WO2021141748A1 - Compositions et procédés de régénération tissulaire - Google Patents

Compositions et procédés de régénération tissulaire Download PDF

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WO2021141748A1
WO2021141748A1 PCT/US2020/065650 US2020065650W WO2021141748A1 WO 2021141748 A1 WO2021141748 A1 WO 2021141748A1 US 2020065650 W US2020065650 W US 2020065650W WO 2021141748 A1 WO2021141748 A1 WO 2021141748A1
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wound
cmc
bis
subject
compositions
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PCT/US2020/065650
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English (en)
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Vicky Feng
Stephen Grubb
Guiting Lin
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Lifescienceplus, Inc.
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    • 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
    • A61L26/0004—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form containing inorganic materials
    • 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
    • A61K47/02—Inorganic compounds
    • 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/14—Esters of carboxylic acids, e.g. fatty acid monoglycerides, medium-chain triglycerides, parabens or PEG fatty acid esters
    • 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
    • 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
    • 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
    • A61K9/08—Solutions
    • 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
    • A61L26/0023—Polysaccharides

Definitions

  • the invention generally relates to hemostatic agents, wound care, bacteria static and tissue regeneration. More particularly, the invention relates to novel compositions for cell proliferation and tissue regeneration, and methods for their preparation and use thereof.
  • tissue repair is particularly relevant to bedridden or diabetic patients who develop severe non-healing ulcers or patient with gum ulcers or stomach ulcers. Patients suffering from internal lesions, such as those associated with the digestive tract, are particularly susceptible to the effects of non or slow-healing tissue damage.
  • Tissue repair and reconstruction requires the reconstructive material to be biocompatible with the patient.
  • the material must cooperate with the regenerative and repair processes in order for new tissue to suitably form in three-dimensions with adequate strength and stability.
  • many current materials are often poorly suited for proper support of the cell proliferation and tissue reconstruction.
  • Many antimicrobial or antiinfection materials are very toxic on surface wound as they damage good tissue and restrain growth of good tissue.
  • many regenerative processes are inadequate, costly or in turn cause other medical complications.
  • This invention is based on the discovery of a carboxymethyl cellulose (CMC)-based composition specially designed to provide maximum cell proliferation and tissue regeneration functionalities.
  • CMC carboxymethyl cellulose
  • the aqueous formulation of carboxymethyl cellulose and select ingredients and devices made thereof are superior to existing comparable materials and devices.
  • the enhanced cell proliferation and tissue regeneration effect can be achieved quickly and is long-lasting with formation of stable gel to seal the damaged wound injury or infected chronically wounds including the arterial and vein injury.
  • the controlled and sustained tissue regeneration and wound healing offer a novel tool to improve traumatic injury outcomes in both civilian and military settings.
  • a major feature of the invention is that the disclosed compositions and methods allow rapid generation of new tissue while reducing risks of infection, such as infections on stomach ulcers, hemorrhage ulcers, colon ulcers, throat ulcers, oral ulcers, and skin ulcer and chronic wounds, as well as infections associated with acute wounds such as bum, abrasion, surgical or trauma wounds and all other acute and chronic wounds.
  • infections on stomach ulcers, hemorrhage ulcers, colon ulcers, throat ulcers, oral ulcers, and skin ulcer and chronic wounds as well as infections associated with acute wounds such as bum, abrasion, surgical or trauma wounds and all other acute and chronic wounds.
  • compositions and methods are particularly suited for treating wounds, including chronic ulcer wounds, diabetes wounds or pressure ulcer wounds, etc., that require tissue repair and regeneration.
  • the invention generally relates to Wound Healing Tissue Regeneration Material (WHTRM) in a solid, powder, gel or liquid form (e.g., an aqueous composition) for cell proliferation and/or tissue regeneration, comprising: carboxymethyl cellulose (CMC) having a concentration in the range of about 10 mg/L to about 1,000 mg/L, Nad having a concentration in the range of about 6,000 rng/L to about 10,000 mg/1,, KC1 having a concentration in the range of about 100 mg/L to about 300 mg/L), one or both of ⁇ ad iPCL and KH2PQ4, and one or more of Magnesium, Aluminum, Chromium, Manganese, Iron, Nickel, Zinc, Strontium, Molybdenum and Barium.
  • CMC carboxymethyl cellulose
  • Nad having a concentration in the range of about 6,000 rng/L to about 10,000 mg/1
  • KC1 having a concentration in the range of about 100 mg/L to about 300 mg/L
  • the invention generally relates to a unit dosage form comprising any one of the WHTRMs disclosed herein.
  • the invention generally relates to a medical device comprising any one of the WHTRMs disclosed herein.
  • the invention generally relates to a method for promoting cell proliferation, comprising administering to a subject in need thereof an effective amount of any one of the WHTRMs disclosed herein.
  • the invention generally relates to a method for promoting tissue regeneration, comprising administering to a subject in need thereof an effective amount of any one of the WHTRMs disclosed herein.
  • the invention generally relates to a method for treating a wound, comprising administering to the wound of a subject an effective amount of any one of the WHTRMs disclosed herein.
  • FIG. 1 Establishment of pig dermal cells (PDC).
  • FIG. 3 PDCs cell mitosis enhanced by CMC and WHTRM treatment in vitro. Under the treatment, there are 22.2 ⁇ 3.5% cells underwent cellular mitosis in WHTRM 100 pg/mL while 12.5 ⁇ 3.2% cells in CMC 100 pg/mL. Interestingly, there is no significant difference at 1,000 pg/mL of both CMC and WHTRM (13.2 ⁇ 2.2% vs 11.9 ⁇ 3.6%, respectively).
  • FIG. 4. PDCs Cell proliferation affected by CMC and WHTRM at different concentration in vitro.
  • FIG. 5 Differential PDCs Cell proliferation affected by CMC and WHTRM at 10 pg/mL.
  • FIG. 6 Differential PDCs Cell proliferation affected by CMC and WHTRM at 100 pg/mL.
  • FIG. 7 Differential PDCs Cell proliferation affected by CMC and WHTRM at 1,000 pg/mL.
  • FIG. 8 Cellular signaling affected by CMC in both PDCs and PADSCs.
  • CMC enhanced the phosphorylation of AMPK, which leaded to the accumulation of b-catenin and activated the Wnt/ -catenin signal pathway, and leaded to the increased expression of Cyclin D1 in PDCs.
  • b Effect of CMC on PADSCs in vitro.
  • FIG. 9 A hypothetical mechanism of carboxymethylcellulose in activating dermal cells and adipose derived stem cells.
  • FIG. 10 Zone of inhibition (ZOI) for both E. coli strain UTI89 and CFT073 from BloodSTOP disks (arrow) in LB agar plates in vitro.
  • FIG. 11 Inhibition of E. coli by 1.5% BloodSTOP iX in agar dilution LB plate.
  • FIG. 12 Inhibition of E. coli by 1.5% BloodSTOP iX in agar LB plate surface smear test.
  • the invention is based in part on the discovery of novel compositions for cell proliferation and sustained tissue regeneration, and methods for their preparation and use thereof.
  • the compositions of the invention offer significantly improved tissue regeneration and wound healing outcomes and are applicable to various wound and tissue types and conditions.
  • the herein disclosed wound healing tissue regenerative material (WHTRM), an innovative carboxymethylcellulose (CMC)-based formulation, is an efficient and effective wound care and tissue regeneration material.
  • WHTRM wound healing tissue regenerative material
  • CMC carboxymethylcellulose
  • the material can be in solid, powder, gel or liquid form (e.g., as an aqueous composition).
  • Carboxymethyl cellulose is a cellulose derivative with carboxymethyl groups (-CH2- COOH) bound to some of the hydroxyl groups of the glucopyranose monomers that make up the cellulose backbone. It may be used as its sodium salt, sodium carboxymethyl cellulose.
  • the invention generally relates to use of a WHTRM for cell proliferation and/or tissue regeneration, comprising: carboxymethyl cellulose (CMC) having a concentration in solution in the range of about 10 mg/L to about 1,000 mg/L, Nad having a concentration m the range of about 6,000 mg/L to about 10,000 mg/L, KC1 having a concentration in the range of about 100 mg/L to about 300 mg/L), one or both of NayJIPCti and KH2PO4, and one or more of Magnesium, Aluminum, Chromium, Manganese, Iron, Nickel, Zinc, Strontium, Molybdenum and Barium.
  • CMC carboxymethyl cellulose
  • the one or both of NaLIPOi and KH2PO4, if present, have concentrations of about 1,000 mg/L to 1,800 mg/L ( S ⁇ HPOr) and of about 200 rng/L to about 180 mg/L (KH2PO4).
  • the one or more of Magnesium, Aluminum, Chromium, Manganese, Iron, Nickel, Zinc, Strontium, Molybdenum and Barium have concentrations as follows: about 0.066 -6.6 pg/L (Magnesium), about 0.0034 pg/L to about 0.34 pg/L (Aluminum), about 0.0011 pg/L to about 0.01133 pg/L (Chromium), about 0.0020 pg/L to about 0.2 pg/L (Manganese), about 0.019 pg/L to about 1.93 pg/L (Iron), about 0.019 pg/L to about 1.93 pg/L (Nickel), about 0.023 pg/L to about 2.33 pg/L (Zinc), about 0.0044 pg/L to about 0.44 pg/L (Strontium), about 0.001 pg/L to about 0.1
  • the aqueous composition further comprises one or more of Cyclic diethylene glycol adipate, 7-Oxabicyclo[2.2.1]hept-5- ene-2,3-dicarboxylic acid, Bis(2-hydroxyethyl) adipate, D-glucose, 2,3-bis-0-(carboxymethyl), Decanamide, N,N-bis(2- hydroxy ethyl), Diethyl succinate, Glycine, N-(2-hydroxyethyl)- N-[2-[(l-oxohexadecyl) amino]ethyl], Hexadecanamide, Octadecanamide, and Poly(ethylene glycol) monohexadecyl ether.
  • the invention generally relates to a unit dosage form comprising any one of the WHTRMs disclosed herein.
  • the invention generally relates to a medical device comprising any one of the WHTRMs disclosed herein.
  • the invention generally relates to a method for promoting cell proliferation, comprising administering to a subject in need thereof an effective amount of any one of the WHTRM’s disclosed herein.
  • the invention generally relates to a method for promoting tissue regeneration, comprising administering to a subject in need thereof an effective amount of any one of the WHTRM’s disclosed herein, with a reduction of scaring upon wound healing.
  • the invention generally relates to a method for reducing and reversing the effects of skin aging, comprising administering to the skin of a subject an effective amount of any one of the WHTRM forms disclosed herein.
  • the invention generally relates to a method for treating a wound, comprising administering to the wound of a subject an effective amount of any one of the WHTRMs disclosed herein.
  • the subject suffers from a bum wound.
  • the subject suffers from a surgical wound.
  • the subject suffers from a trauma wound.
  • the subject suffers from a diabetic ulcer wound.
  • the subject suffers from an acute wound. [0046] In certain embodiments, the subject suffers from a chronical wound.
  • the subject suffers from a skin lesion.
  • the subject suffers from a skin wound.
  • the subject suffers from skin wrinkles, sagging skin, and general signs of skin aging.
  • the subject suffers from excessive scaring during wound healing.
  • the CMC -based formulation is, in addition to hemostatic application, wound sealing, and protection for skin wounds, can also promote adipose derived stem cell proliferation and enhanced wound healing. It binds to epithelium and promotes cell migration for tissue regeneration.
  • the CMC formulation of the invention thus offers the capability to stimulate dermal and stem cell proliferation and is useful for enhancing skin regeneration, for example, after skin injury or bum.
  • the growth factors are from host blood, the ability of the CMC formulation in promoting cell proliferation was enhanced significantly.
  • Activation of tissue residential stem/progenitor cells is a novel approach for stem cells therapy on the skin. There is, however, no well-established method to achieve this task.
  • the present invention allows application of the disclosed composition to accelerate stem cells activation.
  • the disclosed invention addresses current unmet needs by promoting proliferation of resident stem/progenitor cells, including dermis and adipose derived stem cells, etc. and thus facilitate: regeneration of skin and subcutaneous tissue in bum wound, promotion of nerve regeneration, and facilitation of formation of new blood vessels.
  • the disclosed invention provides a versatile methodology with far-reaching clinical applications.
  • the disclosed composition and materials may be formulated especially for treating bum injuries, nerve regeneration, stem cell activation for both endogenous and exogenous stem cells, and tissue regeneration, including skin antiaging.
  • a new epithelium After injury to skin, whether bums or wounds, or other injury, a new epithelium has to form in order to close the wound, which requires the proliferation of keratinocytes, fibroblasts, dermal stem cells and mesenchymal stem cells (MSCs).
  • the dermal stem cells and MSCs are essential for skin homeostasis and repair of the lesions, by promoting cell differentiation and re- epithelialization.
  • compositions enjoy key advantages over current products due to its non- invasive activation of endogenous stem cells and boosting of cell proliferation and tissue regeneration. It is superior to existing approaches in several important aspects.
  • endogenous stem cells can be activated without their isolation and culturing thus avoiding invasive procedures and the associated costs and delays.
  • boosting, mobilization and retaining of stem cells within the target tissue offer preventive and therapeutic effects.
  • the compositions are well suited for treating diabetes complications, such as skin ulcer with non-invasive and effective method.
  • the disclosed innovation is superior to existing products because of it is a non-invasive product that can activate endogenous stem cells for regeneration, boost stem cells proliferation and for tissue regeneration.
  • WHTRM Wound Heal Tissue Regenerative Material
  • the WHTRM when in solution form contains multiple ingredients.
  • Exemplary formulations include the followings:
  • CMC Carboxymethyl Cellulose
  • CMC or WHTRM activate PVnl b-calenin signal pathway through activating AMP K
  • WHTRM’s contain major component is CMC.
  • the CMC is consisted of glucopyranose subunits that have the ability to bind the glucose transporter (GLTU). When binding to glucose transporters, the CMC will competitively inhibit the intracellular transfer of glucose, which lowers the concentration glucose in cytoplasm, this activated the AMPK pathway. It is well known that the Wnt/ -catenin signal pathway is the pivotal pathway in stem cell activation. To explore the mechanism of CMC promote cell proliferation of PDCs and PADSCs, we checked the Wnt/ -catenin signal pathway and related proteins under the treatment of CMC at different concentrations.
  • CMC enhanced the phosphorylation of AMPK, which leaded to the accumulation of b-catenin and activated the Wnt/ -catenin signal pathway, and leaded to the increased expression of Cyclin D1 in both PDCs and PADSCs.
  • the phosphorylation level of AMPK is 12.89 ⁇ 1.19% in control group, increased to 20.33 ⁇ 1.78% (p ⁇ 0.05) in 10 pg/mL CMC, to 22.27 ⁇ 1.55%(p ⁇ 0.01) in 100 pg/mL CMC, and to 21.69 ⁇ 1.37%(p ⁇ 0.05) in 1,000 pg/mL CMC respectively.
  • the cyclin Dl/ -actin ratio was 0.38 ⁇ 0.03 in control group, increased to 0.61 ⁇ 0.05% (p ⁇ 0.05) in 10 pg/mL CMC, to 0.73 ⁇ 0.04%(p ⁇ 0.01) in 100 pg/mL CMC and 0.67 ⁇ 0.07% (p ⁇ 0.05) in l,000pg/ml CMC (FIG. 8).
  • the cyclin D I/b-actin ratio was 0.38 ⁇ 0.05 in control group, increased to 0.74 ⁇ 0.06% (p ⁇ 0.05) in lOpg/ml CMC, to 0.74 ⁇ 0.05%(p ⁇ 0.01) in 100 pg/mL CMC and 0.83 ⁇ 0.04% (p ⁇ 0.01) in 1,000 pg/mL CMC (FIG. 8b).
  • WHTRM consisting more than 25 components including the critically significant molecule CMC, generated significantly better biological effects in promoting cell proliferation and tissue regeneration due to the beneficial molecules within WHTRM.
  • the WHTRM and CMC related products have the ability to stimulate dermal and stem cell proliferation and thus may be useful for enhancing skin regeneration after skin injury or bum.
  • the WHTRM and CMC enhanced the phosphorylation of AMPK, which lead to the accumulation of b-catenin and activated the Wnt/ -catenin signal pathway, and lead to the increased expression of Cyclin D1 in PDCs, which by binding to glucose transporter (GLTU) on the cell membrane of stem cells and other cells within the tissues.
  • GLTU glucose transporter
  • PDCs Pig Dermal cells
  • fPADSCs Pig adipose tissue derived stem cells
  • a dermal tissue from a pig were cut into small pieces in PBS on ice then add complete Dispase (0.8U/mL) in RPMI1640 for 5 times volume. After incubating at 4°C overnight, the tissues were incubated at 37°C for 2 hours allowing the separation of dermis and epidermis with tweezers. Then the dermis was incubated separately in 0.1% Collagenase D in complete RPMI 1640 medium at 37°C overnight. After vigorously pipetting the dermal solution to single cell suspension, filtered the cell suspension with a 70-micron filter. The cells were then cultured in DMEM containing 10% FBS, 1% Pen/Strep, and 1% ascorbic acid and lOng/ml EGF and FGF2. (FIG. 1).
  • ADSC isolation has been described previously (Lin, et al., Stem Cells Dev 2008 Dec;17(6): 1053-63). Briefly, the adipose tissue was rinsed with PBS containing 1% penicillin and streptomycin, minced into small pieces, and then incubated in a solution containing 0.075% collagenase type IA (Sigma-Aldrich, St. Louis, MO) for 1 hr at 37°C with vigorous shake. The top lipid layer was removed and the remaining liquid portion was centrifuged at 220 g for 10 min at room temperature. The pellet was treated with 160 mM NH4C1 for 10 min to lyse red blood cells.
  • the remaining cells were suspended in DMEM supplemented with 10% fetal bovine serum (FBS), filtered through a 40-um cell strainer (BD Biosciences, Bedford, MA), and plated at a density of 1 x 106 cells in a 10-cm dish. After reaching 80% confluence, the cells were harvested and stored in liquid nitrogen at a density of 5 x 105 cells per ml of freezing media (DMEM, 20% FBS, and 10% DMSO). The frozen cells were thawed for experiments as needed.
  • FBS fetal bovine serum
  • 40-um cell strainer BD Biosciences, Bedford, MA
  • CMC carboxymethylcellulose sodium
  • WHTRM carboxymethylcellulose sodium
  • PDCs cells were seeded in 6- well plates with glass cover slide and cultured in DMEM (supplemented with 10% FBS) for overnight followed by CMC and WHTRM treatment. This concentration of CMC and WHTRM or controls was selected 156 pg/mL. The cells were cultured in test media for 24 hour. At the end of culture, the cells were washed with culture medium extensively, to remove the unbound CMC and WHTRM. The cells were then labeled with lOuM Calcein-AM to view the fluorescence density and cell numbers.
  • the PDCs and PADSCs cells at 80% confluence were rinsed twice with PBS, trypsinized and resuspended in serum-free DMEM (supplemented with 0.1% BSA) at 1 x 10 5 cells/mL.
  • serum-free DMEM supplied with 0.1% BSA
  • the cells were seeded in 6 well dishes with glass cover slides, for cell proliferation assay, the cells were seeded in 96 well dishes; for cellular signaling assay, the cells were seeded in 6 well dishes.
  • the cells were treated with in serum-free DMEM (supplemented with 0.1% BSA) for cell synchronization.
  • the PDCs and PADSCs were then treated with different concentration of CMC (0, 10, 100, l,000pg/mL) or WHTRM (0, 10, 100, l,000pg/mL) for different time point.
  • the cellular protein samples were prepared by homogenization of cells in a lysis buffer containing 1% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% sodium docecyl sulfate, aprotinin (10 mg/mL), leupeptin (10 mg/mL), and PBS.
  • Cell lysates containing 20 pg of protein were electrophoresed in sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to a polyvinylidene fluoride membrane (Millipore Corp, Bedford, MA). The membrane was stained with Ponceau S to verify the integrity of the transferred proteins and to monitor the unbiased transfer of all protein samples.
  • Detection of target proteins on the membranes was performed with an electrochemiluminescence kit (Amersham Life Sciences Inc, Arlington Heights, IL) with the use of primary antibodies for p-AMPK (1:500), AMPK (1:300) (Cell Signaling Technology, Beverly, MA), b-catenin (1:500), cyclin D1 (1:500) and b-actin (1:1000) (Santa Cruz Biotechnology, Santa Cruz, CA). After the hybridization of secondary antibodies, the resulting images were analyzed with Chemilmager 4000 (Alpha Innotec) to determine the integrated density value of each protein band.
  • the cells were fixed with ice-cold methanol for 8min, permeabilized with 0.05% TritonX- 100 for 5min, and blocked with 5% normal HS in PBS for lh at room temperature. Next, they were incubated with rabbit anti-H3P (1:500, Merck Millipore, Billerica, MA, USA) antibody for lh at room temperature. After washing with PBS three times, the cells were incubated with Alexa Fluor- conjugated goat anti-rabbit antibody (1:500, Invitrogen Inc) for 1 hr at room temperature. After three washes with PBS, the cells were stained with DAPI for nuclear staining for 5min. Finally, the Immunofluorescence slides were examined under a fluorescence microscope and photographed.
  • composition of the invention showed significant utility in prevention of bacterial infection and wound management, especially for burn wounds where prevention of infection is critical.
  • the antibacterial activity is mostly evaluated using in vitro tests such as the agar diffusion test (ADT), broth dilution, and agar dilution test.
  • ADT agar diffusion test
  • the agar diffusion test (ADT) can be utilized to measure the effect of an antimicrobial sample against micro- organisms grown on culture plates. If the agent is effective against the microorganisms at a certain concentration, no colonies will grow where the concentration in the agar is greater than or equal to the effective concentration. This is the zone of inhibition (ZOI). Accordingly, the size of the ZOI can be used to rate the agent’s antimicrobial efficacy: the larger the ZOI, the more effective the agent. This method further strictly depends on the diffusion of the antimicrobial agent in the wound dressing.
  • WHTRM wound heal tissue regenerative material
  • E. coli strain UTI89 and CFT073 were used in this study.
  • LB agar plate To prepare LB agar plate, the LB broth as described above but add 15 g of agar. Autoclave and allow the medium to cool to approximately 55 °C. Pour into plates. Store the plates at 4 °C. [0087] The agar diffusion test (ADT) was performed in according with the DIN 58940-3.
  • E. coli strain UTI89 and CFT073 overnight cultures were diluted 1:100 to adjust the working suspensions to a microbial count of app. 1-x 10 6 cfu/mL. 100 uL of these suspensions were plated on LB agar plates and left to dry for 10 min and culture at 37°C for overnight. Afterwards aseptically prepared dressing samples (with a diameter of 4X6 mm) were placed onto the inoculated agar plates. The plates were incubated for 24 h at 37 °C. Afterwards the zone of inhibition (ZOI) was measured in mm and all plates were photographed for documentation.
  • ZOI zone of inhibition
  • WHTRM agar dilution plate 1.5 gm of WHTRM were added to 100 ml agar LB broth at 55 °C, mix well and Pour into plates. Store the plates at 4 °C. E. coli strain UTI89 and CFT073 overnight cultures were diluted 1 : 100 to adjust the working suspensions to a microbial count of app. 1-x 10 6 cfu/mL. 50 uL of these suspensions were plated on LB agar plates and left to dry for 10 min and culture at 37°C for overnight. Afterwards the clones of UTI89 and CFT073 were photographed for documentation.
  • WHTRM exhibit antibacterial activity in the ADT test
  • the WHTRM exhibited a formation of a distinct zone of inhibition (ZOI) for both E. coli strain UTI89 and CFT073. It was further noted, there is no E-coli growth underneath the solid form ofWHTRM (FIG. 10).

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Abstract

L'invention concerne de nouveaux dispositifs, des matériaux pour la cicatrisation, l'antimicrobien ou l'anti-infection, la prolifération cellulaire et la régénération tissulaire, et leurs procédés de préparation et d'utilisation.
PCT/US2020/065650 2020-01-08 2020-12-17 Compositions et procédés de régénération tissulaire WO2021141748A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070051376A1 (en) * 2002-05-24 2007-03-08 Corium International, Inc. Composition for cushions, wounds dressings and other skin-contacting products
US20070053960A1 (en) * 2005-08-31 2007-03-08 Board Of Regents, The University Of Texas System Multiribbon nanocellulose as a matrix for wound healing
US20090233509A1 (en) * 2005-07-22 2009-09-17 Davide Bellini Biomaterials Based On Carboxymethylcellulose Salified With Zinc Associated With Hyaluronic Acid Derivatives
US20090254104A1 (en) * 2006-09-28 2009-10-08 Children's Medical Center Corporation Methods and collagen products for tissue repair

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070051376A1 (en) * 2002-05-24 2007-03-08 Corium International, Inc. Composition for cushions, wounds dressings and other skin-contacting products
US20090233509A1 (en) * 2005-07-22 2009-09-17 Davide Bellini Biomaterials Based On Carboxymethylcellulose Salified With Zinc Associated With Hyaluronic Acid Derivatives
US20070053960A1 (en) * 2005-08-31 2007-03-08 Board Of Regents, The University Of Texas System Multiribbon nanocellulose as a matrix for wound healing
US20090254104A1 (en) * 2006-09-28 2009-10-08 Children's Medical Center Corporation Methods and collagen products for tissue repair

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