WO1998025575A2 - Composition d'hydrogel amelioree - Google Patents

Composition d'hydrogel amelioree Download PDF

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Publication number
WO1998025575A2
WO1998025575A2 PCT/US1997/022860 US9722860W WO9825575A2 WO 1998025575 A2 WO1998025575 A2 WO 1998025575A2 US 9722860 W US9722860 W US 9722860W WO 9825575 A2 WO9825575 A2 WO 9825575A2
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WIPO (PCT)
Prior art keywords
cells
alginate
hydrogel
gel
calcium
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PCT/US1997/022860
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English (en)
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WO1998025575A3 (fr
Inventor
Kermit M. Borland
Tao Zhou
Gordon P. Nelson
Anthony Atala
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Reprogenesis, Inc.
Children's Medical Center Corporation
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Application filed by Reprogenesis, Inc., Children's Medical Center Corporation filed Critical Reprogenesis, Inc.
Priority to AU56015/98A priority Critical patent/AU5601598A/en
Publication of WO1998025575A2 publication Critical patent/WO1998025575A2/fr
Publication of WO1998025575A3 publication Critical patent/WO1998025575A3/fr

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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
    • 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
    • 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
    • A61L27/38—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 containing added animal cells
    • A61L27/3804—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 containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3817—Cartilage-forming cells, e.g. pre-chondrocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0068—General culture methods using substrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B17/00—Surgical instruments, devices or methods, e.g. tourniquets
    • A61B2017/00743—Type of operation; Specification of treatment sites
    • A61B2017/00805—Treatment of female stress urinary incontinence
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/0004—Closure means for urethra or rectum, i.e. anti-incontinence devices or support slings against pelvic prolapse
    • A61F2/0022—Closure means for urethra or rectum, i.e. anti-incontinence devices or support slings against pelvic prolapse placed deep in the body opening
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/0059—Cosmetic or alloplastic implants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2210/00—Particular material properties of prostheses classified in groups A61F2/00 - A61F2/26 or A61F2/82 or A61F9/00 or A61F11/00 or subgroups thereof
    • A61F2210/0085—Particular material properties of prostheses classified in groups A61F2/00 - A61F2/26 or A61F2/82 or A61F9/00 or A61F11/00 or subgroups thereof hardenable in situ, e.g. epoxy resins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2240/00—Manufacturing or designing of prostheses classified in groups A61F2/00 - A61F2/26 or A61F2/82 or A61F9/00 or A61F11/00 or subgroups thereof
    • A61F2240/001—Designing or manufacturing processes
    • A61F2240/002—Designing or making customized prostheses
    • 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
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/06—Flowable or injectable implant compositions
    • 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
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/22—Materials or treatment for tissue regeneration for reconstruction of hollow organs, e.g. bladder, esophagus, urether, uterus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/12—Light metals, i.e. alkali, alkaline earth, Be, Al, Mg
    • C12N2500/14—Calcium; Ca chelators; Calcitonin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/70—Polysaccharides
    • C12N2533/74—Alginate

Definitions

  • the present invention is generally in the area of creating new tissues using polysaccharide hydrogel-cell compositions.
  • Tissue engineering involves the morphogenesis of new tissues from constructs formed of isolated cells and biocompatible polymers. Cells can be adhered onto a polymeric matrix and implanted to form a cartilaginous structure. This can be accomplished, as described in U.S. Patent No. 5,041,138 to Vacanti, et al., by shaping of the matrix prior to implantation to form a desired anatomical structure and surgical implantation of the shaped matrix.
  • a simple method of delivering additional autogenous cartilage or bone to the craniofacial skeleton that is site specific and of controlled dimensions would reduce surgical trauma and eliminate the need for allografts or alloplastic prostheses. If one could transplant by means of injection or simple application to the desired site, and cause to engraft large numbers of isolated cells, one could augment the craniofacial osseocartilaginous skeleton with autogenous tissue but without extensive surgery. Furthermore, successfully implanting isolated cells would create the potential for tissue culture augmentation of the cells.
  • a liquid support matrix that polymerizes to a gel is more easily shaped and molded for custom reconstruction or augmentation. Additionally, a liquid polymer system can potentially be used for injectable delivery, which would be much less invasive than open implantation.
  • Calcium alginate gels have been proposed as a means of delivering large numbers of isolated chondrocytes to promote engraftation and cartilage formation. These initial studies were extended in International Patent Publication No. WO 94/25080 to the formulating of slowly polymerizing calcium alginate gels and to the use of these gels to deliver large numbers of chondrocytes by means of injection, for the purpose of generating new cartilage.
  • Injectable compositions containing cells have been produced by suspending suitable cells in medium Ml 99, and mixing the cell suspension with an equal part of 2% sodium alginate solution, then adding solid calcium sulfate powder to initiate cross-linking of the alginate to form a hydrogel.
  • Such a composition typically will contain 1% sodium alginate in a hydrogel with insoluble calcium sulfate in an amount of about 200 mg per ml of suspension.
  • a small amount of calcium chloride may be provided by the cell suspension, but usually less than 0.1 mg per ml in the final suspension.
  • hydrogel-cell suspensions have a latent period on the order of an hour, followed by a rapid increase in viscosity to produce a relatively hard, even brittle gel within half an hour of the viscosity increase.
  • the consistency of hydrogel-cell suspensions described above is not totally satisfactory for use in injection into patients in need thereof.
  • the hydrogel-cell suspension hardens before it can be injected into the patient.
  • Such gels are useful for preparing preformed structures for implantation into patients to repair structural defects of known shape, but require extensive surgery to open up a cavity of sufficient size to receive the preformed structure.
  • hydrogel-cell suspensions maintain low viscosity until injection into the desired location in the body, but such suspensions assume the shape of the existing cavity or space in the patient. Such gels cannot be used where the defect is absence of a defined structure.
  • a hydrogel-cell suspension with sufficient consistency to maintain its shape when implanted in the patient, but which remains injectable to minimize the extent of surgery necessary for implantation.
  • shortcomings in the present injectable cartilage formulation that would disallow its use in clinical trials include (a) insufficient viscosity to divide the tissue plane and not extravasate upon injection; (b) extended time required prior to achieving high enough viscosity to attempt injection; and (c) inconsistent performance between lots due to poor distribution of components during formulation.
  • a cell suspension (a) having a consistency similar to POLYTEF teflon paste at injection; (b) achieving this consistency immediately upon mixing, and (c) retaining that consistency for a long period of time to allow proper application.
  • this invention provides a method for implanting cells into an animal, the method comprising (1) forming a mixture containing cells, which may be dissociated cells and/or cell aggregates; a biodegradable, biocompatible polymer which forms a hydrogel upon cross-linking by multivalent ions; a soluble salt of a multivalent ion; and a sparingly soluble salt of a multivalent ion, such that the mixture forms a partially hardened, injectable hydrogel in which the cells are uniformly suspended; and (2) implanting the partially hardened hydrogel mixture into the animal, wherein the implanted partially hardened hydrogel forms a fully hardened hydrogel containing the cells in situ.
  • the biocompatible polymer is alginate.
  • the multivalent ions are metal cations other than magnesium; more preferably the metal cations are calcium.
  • the mixture also contains a sequestrant which competes with alginate for binding calcium ions, preferably an anionic sequestrant such as phosphate anion.
  • the soluble salt is calcium chloride and the sparingly soluble salt comprises a cation selected from the group consisting of copper, calcium, duminum, magnesium, strontium, barium, tin, and di-, tri- or tetra-functional organic cations; and an anion selected from the group consisting of low molecular weight dicarboxylic acids, sulfate ions, carbonate ions, phosphate ions and citrate ions.
  • the sparingly soluble salt is calcium sulfate and the mixture contains calcium chloride/calcium sulfate in ratio of from 0.001 to 100, preferably in ratio of from 0.0075 to 1.0 by weight.
  • the mixture contains at least 0.5 % alginate by weight and at least about O.OOlg calcium chloride per gram of alginate.
  • Suitable cells are selected from the group consisting of chondrocytes and other cells that form cartilage, osteoblasts and other cells that form bone, muscle cells, fibroblasts, and organ cells. In preferred embodiment, the cells are chondrocytes isolated from the patient.
  • this invention provides a method for treating anatomical defects in an animal in need of treatment thereof comprising preparing a suspension of cells uniformly dispersed in a biodegradable, biocompatible hydrogel solution which is partially hardened, and implanting the suspension into the animal at the site of the defect, wherein the hydrogel solution fully hardens after implantation.
  • the partially hardened hydrogel has a viscosity at 25 °C of at least 15,000 cps or the suspension of dissociated cells in the partially hardened hydrogel is suitable for injection through a 23-gauge cannula at a rate of 10 mVmin. without substantially damaging the cells.
  • this invention provides a composition for implanting cells into an animal comprising a hydrogel containing aggregated cells or dissociated cells, such as chondrocytes and other cells that form cartilage, osteoblasts and other cells that form bone, muscle cells, fibroblasts, and organ cells; a biocompatible polymer which forms a hydrogel upon cross-linking by multivalent cations; a highly soluble ionic salt of a first multivalent cation; and a sparingly soluble salt of a second multivalent cation, the composition forming a partially hardened hydrogel at room temperature which is suitable for injection into an animal and which, after injection, will fully harden in vivo.
  • aggregated cells or dissociated cells such as chondrocytes and other cells that form cartilage, osteoblasts and other cells that form bone, muscle cells, fibroblasts, and organ cells
  • a biocompatible polymer which forms a hydrogel upon cross-linking by multivalent cations
  • the first and second multivalent cations may be the same or different and are selected from the group consisting of copper, calcium, aluminum, magnesium, strontium, barium, tin, and di-, tri- or tetra-functional organic cations; and an anion selected from the group consisting of low molecular weight dicarboxylic acids, sulfate ions and carbonate ions.
  • the partially hardened hydrogel is suitable for injection through a 23-gauge cannula at a rate of at least 10 rnl/min. without reducing viability of the cells by more than 25%.
  • the composition contains as the biocompatible polymer, alginate at a concentration of at least about 0.5%, as the highly soluble salt, calcium chloride at a concentration of 1.5 mg/mL, and/or as the sparingly soluble salt, calcium sulfate at a concentration of 15 mg/mL.
  • the ratio for calcium chloride: calcium sulfate is preferably from 0.001 to 100, more preferably from 0.0075 to 1.0 by weight.
  • the compositions contains a sequestrant which competes for binding of the second multivalent cation with alginate. More preferably, the sequestrant is phosphate anion.
  • Suitable cells may be selected from the group consisting of chondrocytes, osteoblasts, muscle cells, fibroblasts, and organ cells; preferably, the cells are chondrocytes.
  • the present invention provides a readily available source of cross- linking cations, generally in the form of calcium chloride, at a level sufficient only to initiate and partially cross-link a biocompatible anionic polymer (e.g., alginate) to a thick paste consistency, preferably in approximately 15 minutes. More preferably, alginate is present in an amount sufficient to incorporate all water in the formulation into the loosely cross- linked gel (typically greater than about 0.5% alginate is present in such injectable cell suspensions). Such a formulation will set up quickly for prompt use in an operating theater, while maintaining good "injectable" consistency for an extended period of time to allow completion of procedures. The consistency of this partially cross-linked hydrogel may be maintained in excess of 24 hours. Furthermore, the suspension has a thick consistency which more easily divides tissues when injected and is less subject to extravasation from the injection site.
  • a biocompatible anionic polymer e.g., alginate
  • Figure 1 shows the time course of gel formation at 25 °C for alginate cross-linked with calcium sulfate powder and calcium chloride/calcium sulfate, respectively.
  • Figure 2 shows the time course of gel formation at 37° C for alginate cross-linked with calcium sulfate powder and calcium chloride/calcium sulfate, respectively.
  • Figure 3 shows the time course of gel formation at 5 °C for alginate cross-linked with calcium sulfate powder and calcium chloride/calcium sulfate, respectively.
  • Cell/polymer systems which form hydrogel compositions for tissue engineering include suspensions of cells such as chondrocytes in alginate solution to which calcium salts are added to initiate hydrogel formation.
  • Typical systems include chondrocyte-calcium alginate solutions created by vortexing an isolated cell suspension with sodium alginate solution (e.g., in 0.1M K 2 HP0 4 , 0.135 M NaCl, pH 7.4) to yield a cellular density of 20 X 10 6 cells/ml (a cellular density of approximately 50 percent of that of native articular bovine cartilage) in a 1.0% alginate solution.
  • the chondrocyte- sodium alginate suspension can be stored on ice at 4°C until use.
  • 0.2 gm of sterilized CaS0 4 powder is added to each milliliter of the cold chondrocyte-alginate solution.
  • Studies on the gel formation properties of formulations using calcium sulfate as the source of cross-linking ion show that the formulation can gel in as little as 16 minutes at human body temperature, but does not gel readily at room temperature.
  • the temperature of the alginate/cell suspension is raised to the level necessary to initiate gel formation within a reasonable time (i.e., on the order of minutes rather than hours in the operating room), a window of only several minutes exists during which the consistency of the composition is suitable for injection.
  • the method of this invention involves formation of hydrogel by multivalent cation cross-linking of acidic biopolymers in which the availability of the multivalent cation is controlled by dissolution of a sparingly soluble salt to replenish soluble cation as the soluble cation is absorbed in cross-links.
  • a portion of the multivalent cation is provided by a fully soluble salt and is freely available ("fast" cross-linking ion), while another portion of the multivalent cation necessary for full cross-linking is provided in a sparingly soluble salt (“slow" cross-linking ion).
  • compositions that also contains a soluble buffering system for the multivalent cation, usually phosphate when the cation is a di- or tri-valent metal ion.
  • Phosphate competes with alginate for the metal, so that cross-linking of the alginate in the composition is forestalled by competition between alginate and phosphate anions for the limited amount of available cation as the components of the composition are mixed.
  • the biocompatible acidic biopolymer alginate is cross-linked by a minor portion of fully-soluble calcium chloride and a major amount of the much-less-soluble calcium sulfate.
  • the composition from which the hydrogel is formed contains phosphate anion in relatively high concentration to buffer the soluble calcium ion concentration and forestall complete cross-linking of the alginate. Once the composition is injected at the desired site, phosphate anion is diluted out by diffusion into the surrounding biological fluids, and the concentration of soluble calcium ion increases locally. As the soluble calcium ion concentration increases, more calcium cross-links are formed in the alginate, and the hydrogel hardens. Similar effects may be obtained for analogous metal ions by buffering with phosphate anion or other similar systems, e.g., biologically compatible sequestrants, which buffer soluble cation concentration in an analogous manner. Sources of Cells.
  • This technique can be used to provide multiple cell types, including genetically altered cells, within a three-dimensional scaffolding for the efficient transfer of large number of cells and the promotion of transplant engraftment for the purpose of creating a new tissue or tissue equivalent. It can also be used for immunoprotection of cell transplants while a new tissue or tissue equivalent is growing by excluding the host immune system.
  • cells which can be implanted as described herein include chondrocytes and other cells that form cartilage, osteoblasts and other cells that form bone, muscle cells, fibroblasts, and organ cells, and also includes expanded populations of isolated stem cells.
  • organ cells includes hepatocytes, islet cells, cells of intestinal origin, cells derived from the kidney, and other cells acting primarily to synthesize and secret, or to metabolize materials.
  • the cells to be encased in the injectable hydrogel can be obtained directly from a donor, from cell culture of cells from a donor, or from established cell culture lines.
  • cells are obtained directly from a donor, washed and implanted directly in combination with the polymeric material.
  • Cellular material may be dissociated single cells, minced tissue (i.e., clumps of aggregated cells) or cell aggregates generated in vitro from dissociated cells.
  • the cells may be cultured using techniques known to those skilled in the art of tissue culture. Cell persistence and viability after injection can be assessed using scanning electron microscopy, histology, and quantitative assessment with radioisotopes.
  • the function of the implanted cells can be determined using a combination of the above-techniques and functional assays.
  • in vivo liver function studies can be performed by placing a cannula into the recipient's common bile duct. Bile can then be collected in increments. Bile pigments can be analyzed by high pressure liquid chromatography looking for underivatized tetrapyrroles or by thin layer chromatography after being converted to azodipyrroles by reaction with diazotized azodipyrroles ethylanthranilate either with or without treatment with P-glucuronidase.
  • Diconjugated and monoconjugated bilirubin can also be determined by thin layer chromatography after alkalinemethanolysis of conjugated bile pigments. In general, as the number of functioning transplanted hepatocytes increases, the levels of conjugated bilirubin will increase. Simple liver function tests can also be done on blood samples, such as albumin production. Analogous organ function studies can be conducted using techniques known to those skilled in the art, as required to determine the extent of cell function after implantation. For example, islet cells of the pancreas may be delivered in a similar fashion to that specifically used to implant hepatocytes, to achieve glucose regulation by appropriate secretion of insulin to cure diabetes. Other endocrine tissues can also be implanted.
  • chondrocytes function is defined as synthesis of cartilage matrix components (e.g., proteoglycans, collagens) which can provide appropriate structural support for the surrounding attached tissues.
  • Precursor cells of chondrocytes can also be used in place of the chondrocytes.
  • chondrocytes include such chondrocyte precursor cells.
  • Polymer The polymeric material which is mixed with cells for implantation into the body should form a hydrogel.
  • a hydrogel is defined as a substance formed when an organic polymer (natural or synthetic) with charged side groups is cross-linked via ionic bonds to create a three-dimensional open- lattice structure which entraps water molecules to form a gel.
  • materials which can be used to form a hydrogel include polysaccharides such as carrageenan or alginate, the latter being the preferred biopolymer for formation of cell-containing hydrogels of this invention.
  • Alginate is a copolymer of stretches of polymannuronate and polyguluronate obtained from seaweed. Suitable alginate is available from commercial sources. Individual alginate preparations will have a determinable capacity for binding calcium which is a function of the mannuronate:guluronate (M/G) ratio. Effects resulting form varying the ratio between mannuronate and guluronate residues are described in U.S. Patent No. 4,950,600, incorporated herein by reference.
  • the method of this invention for controlling gelation characteristics and final gel properties using multiple cation sources to supply cross-linking cations, where the cation courses are chosen for disparate solubilities, could also apply to other polymers, such as the biopolymer K-carrageenan.
  • Cross-linking Agent for controlling gelation characteristics and final gel properties using multiple cation sources to supply cross-linking cations, where the cation courses are chosen for disparate solubilities, could also apply to other polymers, such as the biopolymer K-carrageenan.
  • the water soluble polymer with charged side groups is cross-linked by reacting the polymer with an aqueous solution/suspension containing multivalent ions of the opposite charge, either multivalent cations if the polymer has acidic side groups or multivalent anions if the polymer has basic side groups.
  • the preferred cations for cross-linking of the polymers with acidic side groups to form a hydrogel are divalent and trivalent cations such as copper, calcium, aluminum, magnesium, strontium, barium, and tin, although di-, tri- or tetra-functional organic cations such as alkylammonium salts, e.g., R 3 N+ -VW- + NR 3 can also be used.
  • Aqueous solutions of the salts of these cations are added to the polymers to form soft, highly swollen hydrogels and membranes.
  • concentration of cation or the higher the valence, the greater the degree of cross-linking of the polymer. Concentrations from as low as 0.005 M have been demonstrated to cross-link these polymers. Higher concentrations are limited by the solubility of the salt.
  • the preferred multivalent ion for use with the preferred biopolymer (alginate) is calcium.
  • the method of this invention is described herein using calcium and calcium salts as representative cross-linkers.
  • Other multi-valent ions which perform similarly are, of course, within the contemplation of this invention.
  • this invention provides a hydrogel forming composition containing two sources of cross-linking polyvalent cation: a minor amount of one cation source which provides fully available cation and a major amount of another cation source which provides slow release of the cross-linking ion as the gel is formed.
  • the first cation source is a highly water soluble salt - i.e., solubility of at least lg per 100 ml, preferably at least 3 Og/ 100ml, more preferably at least 60g/ 100ml.
  • Particularly preferred is a calcium salt such as calcium chloride.
  • the second cation source is a sparingly soluble salt - i.e., less than lg/lOOml, preferably less than 0.5g/100ml, more preferably less than 0.3g/100ml.
  • a calcium salt such as calcium sulfate. Any two salts of multivalent cations having similar water solubilities to calcium chloride and calcium sulfate, respectively, are also suitable for use in this invention. While either salt alone may provide sufficient cross-linking cation to produce a fully hardened alginate hydrogel, use of two salts spreads the cross-linking action over time, thereby expanding the time window during which a partially hardened hydrogel is of suitable consistency for injection through a cannula.
  • Calcium chloride is one preferred "fast" Ca +2 source to speed up the alginate gelling because of its high solubility.
  • Other calcium salts of equivalent solubility work in a similar fashion.
  • Experiments were run to determine if CaCl 2 alone could work. A concentration range of 1.0 to 6.6 mg/ml was tried, and it was found that CaCl 2 alone was not suitable to crosslink the cell-containing hydrogel, since the alginate gelled up immediately at the interface of alginate and CaCl 2 solutions when the concentration of CaCl 2 -2H 2 0 is equal to or higher than 3.3 mg/ml, but would not gel up at all when equal to or lower than 2.7 mg/ml when alginate was used at a final concentration of 1.5% (w/v).
  • CaCl 2 may range from 0.5mg/ml to 3.0 mg/ml.
  • the amount of CaSCy2H 2 0 required to react stoichiometrically with sodium alginate is 0.309 mg per mg of alginate.
  • a typical known calcium sulfate cross-linked formulation uses 20 mg CaS0 4 -2H 2 0 per mg of sodium alginate, or approximately 65 times the amount required.
  • the solubilities of calcium sulfate dihydrate (CaS0 4 -2H 2 0) in cold and hot (100°C) water are 2.41 and 2.22 mg/ml, respectively.
  • the ratio of CaCl 2 .2H 2 0/CaS0 4 .2H 2 0/alginate can be 1 : 10: 10 when certain amount of phosphate presents in the formulation.
  • suitable compositions may have proportions which produce a gel of acceptable consistency in approximately 14 minutes at room temperature.
  • the ratio of CaS0 4 /CaCl 2 which provides about 3 mg calcium chloride and 30 mg of calcium sulfate per 30 mg of alginate is suitable for use with alginate which binds 0.309 g of CaS0 4 *2H 2 0 per g of sodium alginate.
  • the stoichiometry for alginate with a different calcium binding capacity can be readily determined by one skilled in the art, and similar mole ratios of sparingly soluble and freely soluble salts can be calculated to determine suitable amounts of these alternative salts.
  • M/G ratio of the alginate determines the Ca-binding capacity.
  • the relative amounts of alginate, fully soluble salt and sparingly soluble salt, and optionally soluble sequestrant will be such that the mixture forms a gel within 20 minutes that will hold its shape against the force of gravity, but may be injected without clogging the injection canula for at least 4 hours.
  • formulations using only slowly soluble cation sources produce a partitioning of components into alginate-encapsulated clods of undissolved salt and a more liquid portion that could be collected and injected.
  • Such a product is poorly reproducible in consistency, cannot be well-characterized, well-controlled, or altered to give the final formulation a range of properties.
  • Use of only rapidly soluble cation sources can produce partitioning as well, if the cation cannot be distributed throughout the mixture, as portions of fully cross-linked gel will be produced and are not easily injected. Addition of both a sparingly soluble and a freely available multivalent cation source may circumvent these problems.
  • the hydrogel forming composition according to this invention optionally contains a component which will buffer the amount of cross-linking cation available to the biopolymer.
  • the buffering component is usually an anion sequestrant such as phosphate anion. Rapid and complete cross-linking of the alginate is prevented by the P0 4 anions in the formula competing with alginate in binding of Ca +2 , which greatly reduces the gelation speed. Phosphate anion is typically present at about 0. IM, but the phosphate concentration may be varied from 0.03M to 0.3M to control gelling time.
  • Other biologically compatible sequestrants which will complete with alginate for calcium, and thus prevent formation of alginate encapsulated salt particles in a similar manner, are also contemplated by this invention.
  • Substantial variation is permitted in the order of addition for the components which react to form the ceU-containing hydrogel.
  • the cells are added before the hydrogel reaches its partially hardened consistency.
  • this means that the cells are introduced into a premixture containing either the alginate or one or more of the cation sources before the cation sources and the alginate are combined.
  • cells are first mixed with alginate, optionally including the sequestering anion, and then the cation sources are added in one or more steps.
  • chondrocytes are harvested, grown to confluence, passaged as needed, then mixed with a biodegradable liquid polymer such as alginate which is designed to solidify at a controlled rate upon subsequent mixing with multivalent ionic salts.
  • a biodegradable liquid polymer such as alginate which is designed to solidify at a controlled rate upon subsequent mixing with multivalent ionic salts.
  • Variations in the levels of individual components, or conditions of mixing and incubation can be modified to a) control the consistency of the material as injected; b) control the time required for attaining an injectable consistency; c) and controlling the properties of the final gel in vivo to accommodate particular requirements of the transplanted cells for engaftment and function or of the receiving tissue site for appropriate texture and dimensional retention.
  • Levels of individual components can be modified to alter different properties of the formulation both before and after application so as to accommodate particular requirements a) for injection and application, b) for individual cell viability or successful engraftment and creation of required properties and function of the final gel and replacement tissue, or c) for the manufacture, distribution, and application to patients of the formulation.
  • injection of a cell/gel formulation into compact tissues e.g. muscle, submucosa
  • compact tissues e.g. muscle, submucosa
  • preformed structural elements vascular grafts, stents, polymer scaffolds, etc.
  • Alterations of viscosity can be achieved by a number of mechanism either singly or in concert, such as (1) selection of the viscosity of the raw material (e.g. low, medium, or high viscosity alginate); (2) concentration of gel (e.g. a range of 0.3% to 3.0% alginate can be used to achieve a broad range of gel viscosities); (3) amount of highly soluble multivalent cation source to control degree of partial cross- linking; or (4) level of anions provided to compete with alginate for cation binding.
  • Successful engraftment of different cell types, or the nature of the desired replacement tissue to be created may require alterations of the formulations components. For example, creation of a firm, compact tissue (e.g.
  • liver, cartilage requires a gel of higher polymer chain length or concentration (e.g. >1.5% alginate, high guluronic content alginate), while simply coating a surface such as bone surface augmentation using a gel/osteoclast formulation is more successful with a lower concentration of polymer.
  • Controlling the duration of injectability and properties of a formulation prior to injection would aid the manufacture, distribution, and apphcation of gel/cell therapies.
  • the working time of the final formulation, or the time required for materials to setup to an injectable consistency can be controlled through multiple methods, such as by the amount of highly soluble cation provided and/or the level of anion provided to compete for cation binding, in addition to temperature regulation.
  • duration of an injectable consistency can be controlled through temperature so as to allow a working time of minutes (34-38 °C) or beyond one month (4-8 °C). Temperature plays a very important role in the gel formation speed.
  • the material Upon increasing the temperature, as will occur, for instance, when the material is injected into tissue having a temperature of 37° C, the material will proceed to harden into a fully cross-linked hydrogel.
  • the partially hardened gel could set up in 32 minute at body temperature and remain injectable for at least 90 minutes.
  • This best case scenario of CaS0 4 at 37 °C still requires too much time to reach injectable consistency, and is both more complicated and more inconsistent than desired.
  • Cation sources can be chosen to control gelling and gel properties, and also combined to control degradation.
  • the composition according to this invention having both a fast and slow source of cross-linking agent, can provide both more rapid viscosity increase, and a longer time before becoming fully cross-linked compared to the formulation with calcium sulfate alone.
  • the mixture which forms partially hardened hydrogel will also include a significant amount of phosphate anion. Typically, the phosphate concentration will be approximately 0.1 molar. Increasing the concentration of alginate is also helpful for the gel to hold more water, resulting in a thicker paste and a stronger gel, and also in accelerating the gel formation.
  • Preferred alginate level is at least 0.75%, more preferably between 1.5% and 2.5% and up to 3.0%, in the final cell- containing suspension which forms the hydrogel.
  • the acidic biopolymer is sodium alginate
  • the fully soluble salt is calcium chloride
  • the sparingly soluble salt is calcium sulfate.
  • the alginate is present in the amount of at least 0.75% by weight of solution, preferably about 1.5%.
  • the freely soluble salt provides 10 to 15 mM calcium ion, while the sparingly soluble salt is provided as a powder dispersed in the alginate solution in an amount which would supply approximately 8 times more calcium ion if it were completely dissolved.
  • calcium is supplied as the divalent metal cross-linking agent by two salts, calcium chloride and calcium sulfate, in a ratio of 1 part to 10 parts by weight.
  • a suitable cell suspension medium such as M- 199
  • 1 part of the same cell suspension medium containing 1.8% anhydrous calcium chloride with 18% calcium sulfate suspended therein is added to the alginate-cell mixture.
  • the final mixture is mixed thoroughly, taking adequate precautions to avoid damaging the cells, and used for injection into tissue. A rapid increase in viscosity may be expected within 15 minutes of mixing all of the components together.
  • the consistency of this mixture should be sufficient for the mixture to hold its shape against gravity, but will remain injectable at room temperature for at least 24 hours.
  • a preferred viscosity for injectable apphcation varies by intended use, but is a balance of parameters that include (a) the formulation reservoir (e.g., syringe size, piston diameter, resistance required for proper "touch” or apphcation rate), (b) apphcation device (catheter or needle length, diameter, composition), (c) receiving tissue resistance to division, and (d) type of distribution required (e.g., topical application to bone or organ surface, injection within tissues without extravasation, etc.).
  • the formulation reservoir e.g., syringe size, piston diameter, resistance required for proper "touch” or apphcation rate
  • apphcation device catheter or needle length, diameter, composition
  • receiving tissue resistance to division e.g., topical application to bone or organ surface, injection within tissues without extravasation, etc.
  • the composition is formulated to form a partially hardened hydrogel within thirty minutes, preferably within 10-15 minutes at room temperature, the consistency of the partially hardened hydrogel having a viscosity of at least 15,000 centipoise at 25 °C, or alternatively being fluid enough for injection through a 23-gauge cannula at a rate of about 10 ml/minute without substantially damaging the cells (i.e., viability of the cells reduced by no more than 25%).
  • cells are suspended in a hydrogel solution and injected directly into a site in a patient, where the hydrogel hardens into a matrix having cells dispersed therein.
  • cells may be suspended in a hydrogel solution which is poured or injected into a rigid or inflatable mold having a desired anatomical shape, then hardened to form a matrix having cells dispersed therein which can be implanted into a patient. After implantation, the hydrogel ultimately degrades, leaving only the resulting tissue.
  • a hydrogel solution which is poured or injected into a rigid or inflatable mold having a desired anatomical shape, then hardened to form a matrix having cells dispersed therein which can be implanted into a patient. After implantation, the hydrogel ultimately degrades, leaving only the resulting tissue.
  • Such hydrogel-cell mixtures can be used for a variety of reconstructive procedures, including custom molding of cell implants to reconstruct three dimensional tissue defects, filling pre-inserted inflatable molds or scaffolds, as well as implantation of tissues generally.
  • chondrocytes preferably autologous chondrocytes
  • a liquid biodegradable biocompatible polymeric material such as alginate which can be solidified in vivo, or other carrier to form a cell suspension.
  • the cell suspension is injected into the area where reflux is occurring, or where a bulking agent is required, in an amount effective to yield cartilage that provides the required control over the passage of urine.
  • the cell suspension contains chondrocytes, harvested, grown to confluence, passaged as needed, and then mixed with a biodegradable liquid polymer such as alginate, a copolymer of guluronic and mannuronic acid, which is designed to sohdify at a controlled rate when contacted with calcium salts.
  • a biodegradable liquid polymer such as alginate, a copolymer of guluronic and mannuronic acid, which is designed to sohdify at a controlled rate when contacted with calcium salts.
  • the cells are mixed with hydrogel forming compositions and then injected at the desired site where they proliferate and correct the defect.
  • chondrocyte cells are mixed with a liquid polymeric material, such as alginate which can be solidified in vivo, to form a cell suspension, and injected into the area of the defect, in an amount effective to yield an elevation of the bladder mucosa that corrects the defect, for example, which provides the required control over the passage of urine.
  • a biodegradable polymer, embedded with cells serves as a synthetic substrate for the injectable delivery and maintenance of tissue architecture in humans that satisfies all the requirements for an ideal injectable substance.
  • a biopsy of auricular cartilage can be easily and quickly performed followed by chondrocyte cell processing and endoscopic injection of the autologous cell/polymer suspension for the treatment of reflux incontinence and other defects. Studies show that chondrocyte cells can be easily harvested and combined with alginate in vitro, the suspension can be easily injected cystoscopically, and the tissue formed is able to correct vesicoureteral reflux without any evidence of obstruction.
  • the ideal injectable substance for the endoscopic treatment of reflux should be a natural bulking agent which is non-antigenic, non-migratory, and volume stable.
  • Autologous chondrocyte cells seem to fulfill all of these requirements. Since the cells are autologous, this method of treatment does not require FDA approval. The procedure can be performed under 15 minutes, with a short period of a mask anesthetic, in the outpatient unit, without any need for a hospital stay. Neither vesical nor perivesical drainage is required. Since the whole procedure is done endoscopically and the bladder is not entered surgically, there is no postoperative discomfort whatsoever. The patient can return to a normal level of activity almost immediately.
  • CaS04 particles and resulting gel formed around these particles Each sample contains 1.5 ml 2% Sodium Alginate in pH 7.4 buffer, 1.5 ml M199 Medium (no cells), and indicated amounts of CaS0 4 powder. Materials are stored on ice prior to mixing. After mixing, samples are stored at the indicated temperature, and injected at the indicated time onto mylar sheet.
  • Example 2 Mixtures of Calcium Chloride and Calcium Sulfate.
  • sodium alginate is dissolved in pH 7.4
  • K 2 HP0 4 buffer Cells are supplied in Ml 99 medium and then suspended in the alginate solution. Calcium sulfate powder is stored in a 1.5 ml vial. Calcium chloride is dissolved in M199 medium with concentration of 18 mg/ml and stored in another vial. In the application, the CaCl 2 solution will be mixed with CaS04 powder, and then mixed with alginate, taken into syringe and wait for about 15 minutes, the formula is ready to be injected.
  • alginate used is UP MVG grade, dissolved in K 2 HP0 4 buffer 2 CaCl 2 is dissolved in M199 medium.
  • the time for gel formation and the period during which the consistency remained suitable for injection are shown in Table 3.
  • the amount of each component is indicated in the table as M for the base value, H for 16% above the base value, and L for 16% below the base value.
  • the gel time was 18 minutes plus or minus about 15%, indicating little effect of variation in any particular component within this range. Although all of these solutions gelled within about 20 minutes, the consistency of the alginate gel was still acceptable for injection beyond 24 hours later. Samples of this formulation prepared in a similar manner demonstrated suitable injectabihty properties over one month after formulation.
  • Alginate concentration is 2% by weight/volume
  • CaCl 2 concentration is 18mg/ml in M199 medium
  • Alginate gelled by CaS0 4 powder was prepared as described for Example 1, and alginate gelled by a mixture of CaCl 2 /CaS0 4 was prepared as described for Example 2.
  • the CaCl 2 /CaS0 4 samples were prepared with syringe mixing, and CaS0 4 powder samples were prepared with vortex mixing method. Syringe mixing allows a very simple procedure that is easy to perform and yields a very consistent product. It also permits the use of approved biomedical devices, and creates a closed system to segregate product from environmental exposure. Viscosity changes along the gelling course of the two systems at temperatures of 5 °C, 25 °C and 37 °C were measured and the result are plotted in Figure 1 through Figure 3.
  • the CaC CaS0 4 sample gelled to an injectable set point 1 at 13 minutes, while the CaS0 4 -only sample did not set the gel until minute 56.
  • the viscosity of the CaCl 2 /CaS0 4 sample before gelling is
  • Injectable set point consistency of gel that can be stacked without collapsing. about 665 cP, and CaS0 4 -only sample is 202 cP; the former is approximately 3.3 times as thick as that of the latter.
  • Fig. 1 When the alginate starts gelling, its viscosity increases abruptly and reaches very high values (Fig. 1), going from 1100 cP to 30,000 cP in 2 minutes.
  • the viscosity of the CaCl 2 /CaS0 4 sample is about 2 times that of CaSO -pnly sample (about 40,000 cP vs. 20,000 CP).

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Abstract

L'invention porte sur des compositions destinées à implanter des cellules chez un animal (ces cellules pouvant être des cellules dissociées et/ou des agrégats cellulaires) et comprenant: un polymère biodégradable, biocompatible, qui forme un hydrogel lors de sa réticulation par des ions multivalents; un sel soluble d'un ion multivalent; et un sel modérément soluble d'un ion multivalent, ces composants étant combinés pour obtenir un mélange formant un hydrogel injectable, partiellement durci dans lequel les cellules sont uniformément en suspension, la consistance du mélange étant appropriée pour implanter le mélange d'hydrogel partiellement durci chez un animal. Cet hydrogel implanté, partiellement durci, forme in situ un hydrogel totalement durci renfermant les cellules. De préférence, le mélange contient également un agent séquestrant biocompatible qui rivalise avec le polymère biocompatible dans la liaison de l'ion de réticulation multivalent. L'invention porte également sur des procédés d'implantation des cellules chez un animal à l'aide de cette composition.
PCT/US1997/022860 1996-12-10 1997-12-10 Composition d'hydrogel amelioree WO1998025575A2 (fr)

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Cited By (2)

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Publication number Priority date Publication date Assignee Title
DE102005035986A1 (de) * 2005-07-28 2007-02-08 Dr. Gerhard Mann Chem.-Pharm. Fabrik Gmbh Steriles tropfbares mehrphasiges emulgatorfreies ophthalmisches Präparat
US7678146B2 (en) 2000-08-25 2010-03-16 Contura A/S Polyacrylamide hydrogel and its use as an endoprosthesis

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US4673566A (en) * 1983-06-01 1987-06-16 Connaught Laboratories Limited Microencapsulation of living tissue and cells
US5336263A (en) * 1992-04-06 1994-08-09 Robert A. Ersek Treatment of urological and gastric fluid reflux disorders by injection of mmicro particles
US5516532A (en) * 1994-08-05 1996-05-14 Children's Medical Center Corporation Injectable non-immunogenic cartilage and bone preparation
US5575815A (en) * 1988-08-24 1996-11-19 Endoluminal Therapeutics, Inc. Local polymeric gel therapy
US5578086A (en) * 1989-02-15 1996-11-26 Xomed, Inc. Prosthesis using biocompatible composite material

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4673566A (en) * 1983-06-01 1987-06-16 Connaught Laboratories Limited Microencapsulation of living tissue and cells
US5575815A (en) * 1988-08-24 1996-11-19 Endoluminal Therapeutics, Inc. Local polymeric gel therapy
US5578086A (en) * 1989-02-15 1996-11-26 Xomed, Inc. Prosthesis using biocompatible composite material
US5336263A (en) * 1992-04-06 1994-08-09 Robert A. Ersek Treatment of urological and gastric fluid reflux disorders by injection of mmicro particles
US5516532A (en) * 1994-08-05 1996-05-14 Children's Medical Center Corporation Injectable non-immunogenic cartilage and bone preparation

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7678146B2 (en) 2000-08-25 2010-03-16 Contura A/S Polyacrylamide hydrogel and its use as an endoprosthesis
US7780958B2 (en) 2000-08-25 2010-08-24 Contura Sa Polyacrylamide hydrogel for the treatment of incontinence and vesicouretal reflux
US7790194B2 (en) 2000-08-25 2010-09-07 Contura A/S Polyacrylamide hydrogel as a soft tissue filler endoprosthesis
US7935361B2 (en) 2000-08-25 2011-05-03 Contura A/S Polyacrylamide hydrogel as a soft tissue filler endoprosthesis
US8216561B2 (en) 2000-08-25 2012-07-10 Contura A/S Polyacrylamide hydrogel for the treatment of incontinence and vesicouretal reflex
DE102005035986A1 (de) * 2005-07-28 2007-02-08 Dr. Gerhard Mann Chem.-Pharm. Fabrik Gmbh Steriles tropfbares mehrphasiges emulgatorfreies ophthalmisches Präparat
DE102005035986B4 (de) * 2005-07-28 2009-10-15 Bausch & Lomb Incorporated Steriles tropfbares mehrphasiges emulgatorfreies ophthalmisches Präparat

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