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WO1995026761A1 - Gels de cellules - Google Patents

Gels de cellules Download PDF

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Publication number
WO1995026761A1
WO1995026761A1 PCT/US1995/003991 US9503991W WO9526761A1 WO 1995026761 A1 WO1995026761 A1 WO 1995026761A1 US 9503991 W US9503991 W US 9503991W WO 9526761 A1 WO9526761 A1 WO 9526761A1
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WO
WIPO (PCT)
Prior art keywords
cross
cell
cells
collagen
linking agent
Prior art date
Application number
PCT/US1995/003991
Other languages
English (en)
Inventor
Trudy D. Estridge
Prema R. Rao
Original Assignee
Collagen Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Collagen Corporation filed Critical Collagen Corporation
Priority to AU22029/95A priority Critical patent/AU682266B2/en
Priority to JP7525855A priority patent/JPH10501706A/ja
Priority to EP95914983A priority patent/EP0754065A1/fr
Publication of WO1995026761A1 publication Critical patent/WO1995026761A1/fr
Priority to MXPA/A/1996/004587A priority patent/MXPA96004587A/xx

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • 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
    • 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
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/24—Collagen
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L71/00—Compositions of polyethers obtained by reactions forming an ether link in the main chain; Compositions of derivatives of such polymers
    • C08L71/02—Polyalkylene oxides
    • 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
    • A61F2310/00—Prostheses classified in A61F2/28 or A61F2/30 - A61F2/44 being constructed from or coated with a particular material
    • A61F2310/00005—The prosthesis being constructed from a particular material
    • A61F2310/00365—Proteins; Polypeptides; Degradation products thereof

Definitions

  • This invention relates to novel "cell-gel” formulations and compositions suitable for augmenting living tissue, comprising a plurality of living cells contained within a cross-linked matrix material, such as cross-linked collagen.
  • Daniels et al . (U.S. Pat. No. 3,949,073) disclose, inter alia , the preparation and injection of soluble collagen into suitable locations of a subject with a fibril-formation promoter (described as a polymerization promoter in the patent) to form fibrous collagen implants in situ , for augmenting hard or soft tissue. Such implants are rapidly colonized by host cells and vascularized. This material is now commercially available from Collagen
  • microcarriers based on gelatin, dextran, cellulose, acrylamide, fluorocarbon-polylysine, and polystyrene have been developed (see, for example, Reuveny, Advances in Cell Culture, 1985, Vol. 4, pp.213-247, issemann et al. , In Vitro Cellular and Developmental Biology. 1985, Vol. 21, No. 7, pp.391-401 and references therein).
  • An important application of such microcarriers is in the culturing of anchorage dependent cells, wherein such cells attach to and proliferate on the surfaces of pre-formed microcarriers.
  • Cell attachment and proliferation may be limited to the outer surface of the microcarrier, or for porous microcarriers, ingrowth and proliferation within interior pore spaces may occur.
  • Nilsson et al . Biotechnology. 1986, Vol. 4, pp. 989-990
  • porous microcarriers from gelatin (denatured collagen) which permit cells to grow in the interior of the porous gelatin bead.
  • Ade a et al . (BioPharm. 1990, Vol. 3, No. 7, pp.20-23) disclose the preparation of strengthened porous microcarriers from a glutaraldehyde cross-linked collagen-glycosaminoglycan (GAG) copolymer.
  • GAG glutaraldehyde cross-linked collagen-glycosaminoglycan
  • Eisenberg U.S. Patent 5,282,859 discloses a living skin equivalent comprising, inter alia , a dermal layer of cultured fibroblast cells in a porous, cross-linked collagen sponge prepared by inoculating commercially available cross-linked, bovine collagen sponge membranes.
  • Saintigny et al . Acta. Derm. Venereol. (Stockholm). 1993, Vol. 73, pp.175-180
  • Weinberg et al . disclose fibrin-containing tissue equivalents comprising collagen, fibrin, and embedded cells (as "contractile agents") .
  • the tissue equivalents may further include an agent which can cross-link fibrin and collagen, for example, Factor XIII (a naturally occurring blood coagulation factor) , to enhance strength and stability.
  • Weinberg also discloses a method for preparing tissue equivalents which includes, inter alia , the step of contemporaneously mixing collagen, fibrin (obtained in situ from reaction of fibrinogen with thrombin) , and cells to form a gel.
  • Freeman (Methods in Enzymology, 1987, Volume 135, pp. 216-222) discloses, inter alia , cell immobilization by gel entrapment of whole cells in cross-linked prepolymerized polyacrylamide-hydrazide gels. This entrapment procedure is based on suspending the cells in an aqueous solution of a linear, water-soluble synthetic polymer, which is then cross-linked, in the presence of cells and under mild physiological conditions, by the addition of a dialdehyde such as glyoxal.
  • a dialdehyde such as glyoxal
  • Biomedical Applications ed. J. Milton Harris, Plenum Press, New York, 1992, pp. 183-1978 disclose collagen- polymer conjugates in which collagen, preferably reconstituted atelopeptide collagen, is chemically bonded to a synthetic hydrophilic polymer, preferably polyethylene glycol. By employing polyfunctional polymers, cross-linked collagen is obtained.
  • One aspect of the present invention relates to a cell- gel composition
  • a cell- gel composition comprising a plurality of cells contained within a matrix material cross-linked with a synthetic polymeric cross-linking agent.
  • Another aspect of the invention relates to a method of making a cell-gel composition
  • a method of making a cell-gel composition comprising:
  • Still another aspect of the invention relates to a method for augmenting tissue at a site within a living mammal comprising placing the above-described cell-gel composition at said site.
  • Figure 1(a) is a graph of relative absorbance data recorded for the collagen-SPEG cell-gels of Example 1.
  • Figure 1(b) is a graph of normalized relative absorbance data recorded for the collagen-SPEG cell-gels of Example 1.
  • cell-gel as used herein relates cell-gel compositions and formulations comprising a plurality of living cells contained within a matrix material which has been cross-linked with a cross-linking agent which are useful for augmenting living tissue.
  • tissueing relates to repairing, preventing, or alleviating defects, particularly defects due to loss or absence of hard or soft tissue, by providing, augmenting, or replacing such tissue.
  • matrix forming material as used herein relates to cross-linkable polymers. That is, polymers which have functional groups which permit the polymers to be cross-linked.
  • matrix forming materials include collagen, fibrin, fibrinogen, chitin, chitosan, their derivatives and analogs, and mixtures thereof, whether obtained from natural sources or synthetically.
  • the matrix forming material comprises collagen or collagen derivatives.
  • Collagen is the major protein component of bone, cartilage, skin, and connective tissue in animals. Collagen is typically isolated from natural sources, such as human placentas, bovine hide, cartilage, or bones.
  • Bones are usually dried, defatted, crushed, and demineralized to extract collagen, while hide and cartilage are usually minced and digested with proteolytic enzymes (other than collagenase) .
  • proteolytic enzymes other than collagenase
  • this procedure conveniently serves to remove most of the contaminating protein found with collagen.
  • Collagen may be denatured by boiling, which produces the familiar product gelatin.
  • collagen refers to all forms of collagen, including native collagens which have been processed or otherwise modified and collagens that have been produced by genetic engineering (ie. recombinant collagen) .
  • Suitable collagens include all types, preferably types I, II and III.
  • Collagens may be soluble (for example, commercially available Vitrogen® 100 collagen-in-solution) , and may have or omit the telopeptide regions.
  • the collagen will ⁇ be reconstituted fibrillar atelopeptide collagen, for example Zyderm® I Collagen Implant (ZCI) or atelopeptide collagen in solution (CIS) .
  • ZCI Zyderm® I Collagen Implant
  • CIS atelopeptide collagen in solution
  • Various forms of collagen are available commercially, or may be prepared by the processes described in, for example, U.S. Patents 3,949,073; 4,488,911; 4,424,208; 4,582,640; 4,642,117; 4,557,764; and 4,689,399, all incorporated herein by reference.
  • cross-linked matrix material as used herein relates to a matrix material in which one or more cross-linkable polymers have been cross-linked by chemical reaction with one or more cross-linking agents to form covalent bonds therewith.
  • cross-linking agent as used herein relates to compounds which (i) have functional groups which are able to react chemically with functional groups of the matrix forming material to form covalent bonds, and (ii) are nominally non-cytotoxic.
  • functional groups which are able to react chemically includes functional groups which can be activated or derivatized so as to be able to react chemically with functional groups of the matrix forming material to form covalent bonds.
  • synthetic cross-linking agent as used herein relates to cross-linking agents which are not naturally occurring. In one embodiment, the synthetic cross-linking agent is derived from a polymeric compound. Examples of synthetic polymeric cross-linking agents include activated polyfunctional polyethylene glycols.
  • difunctional polyethylene glycol is first reacted with a dicarboxylic acid anhydride, such as glutaric anhydride, and subsequently reacted with an activating agent, such as N-hydroxysuccinimide, to form succinimidylpoly(ethylene glycol) glutarate (herein denoted SPEG) , a synthetic polymeric cross-linking agent.
  • a dicarboxylic acid anhydride such as glutaric anhydride
  • an activating agent such as N-hydroxysuccinimide
  • SPEG succinimidylpoly(ethylene glycol) glutarate
  • dicarboxylic acid anhydrides include oxalic anhydride, malonic anhydride, succinic anhydride, glutaric anhydride, adipic anhydride, 1,8-naphthalene dicarboxylic anhydride, 1,4,5,8-naphthalenetetracarboxylic dianhydride and the like.
  • Activating agents are those compounds which react with carboxylic acids to yield activated esters, -C00R, wherein R is a good leaving group.
  • activating groups include N-hydroxysuccinimide, N,N , -disuccinimidyl oxalate, N,N , -disuccinimidyl carbonate, and the like. See also U.S. Patent No. 4,179,337 issued to Davis for additional linking groups.
  • Cross-linking agents react chemically with the matrix forming material to form cross-links. Any known method for derivatizing and subsequently reacting the cross-linking agent and matrix forming material to form cross-links may be used.
  • collagen molecules which possess a number of available lysine groups with free amino (-NH 2 ) groups, may be cross-linked with active ester moieties, such as those of succinimidylpoly(ethylene glycol) glutarate, to form amide (-CONH-) linkages.
  • active ester moieties such as those of succinimidylpoly(ethylene glycol) glutarate
  • the degree of cross-linking may be expressed as the number of functional groups per (initial) molecule of matrix forming material which are involved in cross-linking.
  • the number of available lysines involved in cross-linking may vary from a single residue to 100% of the lysines, preferably 10%-50%, and more preferably 20%-30%.
  • the number of reactive lysine residues may be determined by standard methods, for example by reaction with TNBS (2,3,4-trinitrobenzensulfonic acid).
  • TNBS 2,3,4-trinitrobenzensulfonic acid.
  • the term "nominally non-cytotoxic" as used herein relates to cross-linking agents which, when added to a cell's environment (i.e. added to a cell suspension) at concentrations useful for effecting cross-linking, do not substantially reduce cell viability, for example, does not reduce cell viability by more than 50% over 7 days, and are not physiologically detrimental.
  • cells as used herein relates to living cells, preferably mammalian cells, including, for example, human cells.
  • Cells may be autogeneic, isogeneic, allogeneic, or xenogeneic, more preferably autogeneic or allogeneic. Included are cells which have been genetically engineered.
  • Cell-gels may contain different cell types, which may be chosen to act synergistically, for example, in the formation of tissue. Examples of types of cells include muscle cells, nerve cells, epithelial cells, connective tissue cells, and organ cells. Examples of cells include fibroblast cells, smooth muscle cells.
  • striated muscle cells heart muscle cells, nerve cells, epithelial cells, endothelial cells, bone cells, bone progenitor cells, bone marrow cells, blood cells, brain cells, kidney cells, liver cells, lung cells, pancreatic cells, spleen cells, breast cells, foreskin cells, ovary cells, testis cells, and prostate cells.
  • Other mammalian cells are useful in the practice of the invention and are not excluded from consideration here.
  • cell- gels may be prepared using non-mammalian eucaryotic cells, procaryotic cells, or viruses.
  • the cell-gel compositions and formulations of the present invention further have the properties that they are (i) nominally non-immunogenic and (ii) bioerodable.
  • nominally non-immunogenic as used herein relates to materials which provoke no substantial immune response, inflammation, or foreign body reaction when administered.
  • bioerodable as used herein relates to the potential for a material to be eroded or degraded by the action of enzymes (including, for example, proteinases such as collagenase) , or other biological processes, to yield non-toxic substances or byproducts which are compatible with normal bodily processes.
  • the degree to which a material is bioerodable may be indicated by its bioerosion period.
  • bioerosion period relates to the period of time after which substantial bioerosion of the cross-linked matrix material has occurred.
  • the bioerosion period may vary for the particular indication, and will reflect needs to initially locate, support, and house the entrapped cells, to permit the growth and proliferation of those cells, and to ultimately permit the complete or nearly complete erosion of the original cross-linked matrix material.
  • Examples of bioerosion periods for cell-gels include 20-45 days, for dermal- associated cell-gels, 30-90 days for bone-associated cell- gels, 10-30 days for nerve-associated cell gels.
  • administer as used herein is generic to methods of applying, attaching, implanting, injecting, and the like. If the cell-gel material is a suspension, injection is the preferred method for administration.
  • the cell-gel compositions and formulations of the invention may be prepared by a one-step method comprising contemporaneous mixing of matrix forming material, cross- linking agent, and cell suspension.
  • cell-gel compositions and formulations may be prepared by a number of two-step methods, such as premixing of cell suspension and matrix forming material, followed addition of cross-linking agent; premixing of cell suspension and cross-linking agent, followed by addition of matrix forming material; and premixing of matrix forming material and cross-linking agent, followed by addition of a cell suspension.
  • the physical properties of the resulting cell-gel compositions or formulations may be adjusted by varying reactant concentrations, reaction conditions, reaction time, or other factors.
  • the term "physical properties" as used herein includes, for example, viscosity, consistency, texture, modulus of elasticity, surface properties, surface roughness, pore size, pore shape, pore interconnection, and the like.
  • the viscosity of cell-gels may be increased by increasing the concentration of matrix forming material in the reaction mixture.
  • preferred collagen concentrations are 5-100 mg/mL, more preferably about 10-75 mg/mL, most preferably 30-60 mg/mL.
  • the degree of cross-linking present in cell-gels may be varied according to the molar ratio of matrix forming material to cross-linking agent. Increasing concentrations of cross-linking agent relative to matrix forming material concentrations yields cell-gels with higher viscosities which may be characterized as gel-like, plastic, semi- solid, or solid. Conversely, decreasing the relative concentration of cross-linking agent yields cell-gels with lower viscosities which may be characterized as fluid or liquid. A continuum of viscosities, textures, and consistencies may be obtained.
  • collagen:SPEG molar ratios of about 200:1 to about 5:1 yield cell-gels which are fluid and injectable, whereas ratios of about 5:1 to about 1:10 yield semi-solid cell-gels, and larger ratios of about 1:10 to about 1:75 yield more rigid, heavily cross-linked cell- gels.
  • Ratios of about 1:50 typically lead to cell-gels wherein all available lysines of collagen are involved in cross-linking.
  • the degree of cross-linking present in cell-gels may further be controlled by adjusting reaction temperature and reaction time. Increased reaction temperature (up to, but not greatly exceeding 37°C) will increase the rate of formation of cross-links. Conversely, reducing the reaction temperature will decrease the rate of formation of cross-links. For example, reaction of collagen and SPEG to form cross-links is rapid at room temperature, but substantially slower at 5°C. During reaction, the degree of cross-linking increases with increasing reaction times; by adjusting other conditions, such as concentrations and temperature, suitable reaction rates (and therefore reaction times) may be obtained.
  • cell-gel properties may be adjusted to vary cell-gel properties.
  • a wide range of fibrillar collagen content may be obtained by varying the pH of the reaction mixture.
  • a particulate microgel material may be obtained by agitating a cell-gel reaction mixture comprising, for example, collagen in solution and activated PEG, during cross-linking (e.g., by stirring or passing between syringes) .
  • the salt concentration of the reaction mixture may also be adjusted to control cell-gel properties.
  • Reactants such as the matrix forming material, the cross-linking agent, and the cell suspension
  • a pharmaceutically acceptable carrier for example, since SPEG is subject to hydrolysis, it is typically stored desiccated at -20°C prior to use, and prepared as an aqueous mixture immediately prior to use.
  • SPEG may be prepared as a non-aqueous suspension (using, for example, glycerol, PEG, triglycerides, DMSO, and the like) or as a suspension with reduced water concentrations.
  • non-aqueous or reduced- aqueous suspensions may be used to further control reaction rates and times.
  • non-aqueous or reduced-aqueous cell suspensions may be prepared to control cell-gel properties.
  • cell suspension as used herein relates to living cells suspended in a liquid, preferably aqueous, medium.
  • appropriate liquids include, for example, physiologically buffered salt solutions and cell culture media, and may include such components as glycerol, DMSO, triglycerides, and the like, and may further contain media supplements known in the art, including for example, serum, growth factors, hormones, sugars, amino acids, vitamins, etalloproteins, lipoproteins, and the like.
  • media supplements including for example, serum, growth factors, hormones, sugars, amino acids, vitamins, etalloproteins, lipoproteins, and the like.
  • a common method involves treatment of a cellular aggregate with EDTA, or an enzyme, such as trypsin, collagenase, and the like, which causes cells to become detached from other cells or from solid surfaces.
  • Cell suspension concentrations may be chosen to optimize cell-gel texture and viscosity, the rate of subsequent colonization of the gel, and/or viability of cells within the gel. At present, cell suspension concentrations which result in reaction mixture concentrations of about lxlO 4 to lxlO 6 cells/mL are preferred, and concentrations of about 1x10 s cell/mL are more preferred.
  • Cell-gels of the invention may additionally include biologically active factors to aid in healing or regrowth of normal tissue.
  • biologically active factors such as heparin , epidermal growth factor (EGF) , transforming growth factor (TGF) alpha, TGF-/3 (including any combination of TGF- ⁇ s) , TGF-01, TGF-/32, platelet derived growth factor (PDGF-AA, PDGF-AB, PDGF-BB) , acidic fibroblast growth factor (FGF) , basic FGF, connective tissue activating peptides (CTAP) , ⁇ -thromboglobulin, insulin-like growth factors, tumor necrosis factors (TNF) , interleukins, colony stimulating factors (CSFs) , erythro- poietin (EPO) , nerve growth factor (NGF) , interferons (IFN) , osteogenic factors, and the like. Incorporation of such factors, and appropriate combinations of factors, can facilitate the transformation of the cell-gel
  • Cell-gels of the invention which contain growth fac ⁇ tors are particularly suited for sustained administration of factors, as in the case of wound healing promotion.
  • Osteoinductive factors and cofactors may advantageously be incorporated into compositions destined for bone replacement, augmentation, and/or defect repair.
  • Cell-gels of the invention containing biological growth factors such as EGF and TGF-/3 are prepared by mixing an appropriate amount of the factor into the composition, or by incorporating the factor into the matrix forming material prior to treatment with the cross-linking agent.
  • Cell-gels of the invention containing biological growth factors such as EGF and TGF-/3 are prepared by mixing an appropriate amount of the factor into the composition.
  • One may chemically link the factors to the matrix forming material or to the cross-linked matrix material, for example, by employing a suitable amount of cross-linking agent during preparation of the cell-gel.
  • the factors may be covalently attached to the matrix forming material in the same manner that the matrix forming material is cross-linked.
  • the effective amount of factor is substantially reduced.
  • the term "effective amount” refers to the amount of composition required in order to obtain the effect desired.
  • a "tissue growth promoting amount" of a composition containing a growth factor refers to the amount of factor needed in order to stimulate tissue growth to a detectable degree.
  • Tissue in this context, includes connective tissue, bone, cartilage, epidermis and dermis, blood, and other tissues.
  • factors covalently tethered to the matrix forming material serve as effective controlled-release drug delivery matrices.
  • factors may be chemically linked to collagen using activated PEG: the factor is first reacted with a molar excess of activated PEG in a dilute solution over a period of about 5 min to about 1 hour.
  • the factor is preferably provided at a concentration of about l ⁇ g/mL to about 5 mg/mL, while the activated PEG is preferably added to a final concentration providing a 30 to 80-fold molar excess.
  • the resulting conjugated factor is then added to an aqueous collagen mixture (about 1 to about 60 mg/mL) at pH 7-8 and allowed to react further.
  • the resulting composition is allowed to stand overnight at ambient temperature.
  • the pellet is collected by centrifugation, and is washed with PBS by vigorous vortexing in order to remove non-bound factor.
  • the resulting collagen-factor material is then used at the matrix forming material in the preparation of a cell-gel.
  • particulate materials in the cell-gel for example hydrogel or collagen-dPEG beads, hydroxyapatite/tricalcium phosphate particles, polylactic acid/polyglycolic acid (PLA/PGA) particulates, or teflon beads, to provide a bulkier or more rigid cell-gel after cross-linking.
  • Formulations suitable for repair of bone defects or nonunions may be prepared by providing cell-gels with high concentration of matrix forming material, high concentrations of cross-linking agent, or by admixture with suitable particulate materials.
  • suitable particulate material refers to a particulate material which is substantially insoluble in water, which is biocompatible, and which is immiscible with matrix forming material or cross-linked matrix material.
  • the particles of particulate material may be fibrillar, or may range in size from about 1 to 500 ⁇ m in diameter and be bead-like or irregular in shape. For example, for injectable cell-gels, such as those useful for soft tissue augmentation, preferred particles sizes are less than about 150 ⁇ m.
  • preferred particles sizes are greater than about 100 ⁇ m.
  • Exemplary particulate materials include without limitation fibrillar cross-linked collagen, gelatin beads, cross-linked collagen-PEG particles, poly ⁇ tetrafluoroethylene beads, silicone rubber beads, hydrogel beads, silicon carbide beads, glass beads, carbon fibers, PLA/PGA fibers, and polyethylene terephthalate (PET) fibers.
  • Presently-preferred particulate materials are hydroxyapatite and tricalcium phosphate.
  • Malleable, plastic cell-gel compositions which are injectable may be prepared by adjusting reaction parameters as indicated above, or by the addition of a sufficient amount of a pharmaceutically acceptable carrier, such as water or glycerol.
  • a pharmaceutically acceptable carrier such as water or glycerol.
  • the term "sufficient amount” as used herein is applied to the amount of carrier used in combination with the cell-gels of the invention. A sufficient amount is that amount which when mixed with the cell-gel renders it in the physical form desired, for example, injectable solution, injectable suspension, plastic or malleable implant, rigid implant, and so forth.
  • injectable as used herein relates to materials having a texture and viscosity which permits their flow through a suitable surgical needle by employing typical injection pressures. For example, an injectable material may be forced through a 32-gauge needle under normal pressure. The mixture is injected directly into the site in need of augmentation, such as tendon or cartilage and causes essentially no detectable inflammation or foreign body reaction.
  • Injectable cell-gel formulations are useful for dermal augmentation, for example for filling in dermal creases, and providing support for skin surfaces, sphincter augmentation, (e.g., for restoration of continence), tissue revascularization, depot cell delivery, tumor blood vessel blockage, therapy, and contraception/infertility treatments.
  • an aqueous mixture matrix forming material and cell suspension is combined with a low-concentration solution containing the cross-linking agent, mixed, and the reaction mixture injected or applied before the viscosity increases sufficiently to render injection difficult (usually about 10 minutes) .
  • Mixing may be accomplished by passing the mixture between two syringes equipped with Luer lock hubs, or through a single syringe having dual compartments (e.g., double barrel) .
  • the reaction mixture reacts to form cross- -links in situ (that is, at the site in need of augmentation) , and may additionally cross-link to the endogenous tissue, anchoring the cell-gel in place.
  • denser more viscous formulations may be cast or molded into any desired shape, for example into sheets or membranes, into meshes, into tubes or cylinders, into hooks, cords or ropes, and the like.
  • Flexible sheets or membranous forms of cell-gels may be prepared by methods analogous to those known in the art for the preparation of gel membranes (see, for example, U.S. Patent Nos. 4,600,533; 4,412,947; and 4,242,291).
  • a mixture of a high concentration (10-100 mg/mL) CIS or fibrillar collagen (preferably atelopeptide fibrillar collagen, such as ZCI) , activated PEG (having a molecular weight of approximately 3,400), and cell suspension is cast into a flat sheet container, and allowed to react for 2-3 hours at 37°C.
  • the resulting collagen- cell-gel is removed from the excess reaction solution using a sterile spatula or the like, and may be washed with PBS to remove excess unreacted cross-linking agent. More flexible membranous forms are achieved by using lower collagen concentrations and higher cross-linking agent concentrations as starting materials.
  • sheaths or tubular forms of cell-gel compositions may be prepared which are useful for replacing or augmenting vascular structures, such as blood vessels, or as a nerve tissue sheath.
  • compositions of the invention may be prepared in a form that is dense and rigid enough to substitute for car- tilage or non-weight bearing bones, for example, finger bones. These compositions are useful for repairing and supporting tissue which require some degree of structure, for example in reconstruction of the nose, ear, knee, larynx, tracheal rings, and joint surfaces. One can also replace tendon, ligament and blood vessel tissue using appropriately formed cartilaginoid material.
  • the cell-gel is generally cast or molded into a shape; in the case of tendons and ligaments, it may be preferable to form filaments for weaving into cords or ropes.
  • Human dermal fibroblast cells at the 5th passage were used.
  • the human dermal fibroblast cells were subcultured from those on deposit with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852, USA, under ATCC Accession Number CRL 1885.
  • cells were trypsinized using 25 mg/mL trypsin in EDTA (2 mM) and the cells were pelleted by centrifuging at 150-200 g for 10 min at room temperature. The supernatant was discarded, and the pellet resuspended in 5 mL of DME media (Dulbecco's Modified Eagle's Medium with 4.00 mM L-glutamine, 1000 mg/L glucose, 100 mg/L sodium pyruvate) . The cell concentration was determined using a hemocytometer. The cell suspension was diluted with DME media to a concentration of lxlO 5 cells/mL.
  • Controls were prepared using aliquots of 1 ⁇ L, 10 ⁇ L, and 100 ⁇ L of stock cell suspension solution were used to obtain concentrations of 100 cells/well (1A1, 1A2, 2A1, 2A2), 1,000 cells/well (1A3, 1A4, 2A3, 2A4) , and 10,000 cells/well (1A5, 1A6, 2A5, 2A6) in DME media in the culture studies. (Indices such as 1A3 indicate plate 1, row A, column 3) .
  • a pool of Zyderm I collagen (Collagen Corporation, Palo Alto, CA) was prepared by combining a total of 12 L from different Zyderm I samples and mixing through a sterile bridge (such as a stopcock) to ensure uniformity.
  • Zyderm I is an aqueous mixture of fibrillar collagen (300,000 g/mol) prepared with a concentration of 35 mg/mL or 1.17X10-* mol/L.
  • succinimidyl poly(ethylene glycol) glutarate (SPEG, 3,400 g/mol) was prepared by dissolving 45.0 mg of the activated PEG in 10 mL of PBS (phosphate buffered saline), to give a concentration of 4.5 mg/mL or -1.32X10- 3 mol/L.
  • Zyderm I controls (1B4, 1B5, 1B6) were prepared using 0.5 mL aliquots of Zyderm I pool.
  • Zyderm I/cell cultures were prepared by mixing 1.5 mL of Zyderm I with 30 ⁇ L of stock cell suspension. Aliquots of 0.5 mL were placed in each well (1B1, 1B2, 1B3) , to give 1000 cells/well.
  • a collagen/SPEG (10:1) cell-gel was prepared by mixing 2 mL of Zyderm I pool, 10 ⁇ L of activated PEG solution, and 40 ⁇ L of stock cell suspension. Aliquots of 0.5 mL were placed in each well (1C1, 1C2, 1C3) to give 1000 cells/well.
  • Collagen/SPEG (10:1) controls were prepared by mixing 2 mL of Zyderm I pool with 10 ⁇ L SPEG stock solution. Aliquots of 0.5 mL were placed in each well (1C4, 1C5, 1C6) .
  • a collagen/SPEG (100:1) cell-gel was prepared by mixing 2 mL of collagen stock solution, 1 ⁇ L of activated PEG solution, and 40 ⁇ L of stock cell suspension. Aliquots of 0.5 mL were placed in each well (1D1, 1D2, 1D3) to give 1000 cells/well.
  • Collagen/SPEG (100:1) controls were prepared by mixing 2 mL of collagen stock solution with 10 ⁇ L SPEG stock solution. Aliquots of 0.5 mL were placed in each well (1D4, 1D5, 1D6). Culture plates were centrifuged at 4°C at 150-200 g for
  • the cell culture controls (1A1 though 1A6, 2A1 through 2A6) were trypsinized and counted using a hemocytometer.
  • the l ⁇ 2 cells/well control had too few cells to count.
  • the 10 3 cells/well control had an average count of 9.1X10 3 cells/well.
  • the 10 4 cells/well had an average count of 3.9x10" cells/well.

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Abstract

Gels de cellules composé de cellules contenues dans une matrice réticulée de collagène réticulé avec du polyéthylène glycol bifonctionnel et s'obtenant par réticulation du collagène en présence des cellules. Lesdits gels favorisent la croissance des tissus vivants.
PCT/US1995/003991 1994-04-04 1995-03-31 Gels de cellules WO1995026761A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
AU22029/95A AU682266B2 (en) 1994-04-04 1995-03-31 Cell-gels
JP7525855A JPH10501706A (ja) 1994-04-04 1995-03-31 細胞−ゲル
EP95914983A EP0754065A1 (fr) 1994-04-04 1995-03-31 Gels de cellules
MXPA/A/1996/004587A MXPA96004587A (en) 1994-04-04 1996-10-04 Geles cellula

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US22216094A 1994-04-04 1994-04-04
US08/222,160 1994-04-04

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996040304A1 (fr) * 1995-06-07 1996-12-19 Reprogenesis, Inc. Compositions d'hydrogels injectables
WO2001037889A3 (fr) * 1999-11-24 2002-02-21 Universitaetsklinikum Freiburg Materiau substitut osseux injectable
WO2002030481A1 (fr) * 2000-10-10 2002-04-18 Massachusetts Institute Of Technology Liberation de cellules utilisant des structures gel en maille degradables de maniere controlable
EP0917428A4 (fr) * 1995-10-25 2002-09-18 Transkaryotic Therapies Inc Implants et explants de matrices hybrides
US6533819B1 (en) 1998-02-27 2003-03-18 Bioelastics Research, Ltd. Injectable implants for tissue augmentation and restoration
US6710025B1 (en) 1999-05-26 2004-03-23 The Brigham And Women's Hospital, Inc. Treatment of damaged tissue using agents that modulate the activity of alpha-smooth muscle actin
EP1419792A4 (fr) * 2001-08-21 2005-01-12 Japan Science & Tech Agency Complexe polycation-glycosaminoglycane reticule par un agent de reticulation polyfonctionnel et procede de production associe
DE102006011211A1 (de) * 2006-03-02 2007-09-06 Ossacur Ag Material zur Behandlung von Knochen- und/oder Knorpeldefekten
US8846022B2 (en) 2008-02-13 2014-09-30 Hyperbranch Medical Technology, Inc. Crosslinked polyalkyleneimine hydrogels with tunable degradation rates
US9938378B2 (en) 2011-04-20 2018-04-10 Spheritech Ltd Cross-linked poly-E-lysine non-particulate support
US11590259B2 (en) 2015-04-17 2023-02-28 Rochal Technologies Llc Composition and kits for pseudoplastic microgel matrices

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003500113A (ja) * 1999-05-26 2003-01-07 ザ ブリガム アンド ウイミンズ ホスピタル、 インコーポレイテッド アルファ平滑筋アクチン活性を調節する薬剤の治療的使用
JP2004173941A (ja) * 2002-11-27 2004-06-24 Olympus Corp カルシウム傾斜材料とその製造方法
JPWO2007010924A1 (ja) * 2005-07-21 2009-01-29 国立大学法人富山大学 細胞ゲル化物及び高集積の、細胞又は組織アレイの作製システム

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US4846835A (en) * 1987-06-15 1989-07-11 Grande Daniel A Technique for healing lesions in cartilage
EP0361957A1 (fr) * 1988-09-30 1990-04-04 Organogenesis Inc. Tissu artificiel et procédé de préparation
WO1990005755A1 (fr) * 1988-11-21 1990-05-31 Collagen Corporation Conjugues de collagene-polymere
US5264214A (en) * 1988-11-21 1993-11-23 Collagen Corporation Composition for bone repair
WO1994001483A1 (fr) * 1992-07-02 1994-01-20 Collagen Corporation Conjugues polymeres biocompatibles

Patent Citations (6)

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Publication number Priority date Publication date Assignee Title
US4846835A (en) * 1987-06-15 1989-07-11 Grande Daniel A Technique for healing lesions in cartilage
EP0361957A1 (fr) * 1988-09-30 1990-04-04 Organogenesis Inc. Tissu artificiel et procédé de préparation
WO1990005755A1 (fr) * 1988-11-21 1990-05-31 Collagen Corporation Conjugues de collagene-polymere
US5162430A (en) * 1988-11-21 1992-11-10 Collagen Corporation Collagen-polymer conjugates
US5264214A (en) * 1988-11-21 1993-11-23 Collagen Corporation Composition for bone repair
WO1994001483A1 (fr) * 1992-07-02 1994-01-20 Collagen Corporation Conjugues polymeres biocompatibles

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996040304A1 (fr) * 1995-06-07 1996-12-19 Reprogenesis, Inc. Compositions d'hydrogels injectables
EP0917428A4 (fr) * 1995-10-25 2002-09-18 Transkaryotic Therapies Inc Implants et explants de matrices hybrides
US6582391B2 (en) 1995-10-25 2003-06-24 Transkaryotic Therapies, Inc. Hybrid matrix implants and explants
US6699294B2 (en) 1998-02-27 2004-03-02 Bioelastics Research, Ltd. Injectable implants for tissue augmentation and restoration
US6533819B1 (en) 1998-02-27 2003-03-18 Bioelastics Research, Ltd. Injectable implants for tissue augmentation and restoration
US6710025B1 (en) 1999-05-26 2004-03-23 The Brigham And Women's Hospital, Inc. Treatment of damaged tissue using agents that modulate the activity of alpha-smooth muscle actin
WO2001037889A3 (fr) * 1999-11-24 2002-02-21 Universitaetsklinikum Freiburg Materiau substitut osseux injectable
WO2002030481A1 (fr) * 2000-10-10 2002-04-18 Massachusetts Institute Of Technology Liberation de cellules utilisant des structures gel en maille degradables de maniere controlable
EP1419792A4 (fr) * 2001-08-21 2005-01-12 Japan Science & Tech Agency Complexe polycation-glycosaminoglycane reticule par un agent de reticulation polyfonctionnel et procede de production associe
DE102006011211A1 (de) * 2006-03-02 2007-09-06 Ossacur Ag Material zur Behandlung von Knochen- und/oder Knorpeldefekten
US8846022B2 (en) 2008-02-13 2014-09-30 Hyperbranch Medical Technology, Inc. Crosslinked polyalkyleneimine hydrogels with tunable degradation rates
US9938378B2 (en) 2011-04-20 2018-04-10 Spheritech Ltd Cross-linked poly-E-lysine non-particulate support
US10266652B2 (en) 2011-04-20 2019-04-23 Spheritech Ltd. Cross-linked poly-E-lysine non-particulate support
US11590259B2 (en) 2015-04-17 2023-02-28 Rochal Technologies Llc Composition and kits for pseudoplastic microgel matrices

Also Published As

Publication number Publication date
AU2202995A (en) 1995-10-23
AU682266B2 (en) 1997-09-25
JPH10501706A (ja) 1998-02-17
MX9604587A (es) 1997-11-29
EP0754065A1 (fr) 1997-01-22
CA2187109A1 (fr) 1995-10-12

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