EP4178538A1 - A reaction two-component hydrogel for multifunctional wound dressing and method of its preparation - Google Patents

A reaction two-component hydrogel for multifunctional wound dressing and method of its preparation

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Publication number
EP4178538A1
EP4178538A1 EP21763000.3A EP21763000A EP4178538A1 EP 4178538 A1 EP4178538 A1 EP 4178538A1 EP 21763000 A EP21763000 A EP 21763000A EP 4178538 A1 EP4178538 A1 EP 4178538A1
Authority
EP
European Patent Office
Prior art keywords
hydrogel
component
components
hyaluronic acid
reactive
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.)
Withdrawn
Application number
EP21763000.3A
Other languages
German (de)
French (fr)
Inventor
Marjan MOTIEI
Tomás SÁHA
Petr SÁHA
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.)
Tomas Bata University in Zlín
Original Assignee
Tomas Bata University in Zlín
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Filing date
Publication date
Application filed by Tomas Bata University in Zlín filed Critical Tomas Bata University in Zlín
Publication of EP4178538A1 publication Critical patent/EP4178538A1/en
Withdrawn legal-status Critical Current

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    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/70—Web, sheet or filament bases ; Films; Fibres of the matrix type containing drug
    • A61K9/7007—Drug-containing films, membranes or sheets
    • 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
    • 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
    • A61L15/22—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing macromolecular materials
    • A61L15/28—Polysaccharides or their derivatives
    • 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
    • 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
    • A61L15/42—Use of materials characterised by their function or physical properties
    • A61L15/44—Medicaments
    • 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
    • A61L26/0009—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form containing macromolecular materials
    • A61L26/0052—Mixtures of macromolecular compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • C08B37/0009—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof
    • C08B37/0021—Dextran, i.e. (alpha-1,4)-D-glucan; Derivatives thereof, e.g. Sephadex, i.e. crosslinked dextran
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
    • C08B37/0027—2-Acetamido-2-deoxy-beta-glucans; Derivatives thereof
    • C08B37/003—Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C08B37/0072—Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
    • C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
    • C08J3/075—Macromolecular gels
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
    • C08L5/08—Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives 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
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/23—Carbohydrates
    • A61L2300/232—Monosaccharides, disaccharides, polysaccharides, lipopolysaccharides
    • 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
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/60—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
    • A61L2300/62—Encapsulated active agents, e.g. emulsified droplets
    • A61L2300/624—Nanocapsules
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2305/00—Characterised by the use of polysaccharides or of their derivatives not provided for in groups C08J2301/00 or C08J2303/00
    • C08J2305/08—Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2405/00—Characterised by the use of polysaccharides or of their derivatives not provided for in groups C08J2401/00 or C08J2403/00
    • C08J2405/08—Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof

Definitions

  • the invention relates to a reactive two -component hydrogel for multifunctional wound dressings with a sensitive effect for promoting the healing result, in particular of chronic wounds with an alkaline pH.
  • the invention further relates to a process of the preparation of this reactive two-component hydrogel.
  • the skin is the first defensive line of the human body.
  • When the skin is damaged leakage from the microvessels causes an increase in the pH of the wound surface to physiological pH (7.4), which gradually increases with increasing wound depth due to the presence of ammonia released from the urea.
  • the alkaline pH of chronic wounds in the range of 7.15 to 8.9 causes slow healing due to the stimulation of numerous proteases that cleave proteins, form toxic end products and promote polymicrobial infections, especially anoxic bacteria.
  • the target product should have several attributes, namely: (1) should be easy to apply and remove, (2) provide protection against pathogens, (3) maintain a microenvironment for optimal epithelial cell growth, (4) provide wound hydration and stimulate its healing, (5) remove problems related to wound healing, such as creating of scars.
  • the chitosan hydrogel dressing is prepared from 1 to 8 parts of chitosan or a derivative thereof, from 0.1 to 3 parts of g-polyglutamic acid or a salt thereof and 6 to 10 parts of oxidized hyaluronic acid or a salt thereof.
  • the chitosan hydrogel dressing based on the above invention can be prepared by oxidizing water as a solvent and biological crosslinking agent by the hyaluronic acid based by the Schiff base reaction when no additional crosslinking agents and initiators need to be added. The costs and biological toxicity of the gel are dramatically reduced and the safety of the clinical use of the gel material is ensured.
  • the chitosan dressing has excellent biocompatibility and water absorption, is safe, does not contain toxins and neither the production process nor the degraded dressing pollute the environment.
  • PCT International Patent Application No. W02014161085 deals with a wound dressing comprising a flexible pharmaceutically acceptable carrier material and a hydrogel applied thereon.
  • the hydrogel contains an aldehyde hyaluronic acid prepared by modifying hyaluronic acid with l-amino-3, 3-diethoxypropane and chitosan conjugated to this aldehyde hyaluronic acid through the Schiff base reaction.
  • the supporting material may be a fabric with natural and/or synthetic fibres, optionally the support material may be a polymeric film.
  • the wound dressing may further comprise one or more components of a buffer, emollient and antimicrobial composition.
  • the subject of the Chinese patent No. CN106243410 is a dual- network hydrogel composed of hydroxyethyl chitosan and hyaluronic acid and a process of its preparation. Its preparation comprises the following steps: separate modification of hydroxyethyl chitosan and hyaluronic acid with glycidyl methacrylate to obtain hydroxyethyl chito san-grafted glycidyl methacrylate and hyaluronic acid-grafted glycidyl methacrylate.
  • Glycidyl methacrylate grafted with hydroxyethyl chitosan is photochemically polymerized to obtain a hydroxyethyl chitosan hydrogel of the first network, and the hydroxyethyl chitosan hydrogel is lyophilized, impregnated in an aqueous photoinitiator solution and glycidyl methacrylate grafted with hyaluronic acid and photochemically polymerized; thereby obtaining a hydroxyethyl chitosan hydrogel of the second network.
  • the hydrogel has adjustable mechanical and swelling properties, high molding speed, simple process and good structural stability. It can be used to construct a 3D model of an in vitro tumour, drug delivery, tissue regeneration, etc.
  • hydrogels are prepared on the basis of mixtures (blends) which contain two or more polymers. Chemical reactions are used in the preparation of polymer blends, e.g. using Schiff base or photochemical reactions.
  • the invention of a reactive two-component hydrogel for multifunctional wound dressings with a sensitive effect to promote the healing result, in particular of chronic wounds with an alkaline pH, contributes to a large extent to overcoming the above-mentioned drawbacks of the known solutions listed above.
  • the reactive two-component hydrogel results from an in situ reaction of two oppositely charged hydrogel components, the first component being a chitosan-based hydrogel, which forms a drug delivery system for protection against invading pathogens, and the second component is a hyaluronic acid-based hydrogel forming a supporting structure for mesenchymal stem cells to create a micro-environment for optimal growth of regenerating cells.
  • the first component being a chitosan-based hydrogel, which forms a drug delivery system for protection against invading pathogens
  • the second component is a hyaluronic acid-based hydrogel forming a supporting structure for mesenchymal stem cells to create a micro-environment for optimal growth of regenerating cells.
  • the first component - a chitosan-based hydrogel is a suitable positively charged heat- sensitive hydrogel resulting from the gelation of chitosan in the presence of B-glycerol phosphate.
  • This hydrogel is a matrix for polyelectrolyte nanoparticles based on chitosan, with chitosan being further modified with a hydrophobic amino acid with an aliphatic hydrocarbon side chain, in particular alanine, and forming a system for dosing the antibiotic rifampin, resp. rifampicin.
  • rifampicin is trapped in the core of polyelectrolyte nanoparticles, which contain an amphiphilic chitosan core and an alkaline pH coating based on ionically cross- linked dextran sulfate and polyethyleneimine.
  • the second component - a hyaluronic acid hydrogel is a suitable negatively charged hydrogel that results from the gelation of hyaluronic acid in the presence of guanine and L- ascorbic acid and forms a support structure for mesenchymal stem cells to create a microenvironment for optimal growth of regenerating cells.
  • the process of preparing the reactive two-component hydrogel according to this invention lies in filling each of the hydrogel components into one of separate containers.
  • One reservoir is filled with a chitosan-based hydrogel, which forms a drug delivery system to protect against invading pathogens, and the other is a hyaluronic acid-based hydrogel forming a supporting structure for mesenchymal stem cells to prepare a microenvironment for optimal regenerating cell growth.
  • a sol-gel transition occurs first due to an increase in ambient temperature and then to their interaction due to electrostatic interactions between these oppositely charged hydrogel components and the alkaline pH of the chronic wound.
  • each of the hydrogel components into one of the separate chambers of the two-chamber package, e.g. hyaluronic forming a support structure for mesenchymal stem cells to prepare a microenvironment for optimal growth of regenerating cells.
  • these components are simultaneously expelled from both chambers, they then come into contact in a common container and subsequently interact with each other due to a change in ambient temperature, the opposite charge of both hydrogel components and the alkaline pH of the chronic wound.
  • Chitosan and hyaluronic acid are among the most important biomacromolecules in forming hydrogels, which are able to retain large amounts of water thanks to their hydrophilic structure.
  • Chitosan composed of glucosamine and N-acetylglucosamine is widely used in tissue engineering, wound healing and other biomedical applications because its properties include high biocompatibility, biodegradability, low immunogenicity, antibacterial effects and mucoadhesiveness.
  • the chito san-based hydrogel also has great potential for the use in the area of intelligent hydrogels that respond to changes in pH and surrounding temperature.
  • Chitosan is soluble in an acidic environment with a pH below 6.2 to 6.5 and at higher pH values forms a hydrated gel-like precipitate due to deprotonation of amines and the formation of hydrogen bonds.
  • Heat- sensitive hydrogels can also be formed from high-deacetylated chitosan in the presence of certain phosphates, by grafting or by simple mixing with other polymers.
  • Rifampin is a hydrophobic semi- synthetic antibiotic with a broad spectrum of activity against most gram-positive and some gram- negative bacteria, especially microorganisms adhering to the surface in the form of bio films.
  • the use of rifampin is limited by various complications, such as low bio availability, poor solubility, short biological half-life, modification of the skin microbiome, resistance to this antibiotic and its high hepatotoxicity.
  • polyelectrolyte nanoparticles are used to encapsulate rifampin in order to increase its bio availability by reducing the interaction with the alkaline wound microenvironment.
  • the polyelectrolyte nanoparticles contain an amphiphilic chitosan core for capturing hydrophobic rifampin and a alkaline pH coating based on ionically cross-linked dextran sulphate and polyethyleneimine.
  • chitosan is modified with a hydrophobic amino acid with an aliphatic hydrocarbon side chain, especially alanine.
  • Hyaluronic acid a linear glycosaminoglycan composed of repeating units of N-acetyl- D glucosamine and D-glucuronic acid, shows excellent potential in cell therapeutic therapy. Due to its key physicochemical properties, including biocompatibility, biodegradability, oxygen and nutrient permeability, and tuneable physical and mechanical properties, hyaluronic acid has the potential to control cell migration, growth, and organization. The rapid degradation of hyaluronic acid, which reduces its effectiveness in tissue engineering can be solved by simply modifying the functional groups.
  • the hyaluronic acid hydrogel is suitably modified with guanine due to its hydrophobic structure and its important functions in accelerating wound healing.
  • L-ascorbic acid is used to create functional double filler materials that can be bioactive and contribute to the regeneration and viability of mesenchymal stem cells and to the formation of hydrogels.
  • L- ascorbic acid has various functions in wound healing, and its deficiency results in impaired healing and immune response, decreased collagen synthesis, fibroblast proliferation, angiogenesis, increased capillary fragility, and susceptibility to wound infection.
  • L-ascorbic acid with a pKa of 4.17 also accelerates gel formation in the two-component hydrogel by electrostatic interactions when applied topically.
  • the resulting two-component stimulus-responsive hydrogel is formed in situ by reacting of two oppositely charged chito san-based and hyaluronic acid-based hydrogel components sequentially in three steps. Gel formation is induced above its gelation temperature (i.e. above room temperature) after two oppositely charged hydrogels combine to form an alkaline chronic wound.
  • the chito san-based hydrogel will serve to deliver rifampin-charged polyelectrolyte nanoparticles, the hyaluronic acid hydrogel prepared in the presence of guanine and L-ascorbic acid has a positive effect on mesenchymal stem cell regeneration.
  • the basic difference between the above-mentioned known solutions and the reactive two-component hydrogel for multifunctional wound dressing according to this invention is that while in the known solutions hydrogel based mixtures of two or more polymers are being prepared, according to this invention two separately prepared and stored hydrogels consisting of different polymers come into contact only during the process of application. While the known solutions use chemical reactions (e.g. with Schiff base or photochemical reactions) in the preparation of polymer mixtures, the solution according to the invention uses the electrostatic interaction of two hydrogels which act during application. In contrast to the hydrogels from the known solutions, the reactive two-component hydrogel for multifunctional wound dressing according to this invention reacts and is sensitive to external stimuli.
  • the sol-gel transition of the reactive hydrogel according to this invention takes place in three steps: above its gelation temperature, after contact of two oppositely charged hydrogels and after getting in contact with the basic environment of the chronic wound.
  • the advantage is that the two hydrogel components of the reactive hydrogel are filled in two separate chambers of the package, which eliminates the problems of chemical and physical stability due to the interaction of both components and guarantees a long shelf life.
  • a graft copolymer of alanine and chitosan (A-g-CS) is prepared by grafting alanine onto chitosan using EDC / NHS (1 -ethyl-3- (3-dimethylaminopropyl) carbodiimide / N- hydroxysuccinimide) crosslinking agents in stoichiometric amounts.
  • EDC / NHS (1 -ethyl-3- (3-dimethylaminopropyl) carbodiimide / N- hydroxysuccinimide) crosslinking agents in stoichiometric amounts.
  • the lyophilized products are then stored at -20 ° C.
  • G-HMDA guanine- 1,6-hexamethylenediamine
  • MeCN metal cyanide
  • N-bromosuccinimide is added while constant stirring and the mixture is then filtered.
  • the residual solid substance is transferred in acetone while stirring, then is stored at -20 ° C for 48 hours and filtered again.
  • the orange solid substance is then washed with cold acetone and dries through which 8-bromoguanosine (G-Br) is achieved. It is then dissolved in a solution of 1,6-hexamethylenediamine (HMDA).
  • the pH of the resulting solution is adjusted to 9.8 with concentrated HC1, and the solution is heated up and maintained at 115 ° C at constant stirring. After cooling to room temperature, it is precipitated in distilled water and filtered.
  • the next step is the synthesis of HA-HMDA-G (hyaluronic acid- 1,6- hexamethylenediaminoguanine).
  • EDC / NHS crosslinking agents l-ethyl-3- (3-dimethylaminopropyl) carbodiimide / N- hydroxysuccinimide
  • G-HMDA is added at pH 4.7 while stirring for 24 hours at laboratory temperature .
  • the reaction mixture is dialyzed (MWCO 12 kDa) against dilute hydrochloric acid (pH 3-5) and then against distilled water (dH2018).
  • L-ascorbic acid is then added to the hydrogel at 4 ° C in an optimized molar ratio and the mixture is stirred overnight.
  • MSCs mesenchymal stem cells growth in the hydrogel follows.
  • MSCs are cultured in a-modified minimal essential medium (a-MEM) with 10% fetal bovine serum (FBS) and 1% antibiotics.
  • a-MEM minimal essential medium
  • FBS fetal bovine serum
  • 1% antibiotics 1% antibiotics.
  • Each of the prepared hydrogel components is filled into one of the separate chambers of a two-chamber package - namely a two-chamber squeezing tube - so that one of the chambers contains a chito san-based hydrogel, which forms a drug delivery system to protect against invading pathogens and the other one contains hyaluronic acid based hydrogel forming a support structure for mesenchymal stem cells to prepare a microenvironment for optimal growth of regenerating cells.
  • these components When these components are simultaneously expelled from both chambers, they come into contact in a common outlet of the package and subsequently interact with each other due to the changed ambient temperature, the opposite charge of both hydrogel components and the alkaline pH of the chronic wound. Gel formation is induced above its gelation temperature (i.e. above room temperature) after two oppositely charged hydrogels come into contact and affect the alkaline environment of the chronic wound.

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Abstract

The reactive two-component hydrogel for multifunctional wound dressing with a sensitive effect to support the healing result, especially of chronic wounds with an alkaline pH, is the result of an in situ reaction of two oppositely charged hydrogel-based components. The first component is a chitosan-based hydrogel, which forms a drug delivery system to protect against invading pathogens, and the second component is a hyaluronic acid-based hydrogel forming a support structure for mesenchymal stem cells in the preparation of a microenvironment for optimal regenerating cell growth. In the process for preparing a reactive two-component hydrogel, each of the hydrogel components is filled into one of separate containers. When dosing these components from both reservoirs, they come into contact in a common outlet and subsequently react with each other due to the changed surrounding temperature, the opposite charge of both hydrogel components and the basic pH of the chronic wound.

Description

A REACTION TWO-COMPONENT HYDROGEL FOR MULTIFUNCTIONAL WOUND DRESSING AND A METHOD OF ITS PREPARATION
Field of technology
The invention relates to a reactive two -component hydrogel for multifunctional wound dressings with a sensitive effect for promoting the healing result, in particular of chronic wounds with an alkaline pH. The invention further relates to a process of the preparation of this reactive two-component hydrogel.
State of the Art
Thanks to its dense surface and stratum corneum, the skin is the first defensive line of the human body. As an intact barrier with a naturally acidic pH in the range of 4.2 to 5.6, it plays an important role in homeostasis and protects from invasion of microorganisms. Excessive amounts of physical or chemical factors can, however, disrupt the pH, increase transepidermal water loss, disrupt oxygen levels, protease activities, and the normal flora of the skin's microenvironment. When the skin is damaged, leakage from the microvessels causes an increase in the pH of the wound surface to physiological pH (7.4), which gradually increases with increasing wound depth due to the presence of ammonia released from the urea. The alkaline pH of chronic wounds in the range of 7.15 to 8.9 causes slow healing due to the stimulation of numerous proteases that cleave proteins, form toxic end products and promote polymicrobial infections, especially anoxic bacteria.
To facilitate the wound healing process and to support its good outcome, a multifunctional wound dressing able to meet numerous requirements is desirable. The target product should have several attributes, namely: (1) should be easy to apply and remove, (2) provide protection against pathogens, (3) maintain a microenvironment for optimal epithelial cell growth, (4) provide wound hydration and stimulate its healing, (5) remove problems related to wound healing, such as creating of scars.
Among currently known solutions is the one of the EP2328590 European patent application, which deals with a hydrogel composition for local application on skin and/or wounds. It comprises a suspension or dispersion of particles of at least one excipient, one stabilizer and a compound of one class of tetracyclines together with physiologically acceptable metal salt. The above listed particles are of an average diameter of maximum about 100 [urn] and a cellulose-containing hydrogel is mentioned here as a carrier. The CN107814981 Chinese patent application deals with a chito san-based hydrogel dressing and a process of its preparation. The chitosan hydrogel dressing is prepared from 1 to 8 parts of chitosan or a derivative thereof, from 0.1 to 3 parts of g-polyglutamic acid or a salt thereof and 6 to 10 parts of oxidized hyaluronic acid or a salt thereof. The chitosan hydrogel dressing based on the above invention can be prepared by oxidizing water as a solvent and biological crosslinking agent by the hyaluronic acid based by the Schiff base reaction when no additional crosslinking agents and initiators need to be added. The costs and biological toxicity of the gel are dramatically reduced and the safety of the clinical use of the gel material is ensured. The chitosan dressing has excellent biocompatibility and water absorption, is safe, does not contain toxins and neither the production process nor the degraded dressing pollute the environment.
PCT International Patent Application No. W02014161085 deals with a wound dressing comprising a flexible pharmaceutically acceptable carrier material and a hydrogel applied thereon. The hydrogel contains an aldehyde hyaluronic acid prepared by modifying hyaluronic acid with l-amino-3, 3-diethoxypropane and chitosan conjugated to this aldehyde hyaluronic acid through the Schiff base reaction. The supporting material may be a fabric with natural and/or synthetic fibres, optionally the support material may be a polymeric film. The wound dressing may further comprise one or more components of a buffer, emollient and antimicrobial composition.
The subject of the Chinese patent No. CN106243410 is a dual- network hydrogel composed of hydroxyethyl chitosan and hyaluronic acid and a process of its preparation. Its preparation comprises the following steps: separate modification of hydroxyethyl chitosan and hyaluronic acid with glycidyl methacrylate to obtain hydroxyethyl chito san-grafted glycidyl methacrylate and hyaluronic acid-grafted glycidyl methacrylate. Glycidyl methacrylate grafted with hydroxyethyl chitosan is photochemically polymerized to obtain a hydroxyethyl chitosan hydrogel of the first network, and the hydroxyethyl chitosan hydrogel is lyophilized, impregnated in an aqueous photoinitiator solution and glycidyl methacrylate grafted with hyaluronic acid and photochemically polymerized; thereby obtaining a hydroxyethyl chitosan hydrogel of the second network. The hydrogel has adjustable mechanical and swelling properties, high molding speed, simple process and good structural stability. It can be used to construct a 3D model of an in vitro tumour, drug delivery, tissue regeneration, etc. In all the above listed solutions, hydrogels are prepared on the basis of mixtures (blends) which contain two or more polymers. Chemical reactions are used in the preparation of polymer blends, e.g. using Schiff base or photochemical reactions.
As they are one-component pre-prepared hydrogels, problems with chemical and physical stability can occur when using them.
Grounds of the technical solution
The invention of a reactive two-component hydrogel for multifunctional wound dressings with a sensitive effect to promote the healing result, in particular of chronic wounds with an alkaline pH, contributes to a large extent to overcoming the above-mentioned drawbacks of the known solutions listed above.
The point is that the reactive two-component hydrogel results from an in situ reaction of two oppositely charged hydrogel components, the first component being a chitosan-based hydrogel, which forms a drug delivery system for protection against invading pathogens, and the second component is a hyaluronic acid-based hydrogel forming a supporting structure for mesenchymal stem cells to create a micro-environment for optimal growth of regenerating cells.
The first component - a chitosan-based hydrogel is a suitable positively charged heat- sensitive hydrogel resulting from the gelation of chitosan in the presence of B-glycerol phosphate. This hydrogel is a matrix for polyelectrolyte nanoparticles based on chitosan, with chitosan being further modified with a hydrophobic amino acid with an aliphatic hydrocarbon side chain, in particular alanine, and forming a system for dosing the antibiotic rifampin, resp. rifampicin. In other words, rifampicin is trapped in the core of polyelectrolyte nanoparticles, which contain an amphiphilic chitosan core and an alkaline pH coating based on ionically cross- linked dextran sulfate and polyethyleneimine.
The second component - a hyaluronic acid hydrogel is a suitable negatively charged hydrogel that results from the gelation of hyaluronic acid in the presence of guanine and L- ascorbic acid and forms a support structure for mesenchymal stem cells to create a microenvironment for optimal growth of regenerating cells.
The process of preparing the reactive two-component hydrogel according to this invention lies in filling each of the hydrogel components into one of separate containers. One reservoir is filled with a chitosan-based hydrogel, which forms a drug delivery system to protect against invading pathogens, and the other is a hyaluronic acid-based hydrogel forming a supporting structure for mesenchymal stem cells to prepare a microenvironment for optimal regenerating cell growth. When dispensing these components from both reservoirs, a sol-gel transition occurs first due to an increase in ambient temperature and then to their interaction due to electrostatic interactions between these oppositely charged hydrogel components and the alkaline pH of the chronic wound.
It is most convenient to fill each of the hydrogel components into one of the separate chambers of the two-chamber package, e.g. hyaluronic forming a support structure for mesenchymal stem cells to prepare a microenvironment for optimal growth of regenerating cells. When these components are simultaneously expelled from both chambers, they then come into contact in a common container and subsequently interact with each other due to a change in ambient temperature, the opposite charge of both hydrogel components and the alkaline pH of the chronic wound.
Chitosan and hyaluronic acid are among the most important biomacromolecules in forming hydrogels, which are able to retain large amounts of water thanks to their hydrophilic structure. Chitosan composed of glucosamine and N-acetylglucosamine is widely used in tissue engineering, wound healing and other biomedical applications because its properties include high biocompatibility, biodegradability, low immunogenicity, antibacterial effects and mucoadhesiveness. The chito san-based hydrogel also has great potential for the use in the area of intelligent hydrogels that respond to changes in pH and surrounding temperature. Chitosan is soluble in an acidic environment with a pH below 6.2 to 6.5 and at higher pH values forms a hydrated gel-like precipitate due to deprotonation of amines and the formation of hydrogen bonds. Heat- sensitive hydrogels can also be formed from high-deacetylated chitosan in the presence of certain phosphates, by grafting or by simple mixing with other polymers.
All the above properties also make chitosan a universal platform for drug delivery, especially for sensitive drugs such as rifampin. Rifampin is a hydrophobic semi- synthetic antibiotic with a broad spectrum of activity against most gram-positive and some gram- negative bacteria, especially microorganisms adhering to the surface in the form of bio films. However, the use of rifampin is limited by various complications, such as low bio availability, poor solubility, short biological half-life, modification of the skin microbiome, resistance to this antibiotic and its high hepatotoxicity. To overcome these obstacles, polyelectrolyte nanoparticles are used to encapsulate rifampin in order to increase its bio availability by reducing the interaction with the alkaline wound microenvironment. The polyelectrolyte nanoparticles contain an amphiphilic chitosan core for capturing hydrophobic rifampin and a alkaline pH coating based on ionically cross-linked dextran sulphate and polyethyleneimine. To form the core structure and increase the scavenging capacity of hydrophobic rifampin, chitosan is modified with a hydrophobic amino acid with an aliphatic hydrocarbon side chain, especially alanine.
Hyaluronic acid, a linear glycosaminoglycan composed of repeating units of N-acetyl- D glucosamine and D-glucuronic acid, shows excellent potential in cell therapeutic therapy. Due to its key physicochemical properties, including biocompatibility, biodegradability, oxygen and nutrient permeability, and tuneable physical and mechanical properties, hyaluronic acid has the potential to control cell migration, growth, and organization. The rapid degradation of hyaluronic acid, which reduces its effectiveness in tissue engineering can be solved by simply modifying the functional groups. In the present invention, the hyaluronic acid hydrogel is suitably modified with guanine due to its hydrophobic structure and its important functions in accelerating wound healing. This modification is a key factor that provides an in situ gel with the desired properties for a wide range of uses in regenerative medicine. L-ascorbic acid is used to create functional double filler materials that can be bioactive and contribute to the regeneration and viability of mesenchymal stem cells and to the formation of hydrogels. L- ascorbic acid has various functions in wound healing, and its deficiency results in impaired healing and immune response, decreased collagen synthesis, fibroblast proliferation, angiogenesis, increased capillary fragility, and susceptibility to wound infection. L-ascorbic acid with a pKa of 4.17 also accelerates gel formation in the two-component hydrogel by electrostatic interactions when applied topically.
The resulting two-component stimulus-responsive hydrogel is formed in situ by reacting of two oppositely charged chito san-based and hyaluronic acid-based hydrogel components sequentially in three steps. Gel formation is induced above its gelation temperature (i.e. above room temperature) after two oppositely charged hydrogels combine to form an alkaline chronic wound. The chito san-based hydrogel will serve to deliver rifampin-charged polyelectrolyte nanoparticles, the hyaluronic acid hydrogel prepared in the presence of guanine and L-ascorbic acid has a positive effect on mesenchymal stem cell regeneration.
The basic difference between the above-mentioned known solutions and the reactive two-component hydrogel for multifunctional wound dressing according to this invention is that while in the known solutions hydrogel based mixtures of two or more polymers are being prepared, according to this invention two separately prepared and stored hydrogels consisting of different polymers come into contact only during the process of application. While the known solutions use chemical reactions (e.g. with Schiff base or photochemical reactions) in the preparation of polymer mixtures, the solution according to the invention uses the electrostatic interaction of two hydrogels which act during application. In contrast to the hydrogels from the known solutions, the reactive two-component hydrogel for multifunctional wound dressing according to this invention reacts and is sensitive to external stimuli.
The sol-gel transition of the reactive hydrogel according to this invention takes place in three steps: above its gelation temperature, after contact of two oppositely charged hydrogels and after getting in contact with the basic environment of the chronic wound.
The advantage is that the two hydrogel components of the reactive hydrogel are filled in two separate chambers of the package, which eliminates the problems of chemical and physical stability due to the interaction of both components and guarantees a long shelf life.
Example of implementation of the invention
1. Preparation of a hydrogel component based on chitosan
First, a graft copolymer of alanine and chitosan (A-g-CS) is prepared by grafting alanine onto chitosan using EDC / NHS (1 -ethyl-3- (3-dimethylaminopropyl) carbodiimide / N- hydroxysuccinimide) crosslinking agents in stoichiometric amounts. The lyophilized products are then stored at -20 ° C.
After dissolving A-g-CS in acetic acid (1%), adjusting the pH to 5, mixing with emulsifier Tween 80 (polyoxyethylene sorbitan monooleate at a concentration of 0.5%), rifampin (in dimethyl sulfoxide / dH 2 O) was added at 4 ° C. Due to the basic environment of chronic wounds, the mixture is cross-linked at alkaline pH with dextran sulphate and polyethyleneimine. Both steps take place at constant stirring.
To the clear solution of chitosan in acetic acid, rifampin / polyelectrolyte nanoparticles and filtered P-glycoprotein are added dropwise at 4°C. Electrostatic attraction between oppositely charged P-glycoprotein and chitosan particles leads to more hydrophobic and hydrogen interactions between the chitosan chains, resulting in a sol-gel transition when body temperature is reached upon subsequent heating.
2. Preparation of hydrogel component based on hyaluronic acid
First, G-HMDA (guanine- 1,6-hexamethylenediamine) is prepared by synthesis. After dispersing the guanine on metal cyanide (MeCN) in water, N-bromosuccinimide is added while constant stirring and the mixture is then filtered. The residual solid substance is transferred in acetone while stirring, then is stored at -20 ° C for 48 hours and filtered again. The orange solid substance is then washed with cold acetone and dries through which 8-bromoguanosine (G-Br) is achieved. It is then dissolved in a solution of 1,6-hexamethylenediamine (HMDA). The pH of the resulting solution is adjusted to 9.8 with concentrated HC1, and the solution is heated up and maintained at 115 ° C at constant stirring. After cooling to room temperature, it is precipitated in distilled water and filtered.
The next step is the synthesis of HA-HMDA-G (hyaluronic acid- 1,6- hexamethylenediaminoguanine). After activation of the carboxyl group of hyaluronic acid with EDC / NHS crosslinking agents (l-ethyl-3- (3-dimethylaminopropyl) carbodiimide / N- hydroxysuccinimide), G-HMDA is added at pH 4.7 while stirring for 24 hours at laboratory temperature . The reaction mixture is dialyzed (MWCO 12 kDa) against dilute hydrochloric acid (pH 3-5) and then against distilled water (dH2018). L-ascorbic acid is then added to the hydrogel at 4 ° C in an optimized molar ratio and the mixture is stirred overnight.
Finally, mesenchymal stem cells (MSCs) growth in the hydrogel follows. MSCs are cultured in a-modified minimal essential medium (a-MEM) with 10% fetal bovine serum (FBS) and 1% antibiotics. To encapsulate MSCs labelled with Cell Tracker Green CMFDA dye in a hydrogel, the cell suspension (8 x 106 / ml, 10-12 passages) is mixed on ice with the hydrogel solution. The mixture is transferred to a microcentrifuge tube for gelation at 37°C. After removing the supernatant, culture medium is added to each tube and the tubes are incubated under normal conditions (21% O 2, 5% CO 2, 37 ° C) for 2 weeks.
3. In situ preparation of a two-component hydrogel sensitive to stimuli
Each of the prepared hydrogel components is filled into one of the separate chambers of a two-chamber package - namely a two-chamber squeezing tube - so that one of the chambers contains a chito san-based hydrogel, which forms a drug delivery system to protect against invading pathogens and the other one contains hyaluronic acid based hydrogel forming a support structure for mesenchymal stem cells to prepare a microenvironment for optimal growth of regenerating cells. When these components are simultaneously expelled from both chambers, they come into contact in a common outlet of the package and subsequently interact with each other due to the changed ambient temperature, the opposite charge of both hydrogel components and the alkaline pH of the chronic wound. Gel formation is induced above its gelation temperature (i.e. above room temperature) after two oppositely charged hydrogels come into contact and affect the alkaline environment of the chronic wound.

Claims

P A T E N T C L A I M S
1. A reactive two-component hydrogel for multifunctional wound dressing with a sensitive effect to promote the healing result, in particular of chronic wounds with alkaline pH, characterized in that it results in an in situ reaction of two oppositely charged hydrogel- based components, the first component being a chito san-based hydrogel forming a drug delivery system to protect against invading pathogens, and the second component being a hyaluronic acid-based hydrogel forming a support structure for mesenchymal stem cells in the preparation of a micro-environment for optimal growth of regenerating cells.
2. Reactive two-component hydrogel according to claim 1, characterized in that its first component - chito san-based hydrogel - is a positively charged heat- sensitive hydrogel resulting from gelation of chitosan in the presence of B-glycerol phosphate, chitosan being modified with a hydrophobic amino acid with aliphatic hydrocarbon side chain, especially alanine, and thus forming a system for dosing rifampin nanoparticles, resp. rifampicin entrapped in the form of polyelectrolyte nanoparticles which contain an amphiphilic chitosan core for entrapping hydrophobic rifampin and an alkaline pH coating based on ionically cross-linked dextran sulfate and polyethyleneimine.
3. Reactive two-component hydrogel according to claim 1, characterized in that its second component is a negatively charged hyaluronic acid-based hydrogel which is a product of gelation of hyaluronic acid in the presence of guanine and L-ascorbic acid and forms a support structure for mesenchymal stem cells in microenvironment preparation for optimal growth of regenerating cells.
4. A process for preparing a reactive two-component hydrogel according to claim 1, characterized in that each of the hydrogel components is filled into one of separate containers such that one of the containers contains a chito san-based hydrogel which forms a drug delivery system to protect against invading pathogens and in the second a hyaluronic acid-based hydrogel forming a support structure for mesenchymal stem cells in the preparation of a microenvironment for optimal growth of regenerating cells, where these components from both reservoirs come into contact with each other in the common outlet during dosing and subsequently react mutually with each other due to the change in ambient temperature, the opposite charge of both hydrogel components and the alkaline pH of the chronic wound.
5. A process of preparing a reactive two-component hydrogel according to claim 1, characterized in that each of the hydrogel components is filled into one of the separate chambers of a two-chamber package, e.g. a two-chamber squeeze tube, so that in one of the chambers drug delivery to protect against invading pathogens, and the second contains a hyaluronic acid-based hydrogel forming a support structure for mesenchymal stem cells to prepare a microenvironment for optimal growth of regenerating cells, with these components being simultaneously expelled from the two chambers at a common outlet from the package and their subsequent interaction due to the changed ambient temperature, the opposite charge of both hydrogel components and the alkaline pH of the chronic wound.
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