the treatment composition is formed by adding freeze-dried and ground embryonic tissue to an isotonic solution. This forms a gel that can be used as eye drops.
  • the gel can increase in viscosity within a few, e.g., 3-4 minutes, of application to the eye.
  • the gel may be transparent and can stay on the eye for several hours.
  • These embodiments may be used for ocular surface injuries, cataract incisions, corneal transplants, corneal epithelial cell regeneration, dry eye, etc.
  • the 3-4 minute solidification time, and the ability to control this time via particle size, cross-linkers, thickeners, etc., is useful in many of the applications discussed herein.
  • Some embodiments of the invention include a method of treating a patient, the method comprising: preparing a treatment composition including an isotonic solution and a powder of freeze- dried and ground amniotic fluid; and administrating the treatment composition to the patent as an eyedrop.
  • the treatment composition optionally being configured to form a gel on the outer surface of the patient's eye, the gel lasting on the surface for at least 10, 20, 30, 60, 120 or 180 min.
  • This use of hydrogel supports maintenance and/or regeneration of the corneal epithelial mass, for example of the retinal pigment epithelium.
  • An exemplary hydrogel formulation that may be used for ocular healing (as well as any of the other applications discussed herein) includes: 1) decellularized tissue hydrogel at concentrations of at least 2mg/mL (weight of powder to volume of rehydration fluid), 5mg/mL, lOmg/mL, 20mg/mL or 30mg/mL, or any range between these values; or less than 2mg/mL; and 2) Sodium Chloride and/or Potassium Chloride, 2) e.g., Sodium Chloride at a concentration of at least 0.1% or 0.2%; or between 0.1% and 0.2%; or less than 0.2%; (percentages herein are by weight percent)); 4) e.g., Potassium Chloride at a concentration of at least 0.02% or 0.05%; or between 0.02% and 0.05%; or less than 0.05%.
  • Optional Polyethylene Glycol at a concentration of at least 0.05% or 1%; or between 005% and 1%; or less than 1%.
  • Optional Carboxymethylcellulose Sodium at a concentration of at least 0.05% or 2.5%; or between 0.05% and 2.5%; or less than 2.5%.
  • Optional Sodium Hyaluronate at a concentration of at least 0.1% or 0.5%; or between 0.1% and 0.5%; or less than 0.5%.
  • the rehydration fluid optionally includes purified water.
  • the powder including ground tissue as described elsewhere herein and optionally comprising placenta tissue, such as porcine, bovine or human placenta tissue.
  • This ophthalmic hydrogel may be packaged in a liquid form and delivered sterile.
  • the sterilization is accomplished, for example, through one or more of the following methods: sterile filtration due to the small size particle of the product resulted from cryogrinding of the tissue, or, the product is irradiated by E beam, or irradiation by a radioactive isotope such as Gamma Cobalt 60.
  • a radioactive isotope such as Gamma Cobalt 60.
  • this the tissue derived powder is packaged in a powder form and delivered sterile, and rehydrated with normal purified water or saline upon use.
  • ASD Age-Related Macular Degeneration
  • RPE retinal pigment epithelium
  • the retinal pigment epithelium is a layer of epithelial cells containing melanin between the neural retina and the choroid, which is a single cell layer. Its physiological functions include absorb scattered light; control the fluid and nutrients in the subretinal space; regenerate and synthesize visual pigments; synthesize growth factors and other metabolites; maintain the adhesion of the retina; pinocytosis and digest photoreceptor metabolic waste; maintain electricity Homeostasis; regeneration and repair after trauma and surgery. Many clinical pigment changes in retinal diseases occur in the pigment epithelium.
  • the hydrogel is a rich reservoir for hundreds of proteins such as collagen, elastin and GaGs, etc. to mimic microenvironment for fibroblasts/keratinocytes migration/proliferation, mesenchymal stem cells homing, re-epithelialization, which are considered essential for slowing down Dry AMD (Atrophic AMD), inhibiting inflammatory responses and promoting regeneration of photoreceptor cells, RPE cells and retinal progenitor cells.
  • the hydrogel has a potential in inhibiting the formation of macular choroidal neovascularization (CNV), which is wet AMD.
  • CNV macular choroidal neovascularization
  • the hydrogel also has a potential in treating retinal tears and macular holes, healing the retinal wounds, anti-inflammatory and preventing neovascularization.
  • therapeutic agents which may be included in the hydrogel for these applications include anti inflammatory agents, anti-oxidants, stem cells and/or the like. Any of the other therapeutic agents discussed herein can be included in the hydrogel for these applications.
  • FIGs. 9A & 9B illustrate human retinal pigment epithelial (RPE) cells cultivated in vitro using the highly diluted hydrogel.
  • H&E staining showed that with the extension of culture time, the morphology of the cells changed from short spindle shape in the initial stage to pebble shape in the later stage. Moreover, there were obvious nuclei and bipolar nuclei in the cells and abundant black granules in the cytoplasm. Meanwhile, the proliferation in the hydrogel group was significantly higher than the control group in a dose-dependent way.
  • D Immunohistochemical staining of pigment epithelium-derived factor (PEDF), vascular endothelial growth factor-a (VEGF-a), basic fibroblast growth factor (b-FGF) and transforming growth factor-b (TGF-b). Results showed that with the extension of culture time, the release of vascular growth factor (PEDF) increased gradually, while the release of angiogenic factors (VEGF-a, b-FGF and TGF-b) gradually decreased.
  • E Western blot. Results showed that the expression of vascular growth factor (PEDF) increased in a dose-dependent manner.
  • VEGF-a vascular endothelial growth factor
  • b-FGF vascular endothelial growth factor
  • TGF-b vascular endothelial growth factor
  • tissue derived powder disclosed herein is optionally rehydrated to a paste or hydrogel and used as vaginal and anal treatment and/or as a personal lubricant.
  • the hydrogel may have a pH suitable for penile, anal and/or vaginal application, intended to lubricate and moisturize, to enhance the ease and comfort of intimate sexual activity and supplement the body's natural lubrication.
  • These embodiments are compatible with natural rubber latex and polyisoprene condoms.
  • the vaginal and anal Gel provides a protection layer on the mucosa tissue and provides moisture to promote vaginal and anal wound healing including but not limited to abrasions, surgeries, vaginal or anal ulcers (non-infected or viral, may be caused by chemotherapy or radiotherapy), vaginal or anal stenosis (may be caused by chemotherapy or radiotherapy).
  • the hydrogel may include spermicides, anti-viral, and/or antibacterial agents.
  • An exemplary formulation that may be used as a personal lubricant includes: 1) decellularized tissue hydrogel at a concentration of at least 2mg/mL, 5mg/mL, lOmg/mL, 20mg/mL or 30mg/mL, or any range between these values; or less than 2mg/mL; 2) Polyethylene Glycol at a concentration of at least 0.05%, 1%, 5%, 10%, 15% or 20%, or any range between these values; or less than 20%; 3) Optional hyaluronic acid at a concentration of at least 0.05%, 1%, 5%, 10%, 25%, 50% or 75%, or any range between these values; or less than 75%; 4) optional potassium sorbate; 5) optional saline solution; 6) optional vitamins such as Vitamin C, Vitamin E, Nicotinamide also known as Vitamin B3, Vitamin B12; 7) optional flavoring or sent agent; and 8) optional food grade colorants.
  • the powder may be packaged in a powder form and rehydrated with normal saline or water upon use.
  • the material is sterilized as described elsewhere herein.
  • the rehydrated gel can be delivered by a syringe, irrigator, applicator and/or a catheter to vagina or rectum.
  • the tissue derived powder is packaged in a rehydrated liquid form and can be delivered by a syringe, irrigator, applicator and/or a catheter to vagina or rectum.
  • Embodiments used for vaginal or anal wounding healing may have a higher concentration of ground tissue in the hydrogel.
  • the Vaginal and Anal Gel for personal moisturizing and lubricating has a much less concentration of the ground tissue but a higher concentration of hyaluronic acid which creates more gel viscosity.
  • nicotinamide is used as a whitening agent for the external genitalia.
  • Vaginal and anal wounds often occur during pelvic radiotherapy.
  • the hydrogel can be applied as a vaginal and anal lubricant to manage radiation vaginitis and proctitis, optionally with dilators to manage both vaginal and anal stenosis.
  • the hydrogel forms a protective layer on the vaginal and anal wall, and provides a moist environment for wound healing.
  • the hydrogel demonstrates a strong efficacy in managing vaginal and anal wounds including abrasions, ulcers, lesions, sores and surgical wounds.
  • the low concentration of the hydrogel has a certain anti-inflammatory effect and promotes the growth of epithelial cells. Therefore, it can also be used as a mouthwash to relieve the pain and inflammation in patients with oral ulcers/sores, gingivitis, post-tooth extraction.
  • the hydrogel can also be used as an oral solution to repair esophageal damage caused by radiotherapy in the head and neck, and or form a protective layer on mucosal tissues to manage oral mucositis, stomatitis and esophagitis induced by chemoradiotherapy, immunotherapy or other cancer targeted treatments.
  • the hydrogel can be used to treat the esophagus injuries caused by radiotherapy, acid reflux, ulcers, sores or chemical burns.
  • the hydrogel is optionally combined with a drinkable liquid or provided within a chewable capsule.
  • topical application of the hydrogel including antioxidants such as Hydroxytyrosol, Vitamin C, Vitamin E or Vitamin B12 can also promote wound healing. Any of the other therapeutic agents discussed herein can be included in the hydrogel for these applications. Radiation damage may be treated anywhere on the body.
  • Some embodiments of the invention include a treatment composition including extracellular matrix and carrier matrix as disclosed herein, further having a viscosity appropriate for drinking by a patient or being administered to a patient as an oral or nasal spray.
  • the treatment composition can include a drinkable hydrogel or a hydrogel sprayable into a patient's mouth.
  • Flavoring e.g., strawberry, peppermint or cinnamon flavor, is optionally added to the treatment composition.
  • Some embodiments of the invention include a method of treating a patient, the method comprising: applying a cancer treatment to the patient, the cancer treatment including chemotherapy, radiation therapy or particle beam therapy; preparing a treatment composition including a powder of freeze-dried and ground amniotic fluid; and administrating the treatment composition to the patent.
  • the treatment composition is optionally administered as an oral spray or drop.
  • the treatment composition optionally further includes an antibiotic, an anti-inflammatory agent, an antioxidant, a flavoring compound, a growth hormone, stem cells, antibodies, bacteriophage, and/or any combination thereof.
  • the treatment composition is optionally administered as an oral spray or drop 3-10 times a day.
  • the treatment composition may be used to treat burns, including sunburn, radiation burns, chemical burns and thermal burns. Treated burns may be internal or external to the patient. For example, the treatment composition may be used to treat internal radiation or chemical burns in the throat.
  • the hydrogel forms a protective layer on the skin surface and provides a moist environment for wound healing.
  • the hydrogel demonstrates a strong efficacy in managing cancer treatment induced skin dermatitis and managing pain and relief of pain.
  • FIG. 10 illustrates case studies of real patients using the hydrogel in managing radiation dermatitis and managing pain, according to various embodiments of the invention.
  • a woman with a history of nasopharyngeal carcinoma was treated with radiotherapy combined with chemotherapy (paclitaxel). Skin lesions occurred at the 26 th radiotherapy, and ulceration occurred at the 28 th radiotherapy. The patient has serious Grade IV radiation dermatitis according to the Radiation Therapy Oncology Group (RTOG) Criteria. The hydrogel was applied at Day 0, and radiation dermatitis was significantly alleviated at Day 4 and mostly healed at Day 7.
  • RTOG Radiation Therapy Oncology Group
  • FIG. 11 illustrates case studies of real patients using the hydrogel (spray form) in managing oral mucositis and managing pain, according to various embodiments of the invention ⁇ Oral mucositis had been significantly mitigated by the hydrogel.
  • FIG. 12 Case studies self-reported surveys of real patients using the hydrogel in managing chemoradiotherapy induced radiation dermatitis.
  • the 22 questionnaire results showed that the scores of radiation dermatitis were significantly reduced, suggesting that hydrogel can effectively prevent and manage radiation dermatitis and improve health-related quality of life.
  • FIG. 13 Case studies self-reported surveys of real patients using the hydrogel in managing chemoradiotherapy induced oral mucositis.
  • the 22 questionnaire results showed that the scores of radiation dermatitis were significantly reduced, suggesting that hydrogel can effectively prevent and manage oral mucositis and improve health-related quality of life.
  • FIG. 14 Case studies self-reported surveys of real patients using the hydrogel in managing radiation vaginitis. The 25 questionnaire results showed that the scores of radiation dermatitis were significantly reduced, suggesting that hydrogel can effectively prevent and manage radiation vaginitis and improve health-related quality of life.
  • FIG. 15 Case studies self-reported surveys of radiation proctitis. The 18 questionnaire results showed that the scores of radiation dermatitis were significantly reduced, suggesting that hydrogel can effectively prevent and manage radiation proctitis and improve health-related quality of life.
  • the hydrogel can be used as an ink for 3D printing.
  • the hydrogel has a certain viscosity and adhesiveness while solidifying at 37 °C within 3-5 min with a concentration between 8-10mg/ml.
  • the product printed with the hydrogel exhibits biocompatibility and anti-inflammatory effects and has a function of promotion of local damage repair, which promotes the synthesis of tissues and organs that can match the physiological condition.
  • hydrogel as a daily skin care product can effectively maintain skin moisture, be quickly absorbed by the skin, provide a protective layer against external irritations, reduce local inflammation and promote healing of opened comedo (blackheads).
  • the hydrogel accelerates the process of healing during acne or tattoo laser removal by reducing inflammation and promoting regeneration of new skin.
  • the hydrogel can be applied following laser treatment and/or as a regular (e.g., daily) topical treatment. Any of the other therapeutic agents discussed herein can be included in the hydrogel for these applications.
  • Fig. 16 illustrates a case studies of real patients using the hydrogel in managing acne vulgaris, according to various embodiments. A. Pustules and cysts were significantly diminished at Day 20 after using the hydrogel. B. Inflammation and redness were significantly alleviated at Day 14 after using the hydrogel.
  • the hydrogel has been tested as a subdermal injectable for filling (removing wrinkles) and volumizing the skin.
  • the hydrogel can be adjusted to have a degradation time (1/2 life) of at least 2, 3 or 4 weeks by varying the powder particle size, amount of collagen or other biodegradable thickener.
  • the hydrogel can be used to replace relatively more toxic materials. Rather than being toxic, the hydrogel may serve an antioxidant and/or anti-inflammatory function.
  • the hydrogel has been tested for reducing stretch marks, such as those caused by pregnancy. Stretch mark are caused by relatively deep scaring under the skin.
  • the hydrogel has been found to reducing scaring by improving elasticity and/or reducing adhesion between tissue layers.
  • the hydrogel is optionally applied using a micro-needle roller (dermal roller) during the expansion of the skin.
  • the abdominal bulging causes the skin's elastic fibers and collagen fibers to be damaged or broken.
  • the abdominal skin becomes thinner and thinner, and some pink and purple patterns with different widths and lengths appear. After childbirth, these patterns will gradually disappear, leaving a white or silver-white shiny scar line, which is striae gravidarum. Striae gravidarum appear mainly on the abdominal wall and may also appear in the inner and outer thighs, buttocks, chest, back waist and arms.
  • the main treatment is fractional laser treatment. However, laser can cause skin damage after treatment. If used together with the hydrogel, it not only promotes the repair of epidermal damage, but also further fills the missing collagen fibers in the depression and promotes muscle fiber growth and connection. Any of the other therapeutic agents discussed herein can be included in the hydrogel for these applications. Similar application of the hydrogel may be used following laser treatment to remove tattoos, treat issues "port wine stains" or any other dermatological uses of lasers.
  • the hydrogel can be applied during pregnancy to provide a protective layer of the skin, provide collagen and elastin fibers deep to the damaged skin, and a moist environment to reduce striae gravidarum and striae distensae.
  • the hydrogel provides a natural microenvironment for cell regeneration to further minimize striae gravidarum and striae distensae.
  • the main treatment for post-partum striae gravidarum and striae distensae after its appearance is laser treatment.
  • Applying the hydrogel as a post laser treatment wound care management product it not only promotes the wound healing induced by laser, but also further fills the missing collagen fibers and promotes muscle fiber growth and connection, and thus further minimize striae gravidarum and striae distensae.
  • Any of the other therapeutic agents discussed herein can be included in the hydrogel for these applications.
  • Similar application of the hydrogel may be used following laser treatment to remove tattoos, treat port-wine stains (nevus flammeus) or any other dermatological uses of lasers.
  • FIG. 17 illustrates case studies of real patients using the hydrogel in managing striae gravidarum in combination with laser treatment, according to various embodiments of the invention. Severity of striae gravidarum in two subjects after using the hydrogel in combination with laser treatment was significantly reduced. Black arrows indicate striae gravidarum.
  • Oral and gastrointestinal injuries can be difficult to treat. For example, one cannot merely put a "band-aid" on a wound within the esophagus. Injuries to these tissues may be caused by, for example, cuts, blunt trauma, acid reflux, cancer, inhalation or swallowing of caustic materials, ulcers, etc. In some cases, injuries may be caused by treatments for other ailments. For example, injury to the esophagus and oral tissues may be caused by radiation intended to treat cancer, or by chemotherapy drugs.
  • Embodiments of the invention include a composition intended to be administered to a patient orally.
  • This composition includes therapeutically active materials derived from embryonic tissues, and may also include pharmaceutical agents such as antibiotics, anti-viral agents, anti-cancer drugs, anti inflammatory, and/or the like.
  • Preparation of the treatment composition can include, for example, decellularization, chemical alteration, physical alteration, sterilization, of amniotic tissue and/or addition of therapeutic compounds.
  • the treatment compound can be included in a solid (e.g., freeze dried powder), a gel, a paste, a liquid, and/or the like.
  • the treatment composition may be configured for use in a wide variety of medical applications. For example, the treatment composition may be used to reduce the growth of undesirable tissues (e.g., fibrosis or angiogenesis), to reduce inflammation, to deliver therapeutic compounds (e.g., antibiotics), to treat ocular injuries, to treat burns, and/or the like.
  • undesirable tissues e.g., fibrosis or angiogenesis
  • therapeutic compounds e.g., antibiotics
  • Mesotherapy micro-injections for wrinkle elimination, hydration, toning (brown spots, hyperpigmentation and skin redness minimization), skin filling and volumizing are a non-surgical technique that uses micro-injections of pharmaceutical and homeopathic preparations, plant extracts, vitamins, and other ingredients into subcutaneous fat.
  • the hydrogel carrying abundant nutrition including hundreds of proteins, collagen, elastin, laminin, fibronectin, proteoglycan amino acids, peptides, etc. can be delivered through mesotherapy micro injections along with any of the other therapeutic agents discussed herein, to directly target the dermis.
  • the hydrogel can quickly eliminate wrinkles, deeply hydrate skin, minimize brown spots, hyperpigmentation and skin redness, and provides skin filling and volumizing.
  • the skin filling and volumizing can last up to 4 weeks due to the biodegradability of the hydrogel.
  • the filling and volumizing of the mesotherapy micro-injections of the hydrogel mixing with hyaluronic acid can last up to 6-12 months.
  • the particle size of the hydrogel can be reduced down to 325 Mesh (44 microns) or smaller by multiple cryogrinding processes, which enable the hydrogel to be delivered through mesotherapy micro-injections 34-guage needle.
  • the hydrogel can carry any of the other therapeutic agents discussed herein including vitamins, antioxidants, exosomes, stem cell, etc., to enhance the efficacy of anti-oxidation, anti-aging, anti-wrinkle and moisturizing.
  • FIGs. 18A & 18B illustrate case studies of real patients' satisfaction rate regarding the improvements made by using the hydrogel in mesotherapy micro-injections for skin regeneration, hydration, filling and volumizing, according to various embodiments of the invention. These figures illustrate the rate at which patients report an increase in satisfaction.
  • B-C Two real patients treated with mesotherapy micro-injections of the hydrogel. The results, obtained via VISIA complexion analysis, demonstrated that the patients' brown spots and hyperpigmentation were significantly diminished at Day 14 after the treatment. Meanwhile, it also demonstrated that the treatment also mitigated the skin redness at Day 14.
  • the powders, pastes, hydrogels and solutions discussed herein are optionally added to animal or human food products, health care products, cosmetics, medical devices, and/or medicines.
  • the uses of the powders, pastes, hydrogels and solutions discussed herein are not limited to therapeutic use.
  • they be used for nutritional or cosmetic uses.
  • amniotic tissues are discussed herein by way of example, other tissues may be used in the materials, systems and methods discussed herein. For example, pancreatic, blood, muscle, and/or nerve tissues; or embryotic tissues from animals.
  • cryogenic grinding described is to be used in biomaterial, collagen, fiber or tissue grinding.
  • the embodiments disclosed in can be used cryogenically or with room temperature grinding an applications such as: 1) Environmental agriculture industry: plant seeds (rice, wheat, corn, soybeans, etc.), fast iron-free grinding of rhizomes and leaves; 2) Electronics and materials industry: mechanical alloying, synthesis of amorphous materials, preparation of high-entropy alloys; 3) Chinese and western medicine: medicinal materials (medlar, rehmannia glutinosa, herbs, honeycomb, powder, etc.) are routinely ground to break down cell walls; 4) Textile and paper industry: conventional grinding of multifiber (cotton, linen, paper, cloth, etc.) items; 5) Chemical and pharmaceutical industry: constant temperature ball-milling solid-state reaction, conventional rapid mixing and milling of the materials; and 6) Animal feed industry: components (bone meal, fish meal, forage, etc.) are ground and mixed.
  • Each of these applications may benefit from the preservation of chemical or bioactivity

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US201962925498P 2019-10-24 2019-10-24
CN201911118465.6A CN110787117A (zh) 2019-11-15 2019-11-15 一种皮肤修复液及其制备方法
CN201911118116.4A CN111054486B (zh) 2019-11-15 2019-11-15 一种羊膜凝胶冻干粉加工工艺
US201962944832P 2019-12-06 2019-12-06
US201962946300P 2019-12-10 2019-12-10
CN202010056374.0A CN111000871A (zh) 2020-01-18 2020-01-18 一种口腔喷剂及其制备方法
CN202010056372.1A CN110946814A (zh) 2020-01-18 2020-01-18 一种皮肤中胚层修复液及其制备方法
US202063032729P 2020-06-01 2020-06-01
CN202010975599.6A CN112022880A (zh) 2020-09-16 2020-09-16 一种直肠黏膜损伤及创面修复溶胶液及其制作方法
CN202010975598.1A CN112022879A (zh) 2020-09-16 2020-09-16 一种妇科阴道黏膜损伤及创伤修复溶胶液及其制作方法
PCT/US2020/057431 WO2021081540A1 (en) 2019-10-24 2020-10-26 Preparation and use of therapeutic hydrogels

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