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WO2019119149A1 - Méthodes permettant d'améliorer la formation du tissu cartilagineux - Google Patents

Méthodes permettant d'améliorer la formation du tissu cartilagineux Download PDF

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Publication number
WO2019119149A1
WO2019119149A1 PCT/CA2018/051647 CA2018051647W WO2019119149A1 WO 2019119149 A1 WO2019119149 A1 WO 2019119149A1 CA 2018051647 W CA2018051647 W CA 2018051647W WO 2019119149 A1 WO2019119149 A1 WO 2019119149A1
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Prior art keywords
chondrocytes
media
cartilaginous tissue
glucose
cartilage
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PCT/CA2018/051647
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English (en)
Inventor
Stephen WALDMAN
Roberto TARANTINO
Original Assignee
Waldman Stephen
Tarantino Roberto
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Application filed by Waldman Stephen, Tarantino Roberto filed Critical Waldman Stephen
Publication of WO2019119149A1 publication Critical patent/WO2019119149A1/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
    • A61L27/36—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
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0655—Chondrocytes; Cartilage
    • 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
    • A61L2430/06—Materials or treatment for tissue regeneration for cartilage reconstruction, e.g. meniscus
    • 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
    • C12N2500/02—Atmosphere, e.g. low oxygen conditions
    • 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
    • C12N2500/30—Organic components
    • C12N2500/34—Sugars
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/998—Proteins not provided for elsewhere

Definitions

  • the present disclosure provides methods for improving cartilage tissue engineering. More specifically, the present disclosure provides methods of forming cartilaginous tissue from chondrocyte cell cultures by optimizing nutrient availability.
  • the inventors have shown that increasing glucose availability to isolated chondrocytes seeded on collagen-coated filters results in accelerated cartilaginous tissue deposition. They have also shown that intermediate media volumes elicit the greatest effect. The inventors have further shown that this effect requires stabilization of the transcription factor HIF-1 a. More particularly, the inventors have identified a specific set of glucose conditions which are advantageous for growing tissue engineered cartilage.
  • the present disclosure provides a method of forming cartilaginous tissue comprising: (a) obtaining chondrocytes and (b) culturing the chondrocytes in media to form cartilaginous tissue, wherein the media provides sufficient glucose availability to the chondrocytes such that the chondrocytes are at or around a transition of glucose metabolite overflow from a fermentation pathway to a tricarboxylic acid pathway.
  • the transition of metabolite overflow is indicated by (a) an intracellular pyruvate concentration of at least 4 nmol/pg DNA and/or (b) at least 4-fold higher protein expression of HIF-1 a compared to chondrocytes cultured under low glucose availability.
  • the media comprises 5 to 15 mM glucose and has a volume of 1 ml_ to 3 ml_ per 1 x 10 6 chondrocytes. [0009] In another embodiment, the media has a volume of 1 .5 to 3.0 ml_ per 1 x 10 6 chondrocytes or about 2 to 2.5 ml_ per 1 x 10 6 chondrocytes.
  • the media comprises 3 to 12 mM, or 4 to 10 mM glucose.
  • the media comprises 0.5 to 2 mM glutamine.
  • the disclosure also provides a method of forming cartilaginous tissue comprising:
  • selecting a set of media conditions comprises determining if the chondrocytes are at or around the transition of metabolite overflow as indicated by: increased intracellular pyruvate concentration, decreased PHD2 activity, increased HIF1 -a protein expression and/or increased gene expression of at least one gene target of HIF1 -a.
  • the transition of metabolite overflow is indicated by (a) an intracellular pyruvate concentration of at least 4 nmol/pg DNA and/or (b) at least 4-fold higher protein expression of H IF-1 a compared to chondrocytes cultured under low glucose availability.
  • the disclosure also provides a method of forming cartilaginous tissue comprising: (a) providing chondrocytes and (b) culturing the chondrocytes in media to form cartilaginous tissue, wherein the media comprises 3 to 15 mM glucose and has a volume of 1 ml_ to 3 ml_ per 1 x 10 6 chondrocytes.
  • the chondrocytes are seeded on a collagen-coated substrate, optionally a collagen-coated filter.
  • the cartilaginous tissue is cartilage, optionally articular cartilage.
  • the cartilaginous tissue comprises collagen II.
  • the cartilaginous tissue does not comprise collagen I.
  • the cartilaginous tissue comprises at least 3 times more glycosaminoglycans (GAG) and/or at least 2 times more collagen than cartilaginous tissue obtained from chondrocytes cultured under the same conditions, but in media having a volume of less than 1 ml_ per 1 x 10 6 chondrocytes or more than 3 ml_ per 1 x 10 6 chondrocytes.
  • GAG glycosaminoglycans
  • the chondrocytes are cultured in a bioreactor.
  • the chondrocytes are cultured under continuous flow conditions.
  • the chondrocytes are cultured for at least 2, 3, 4 or 5 weeks to form cartilaginous tissue.
  • At least 0.5 x 10 6 , 1 x 10 6 , 1.5 x 10 6 , 2 x 10 6 , 2.5 x 10 6 , 5 x 10 6 or 10 x 10 6 chondrocytes are provided.
  • the cartilaginous tissue is for use as a cartilage implant.
  • the chondrocytes are human chondrocytes.
  • the media does not comprise serum and/or exogenous growth factors.
  • the disclosure also provides a cartilage implant, wherein the implant comprises cartilaginous tissue produced by the methods as described herein.
  • the implant has an area of 1 -10 cm 2 , optionally 2-6 cm 2 .
  • the disclosure also provides a method of repairing a cartilage defect in a subject in need thereof comprising:
  • the cartilaginous tissue provided to the subject is in the form of a cartilage implant.
  • the cartilage defect is an articular cartilage defect.
  • the disclosure also provides a method of selecting media conditions for optimizing the formation of cartilaginous tissue comprising:
  • the disclosure also provides a method of selecting media conditions for optimizing the formation of cartilaginous tissue comprising:
  • the method further comprises (d), obtaining cartilaginous tissue, wherein the chondrocytes are cultured under the selected set of media conditions.
  • the first set of media conditions and a second set of media conditions differ in the amount of glucose available to the chondrocytes.
  • the notation“a” indicates significantly different (p ⁇ 0.05).
  • Figure 2 shows the histological appearance of developed tissues.
  • GAG stain Safranin-O
  • collagen stain Sirius Red
  • scale bar 150 pm.
  • PYR pyruvate
  • G6P glucose-6-phosphate
  • MAL malate
  • R5P ribose-5- phosphate
  • Figure 6 shows various glucose metabolic pathways utilized by chondrocytes.
  • Figure 7 shows the effect of glucose availability on collagen I and collagen II formation.
  • Figure 8 shows that degradation of H IF-1 a (by YC-1 ) abolishes the effect of glucose availability on cartilage formation.
  • Figure 11 shows an example response surface and corresponding curve fit for collagen deposition.
  • 2nd order response surface fit was determined by least squares regression with only significant terms included (p ⁇ 0.05). Maximal response (at 4.3 ml_, 8.0 mM glucose) was determined from the evaluation of stationary points of the 2nd order (quadratic) response surface.
  • the present inventors have shown that optimizing glucose availability to chondrocytes results in accelerated cartilaginous tissue deposition.
  • the present disclosure provides a method of forming, or optionally enhancing the formation of, cartilaginous tissue comprising: (a) providing chondrocytes and (b) culturing the chondrocytes in media to form cartilaginous tissue, wherein the media provides sufficient glucose to the chondrocytes such that the chondrocytes are at or around the transition of glucose metabolite overflow from the fermentation pathway to the tricarboxylic acid pathway.
  • Cartilage is a connective tissue produced and maintained by chondrocytes.
  • the term“cartilaginous tissue” refers to tissue containing the extracellular matrix constituents of cartilage, namely collagen and proteoglycans (and glycosaminoglycans) and which has the properties and functions of cartilage.
  • “Cartilaginous tissue” includes cartilage. In one embodiment, the cartilage is articular cartilage.
  • the term“enhancing the formation of cartilaginous tissue” includes increasing the amount of cartilaginous tissue formed from a particular number of chondrocytes, increasing the rate of cartilaginous tissue formation and/or increasing desirable qualities of the cartilaginous tissue formed.“Enhancing the formation of cartilaginous tissue” includes increasing desirable qualities, such as, increasing the amount or percentage of glycosaminoglycans (GAGs) and/or collagen deposition in the cartilaginous tissue. “Enhancing the formation of cartilaginous tissue” also includes increasing the thickness of the cartilaginous tissue.
  • GAGs glycosaminoglycans
  • any of the afore-mentioned properties are increased by at least: 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, 150%, 200%, 250%, 300%, 350% or 400% in cartilaginous tissue produced by the methods above compared to cartilaginous tissue produced by chondrocytes cultured under conditions of low glucose availability or cartilaginous tissue of a pre-determ ined standard.
  • enhancing the formation of cartilaginous tissue includes minimizing cellularity of the cartilaginous tissue.
  • the term “low glucose availability” refers to an amount of glucose provided such that the chondrocytes utilize glucose solely, or substantially, in the fermentation pathway. In one embodiment, the term “low glucose availability” refers to culturing the chondrocytes in 10 mM glucose media where the media has a volume of less than 2 ml_ per 2 x 10 6 chondrocytes.
  • chondrocytes are provided. Chondrocytes produce the structural components of cartilage, including collagen and proteoglycans (and glycosaminoglycans). Chondrocytes arise from mesenchymal stem cells (MSCs) and are regulated by a series of cytokine and transcription factor interactions. The chondrocytes are optionally isolated cells. As used herein, the term chondrocytes includes chondrocytes from any source including, but not limited to, bovine, rabbit, rat and mouse chondrocytes. In one embodiment, the chondrocytes are human chondrocytes.
  • “cell” as used herein includes cells in vivo and isolated cells in vitro and includes both one cell and a population of cells.
  • “isolated cells” as used herein refers to cells comprising at least: 30, 40, 50, 60, 70, 80, 90, 95 or 99% of the total cells of the population in which they are present.
  • glucose availability refers to the amount of glucose available to the chondrocytes; specifically the combination of media glucose concentration and the volume of media provided to the chondrocytes.
  • Chondrocytes normally metabolize glucose anaerobically, even in the presence of oxygen, resulting in the formation of lactic acid (or lactate) which is the end-point metabolite of the fermentation pathway.
  • lactic acid or lactate
  • saturation of the fermentation pathway occurs resulting in metabolite overflow to the tricarboxylic acid (TCA) pathway.
  • TCA tricarboxylic acid
  • the term“at or near the transition point of metabolic overflow” refers to the point in chondrocytes where the anaerobic fermentation pathway is saturated and aerobic metabolism via the TCA pathway starts to occur (significantly greater than basal TCA activity).
  • the inventors have further shown that this transition point is evidenced by an accumulation of intracellular pyruvate and results in stabilization of the transcription factor HIF- 1 a (hypoxia induced factor 1 ) which is known to upregulate chondrogenic gene expression (i.e. pseudo-hypoxic effect).
  • the inventors have shown that when 2 x 10 6 chondrocytes are seeded on collagen-coated filters, the transition point of glucose metabolic overflow from the fermentation pathway to the TCA pathway occurs when the chondrocytes are cultured in, or about, 4 ml_ of media comprising 10 mM glucose.
  • the transition point of glucose metabolic overflow from the fermentation pathway to the TCA pathway is indicated by higher chondrocyte intracellular pyruvate concentrations compared to chondrocytes cultured under low glucose availability (for example 2 x 10 6 chondrocytes cultured in 1 to 2 mL of 10 mM glucose).
  • the transition point of glucose metabolic overflow from the fermentation pathway to the TCA pathway is indicated by a chondrocyte intracellular pyruvate concentration of at least 4 nmol/pg or at least 5 nmol/pg DNA.
  • the transition point of glucose metabolic overflow from the fermentation pathway to the TCA pathway is indicated by an increase in chondrocyte intracellular pyruvate concentration, optionally an increase of at least: 20, 30, 40, 50, 60, 70, 80, 90 or 100% compared to chondrocytes cultured under low glucose availability (for example, 2 x 10 6 chondrocytes cultured in less than 2 mL of media containing 10 mM glucose).
  • Methods of determining intracellular pyruvate concentrations are well known in the art. For example, intracellular pyruvate can be measured by chromatography and mass spectrometry (LC-MS/MS).
  • the transition point of glucose metabolic overflow from the fermentation pathway to the TCA pathway is indicated by an decrease in PHD2 activity, optionally a decrease of at least: 20, 30, 40, 50, 60, 70, 80, 90 or 100% compared to chondrocytes cultured under low glucose availability (for example, 2 x 10 6 chondrocytes cultured in less than 2 ml_ of media containing 10 mM glucose).
  • Methods of determining PHD2 activity are well known in the art.
  • PFID2 activity can be measured by a fluorescence based assay, for example as described in Flewitson et al (2007) or McNeill et al (2005).
  • the transition point of glucose metabolic overflow from the fermentation pathway to the TCA pathway is indicated by an increase in expression of SOX9, PDK1 and/or GLUT1 , optionally an increase of at least: 20, 30, 40, 50, 60, 70, 80, 90 or 100%, compared to chondrocytes cultured under low glucose availability (for example, 2 x 10 6 chondrocytes cultured in less than 2 ml_ of media containing 10 mM glucose).
  • Methods of determining gene expression are well known in the art.
  • SOX9, PDK1 and/or GLUT1 can be measured by qPCR.
  • the transition point of glucose metabolic overflow from the fermentation pathway to the TCA pathway is indicated by a higher protein expression of H IF-1 a compared to chondrocytes cultured under low glucose availability (for example 2 x 10 6 chondrocytes cultured in less than 2 mL of 10 mM glucose).
  • the stabilization of H IF-1 a at the transition point of glucose metabolic overflow from the fermentation pathway to the TCA pathway is indicated by at least: 4-fold, 5- fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold higher protein expression of H IF-1 a compared to chondrocytes cultured under low glucose availability (for example 2 x 10 6 chondrocytes cultured in less than 2 mL of media containing 10 mM glucose).
  • Methods of determining expression levels of proteins are well known in the art. For example, protein expression of HIF-1 a may be determined by Western blotting.
  • the media comprises 3 to 12 mM glucose, optionally 4 to 10 mM, or about 4, 5, 6, 7, 8, 9, 9.5 or 10 mM glucose and has a volume of 2 ml_ to 6 ml_, optionally 4 to 6 ml_ or about 3, 3.5, 4, 4.5, 5, 5.5 or 6 ml_ per 2 x 10 6 chondrocytes.
  • the media comprises 3 to 12 mM glucose, optionally 4 to 10 mM, or about 4, 5, 6, 7, 8, 9, 9.5 or 10 mM glucose and has a volume of 2 ml_ to 6 ml_, optionally 4 to 6 ml_ or about 3, 3.5, 4, 4.5, 5, 5.5 or 6 ml_ per 2 x 10 6 chondrocytes initially provided/seeded in the media (i.e., the starting number of chondrocytes).
  • the media comprises 3 to 12 mM glucose, optionally 4 to 10 mM, or about 4, 5, 6, 7, 8, 9, 9.5 or 10 mM glucose and the chondrocytes are cultured in 1 to 3 ml_ of media per 1 x 10 6 chondrocytes, optionally 2 to 3 ml_ or 2 to 2.5 ml_ or about 1 .5, 1 .75. 2, 2.25, 2.5, 2.75 or 3 ml_ of media per 1 x 10 6 chondrocytes.
  • the media comprises 3 to 12 mM glucose, optionally 4 to 10 mM, or about 4, 5, 6, 7, 8, 9, 9.5 or 10 mM glucose and the chondrocytes are cultured in 1 to 3 ml_ of media per 1 x 10 6 chondrocytes, optionally 2 to 3 ml_ or 2 to 2.5 ml_ or about 1 .5, 1 .75. 2, 2.25, 2.5, 2.75 or 3 ml_ of media per 1 x 10 6 chondrocytes initially provided/seeded in the media (i.e., the starting number of chondrocytes).
  • the media provides 0.01 to 0.1 mmols of glucose per 2 x 10 6 chondrocytes to the chondrocytes, optionally 0.02 to 0.06 mmols or 0.03 to 0.05 mmols of glucose per 2 x 10 6 chondrocytes or about 0.04 or 0.04 mmols glucose per 2 x 10 6 chondrocytes to the chondrocytes.
  • the media provides 0.05 to 0.5 mmols of glucose per 1 x 10 6 chondrocytes to the chondrocytes, optionally 0.01 to 0.03 mmols or 0.015 to 0.025 mmols of glucose per 1 x 10 6 chondrocytes or about 0.02 or 0.02 mmols glucose per 1 x 10 6 chondrocytes to the chondrocytes.
  • the number of chondrocytes referred to here is the number of chondrocytes initially provided in the media (i.e. , the starting number of chondrocytes).
  • the media is a commercially available media such as Ham’s F12 media, Dulbecco's Modified Eagle's media (DMEM; high or low glucose) or a mixture of Ham’s F12 and DMEM (for example a 1 : 1 ratio of Ham’s F12 to DMEM).
  • the media optionally contains various supplements, including, but not limited to HEPES, sodium bicarbonate, fetal bovine serum (FBS), ascorbic acid and/or antibiotic/anti- mycotic(s).
  • the media is Ham’s F12 media supplemented with 10 to 30 mM HEPES, optionally about 20 mM HEPES, 10 to 20 mM sodium bicarbonate, optionally about 14 mM sodium bicarbonate, 10 to 30% FBS, optionally about 20% FBS, 50 to 200 pg/mL ascorbic acid, optionally about 100 pg/mL ascorbic acid and 0.5 to 2X, optionally about 1X of an antibiotic/anti-mycotic formulation comprising, for example, penicillin (100 units/mL), streptomycin (100 units/mL) and amphotericin B (0.25 pg/mL).
  • Ascorbic acid can come in several forms such as A2P.
  • the serum is replaced by at least one growth factors, such as (but not limited to), TGFbetas, IGFs, FGFs and/or PDGFs. If the serum is replaced with growth factors, additional supplements may be required such as (but not limited to), insulin, ITS supplements and/or dexamethasone.
  • growth factors such as (but not limited to), TGFbetas, IGFs, FGFs and/or PDGFs.
  • additional supplements may be required such as (but not limited to), insulin, ITS supplements and/or dexamethasone.
  • the media comprises glutamine, optionally 0.5 to 2 mM glutamine, 0.8 to 1.5 mM glutamine, 0.9 to 1.2 mM glutamine or about 1 mM glutamine.
  • the media is a minimal media.
  • minimal media refers to media that contains only the minimum nutrients required for cellular growth.
  • the media is free of serum (serum-free, or has an absence of serum).
  • the media is free of growth factors (growth factor-free, or has an absence of growth factors).
  • the media is both serum-free and growth factor-free.
  • the method comprises seeding, or providing, the chondrocytes in a pellet, in a scaffold, or on a substratum prior to the step of culturing the chondrocytes.
  • the scaffold is an artificial scaffold (for example, a synthetic scaffold such as a polymer based scaffold or a naturally derived scaffold).
  • the scaffold is a substratum (for example, a culture plate insert or any other porous or solid substratum), optionally a collagen-coated substratum.
  • Polytetrafluoroethylene culture plate inserts (MillicellTM) coated with collagen II (from chicken sternum) are available for example.
  • chondrocytes are cultured in a bioreactor, optionally a continuous flow bioreactor.
  • bioreactor refers to a vessel or apparatus for cultivating cells.
  • a “continuous flow bioreactor” is a bioreactor where a constant flow of fresh media into the bioreactor and waste media out of the bioreactor is maintained, thereby allowing a continuous culture.
  • a continuous culture is contrasted with a batch fed culture where new media is provided at discrete time points rather than continuously.
  • the chondrocytes are cultured for at least 2, 3, 4, 5 or 6 weeks to form cartilaginous tissue. In another embodiment, the chondrocytes are cultured for 3 to 5 weeks, optionally about 4 weeks to form cartilaginous tissue.
  • the minimum time needed in culture can depend on the size of the implant to be created as well as the number of cells that are initially seeded.
  • chondrocytes are initially seeded/provided in the culture.
  • the present methods could be scaled up, for example on a semi-commercial or commercial scale (i.e. , higher numbers of chondrocytes could be provided to form a larger amount of cartilaginous tissue).
  • the media is replaced at least, or about, every 6, 12, 24, 48, 60 or 72 hours, optionally every 2-3 days.
  • the media is replaced at least, or about, every 6, 12, 24, 48, 60 or 72 hours, optionally every 2-3 days, with the same volume of media and media containing the same concentration of glucose as originally provided to the chondrocytes.
  • the same media is provided to the chondrocytes throughout the culturing time.
  • the glucose concentration of the culture (as well as the concentration of other nutrients) may decline.
  • the inventors identified that the transition of metabolite overflow from the fermentation pathway to the tricarboxylic acid pathway in chondrocytes can be indicated by increased intracellular pyruvate concentration, decreased PHD2 activity, increased HIF1 -a protein expression and or increased gene expression of at least one gene target of HIF1 -a.
  • the present disclosure also provides a method of increasing the formation of cartilaginous tissue comprising:
  • selecting a set of media conditions comprises determining if the chondrocytes are at or around the transition of metabolite overflow as indicated by: increased intracellular pyruvate concentration, decreased PHD2 activity, increased HIF1 -a protein expression and/or increased gene expression of at least one gene target of HIF1 -a.
  • gene targets of HIF1 -a include, but are not limited to, SOX9, PDK1 and GLUT1.
  • the transition of metabolite overflow is indicated by (a) an intracellular pyruvate concentration of at least 4 nmol/pg DNA and/or (b) at least 4-fold higher protein expression of H IF-1 a compared to chondrocytes cultured under low glucose availability.
  • the disclosure further provides a method comprises a) providing chondrocytes, (b) culturing the chondrocytes under at least a first set of media conditions and a second set of media conditions to obtain cartilaginous tissue, and (c) selecting the set of media conditions wherein the cultured chondrocytes have
  • the method further comprises step (d), obtaining cartilaginous tissue, wherein the chondrocytes are cultured under the selected set of media conditions.
  • increased intracellular pyruvate concentration, decreased PFID2 activity, increased HIF1 -a protein expression and/or increased gene expression of at least one gene target of HIF1 -a refers to increases and decreases compared to chondrocytes that are not at the transition of metabolite overflow, for example chondrocytes that cultured under low glucose availability (for example 2 x 10 6 chondrocytes cultured in less than 2 ml_ of media containing 10 mM glucose).
  • the method comprises culturing the chrondrocytes under multiple sets of media conditions, optionally at least: 3, 4, 5, 6, 7, 8, 9 or 10 sets of media conditions to obtain cartilaginous tissue, and (c) selecting the set of media conditions wherein the cultured chondrocytes have
  • the present disclosure also provides a method of selecting media conditions for optimizing the formation of cartilaginous tissue.
  • the method comprises (a) providing chondrocytes, (b) culturing the chondrocytes under at least a first set of media conditions and a second set of media conditions to obtain cartilaginous tissue, and (c) selecting the set of media conditions wherein the cultured chondrocytes have
  • the method further comprises step (d), obtaining cartilaginous tissue, wherein the chondrocytes are cultured under the selected set of media conditions.
  • the method comprises culturing the chrondrocytes under multiple sets of media conditions, optionally at least: 3, 4, 5, 6, 7, 8, 9 or 10 sets of media conditions to obtain cartilaginous tissue, and (c) selecting the set of media conditions wherein the cultured chondrocytes have
  • lactate production is determined through enzymatic assays of the conditioned culture media.
  • the rate of lactate production is optionally measured in nmol/hr.
  • O2 consumption is determined in the conditioned culture media using p02 sensors and Henry’s law.
  • the rate of O2 consumption is optionally measured in nmol/hr.
  • set of media conditions refers to the type, volume and/or composition of the media.
  • Media conditions include but are not limited to, the amount of glucose available to the chondrocytes in the culture.
  • the first and second set of media conditions differ in the amount of glucose available to the chondrocytes.
  • the first and second set of media conditions vary in the number of moles of glucose available per number of chondrocytes in the culture.
  • the first and second set of media conditions vary in the volume of glucose-containing media per number of chondrocytes. The foregoing also applies to the multiple sets of media conditions described herein, where each set of media conditions in the multiple sets of media conditions differs in the number of moles of glucose or volume of glucose-containing media available to the chondrocytes.
  • the cartilaginous tissue produced by the methods described herein is for use as a cartilage implant by cutting the developed cartilaginous tissue to match the size and shape of the defect or growing the tissue in a mould or vessel with the same/similar size, geometry and/or anatomy of the repair site.
  • the term“cartilage implant” refers to a cartilaginous tissue construct that can be implanted in a subject to correct or prevent cartilage damage.
  • the cartilage damage is articular cartilage damage.
  • the cartilage implant has an area of 1 to 10 cm 2 , optionally 2 to 6 cm 2 .
  • the cartilage implant has an area that is the same, or approximately the same, as the area of a cartilage defect.
  • the cartilage implant has a thickness of 1 to 3 mm.
  • the cartilage implant optionally comprises, consists of, or consists essentially of cartilaginous tissue produced by the methods described herein.
  • the cartilage implant is optionally administered to a subject, or for use in a subject, in need of cartilage repair due to cartilage damage or disease.
  • the term“cartilage damage or disease” refers to any type of injury or disorder affecting cartilage anywhere in the body. Damaged or diseased cartilage, such as damaged or diseased articular cartilage, can cause symptoms such as pain and limited movement. It can also lead to joint damage and deformity. Causes of cartilage damage or disease include, but are not limited to tears and injuries, such as sports injuries, genetic factors and disorders, such as some types of arthritis, osteonecrosis and osteochondritis. Articular cartilage damage may occur in synovial joints, for example the knee joint or the shoulder joint. [0093] In one embodiment, the cartilage damage is a clinically sized defect. As used herein, a clinically sized defect is a cartilage defect with an area of 1 -10 cm 2 , optionally 2-6 cm 2 .
  • cartilage repair examples include focal defect repair and joint resurfacing.
  • Other examples of cartilage repair include, but are not limited to, intervertebral disc repair, external ear reconstruction, rib reconstruction or tracheal reconstruction.
  • the present disclosure is also directed to cartilaginous tissue produced by the methods described herein.
  • the present disclosure is further directed to a cartilage implant produced by the methods described herein.
  • the cartilaginous tissue is hyaline cartilage, optionally articular cartilage.
  • the cartilage is elastic cartilage (e.g. auricular, tracheal, etc.), hyaline cartilage (e.g. articular, nasal, costal, etc.) or fibrocartilage (e.g. meniscus, annulus fibrosis, etc.).
  • the cartilaginous tissue and/or cartilage implant is suitable for implantation in a subject in need thereof.
  • the cartilaginous tissue and/or cartilage is in a biologically compatible form suitable for administration in vivo.
  • biologically compatible form suitable for administration in vivo is meant a form of the composition to be administered in which any toxic effects are outweighed by the therapeutic effects.
  • subject includes all members of the animal kingdom, including a human.
  • the subject is an animal.
  • the subject is a mammal, such as a human.
  • the cartilaginous tissue and/or a cartilage implant as described herein is for use in joint resurfacing, intervertebral disc repair and external ear reconstruction and/or tracheal reconstruction.
  • a use of cartilaginous tissue and/or a cartilage implant as described herein for treating a subject with cartilage damage or disease is provided.
  • a method of treating a subject with cartilage damage or disease comprising administering cartilaginous tissue and/or a cartilage implant as described herein to the subject in need thereof.
  • cartilaginous tissue and/or a cartilage implant as described herein for use in treating a subject with cartilage damage or disease is provided.
  • cartilaginous tissue and/or a cartilage implants described herein are used or administered to a subject to repair cartilage damage in a subject in need thereof.
  • the cartilaginous tissue and/or a cartilage implants described herein are used or administered to a subject as a prophylactic for the prevention of cartilage damage or disease.
  • the cartilaginous tissue and/or a cartilage implants described herein are used or administered to a subject who presents with one or more symptoms of cartilage damage or disease.
  • the cartilaginous tissue and/or a cartilage implants described herein described herein are used prophylactically in a subject in order to prevent or reduce the incidence or recurrence of cartilage damage or disease or symptoms or conditions associated with cartilage damage or disease.
  • scaffold-free articular cartilage constructs can be created from isolated chondrocytes seeded in high-density on ceramic bone substitutes (Waldman et al, 2002; Waldman et al, 2003b; Waldman et al, 2004; Waldman et al, 2006; Waldman et al, 2007; Kandel et al, 2006; Waldman et al, 2003b) (osteochondral construct) and collagen-coated filter inserts (chondral construct) (Fan et al, 2010; Kaupp et al, 2008; Waldman et al, 2004b; Waldman et al, 2008; Waldman et al, 2010; U gleich et al, 2012; Bianchi et al, 2017).
  • Functional articular cartilage implants are important for repairing cartilage defects.
  • Technical hurdles for engineering cartilage tissue remain, including low accumulation of tissue (collagen, proteoglycans) and inferior mechanical properties. These issues are typically addressed using methods that require substantial efforts to optimize and/or limit potential off-target effects.
  • cartilage cells chondrocytes
  • a novel pathway Crabtree- like effect
  • Chondrocytes are typically anaerobic and tend to favour the glycolysis-fermentation pathway (even in the presence of oxygen). However, under increased glucose availability, glycolytic metabolites are shunted down other metabolic pathways ( Figure 6 and Figure 3 - see in particular increased activity of citric acid cycle and pentose phosphate pathway at higher media volumes).
  • HIF-1 a is a subunit of a heterodimeric transcription factor that is considered to be the master transcriptional regulator of the cellular response to hypoxia.
  • Lactate production (nmol/h) and oxygen (O2) consumption (nmol/h) in the culture media correlate with both proteoglycan content and collagen content in developed constructs (Table 2). Thus, these rates can be used as predictors of culture conditions to maximize cartilaginous tissue formation.
  • Crabtree effect is the phenomenon where aerobic cultures (e.g. S. cerevisiae) produce ethanol (a fermentation product) in the presence of oxygen and high external glucose (De Deken et al, 1966). This effect has been explained as an overflow in glucose metabolism caused by the saturation of respiratory capacity (Pronk et al, 1996). In chondrocytes, an inverse pathway is hereby described— under high glucose availability, saturation of the fermentation pathway occurs resulting in overflow to the tricarboxylic acid (TCA) cycle.
  • TCA tricarboxylic acid
  • pyruvate can stabilize the transcription factor HIF-1 a (hypoxia induced factor 1 ) by interfering with proline- hydroxylase-2 (PHD2) (Ren et al, 2011 ; Denko et al, 2008).
  • HIF-1 a Normally, in the presence of oxygen, PHD2 hydroxylates HIF-1 a which initiates degradation by the von Flippel-Lindau (pVFIL)-ubiquitin ligase complex (Lu et al, 2005). After stabilization, HIF-1 a dimerizes with H IF-1 b in the nucleus and the heterodimer then binds to hypoxia response elements (HREs) of its target genes to initiate expression (Dengler et al, 2014).
  • HREs hypoxia response elements
  • HIF-1 a mediated transcription is also regulated by FIH-1 (factor inhibiting HIF) (Masoud et al, 2015).
  • FIH-1 factor inhibiting HIF
  • FIH- 1 hydroxylates HIF-1 a to prevent interaction with p300 to block transcriptional activation (Masoud et al, 2015).
  • pyruvate affects FIH-1 activity (Dalgard et al, 2004; Hewitson et al, 2007) or stabilizes the other known hypoxia-induced factors (Ren et al, 2011 ) (HIF-2a and HIF-3a) which appear to have differential effects to that of HIF-1 a (Dengler et al, 2014).
  • FIG. 9 shows the effect of media volume on the formation of cartilaginous tissue from human donor chondrocytes and Figure 10 shows the effect of media volume on the formation of cartilaginous tissue from rabbit chondrocytes (P2).
  • 2nd order response surface fits were determined by least squares regression with only significant terms included (p ⁇ 0.05). Maximal responses were determined from the evaluation of stationary points of the 2nd order (quadratic) response surfaces.

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Abstract

L'invention concerne des méthodes de formation du tissu cartilagineux. Dans un mode de réalisation, les méthodes comprennent : (a) l'obtention de chondrocytes, (b) la culture des chondrocytes dans des milieux pour obtenir un tissu cartilagineux, où le milieu offre une disponibilité de glucose suffisante aux chondrocytes pour que les chondrocytes soient à, ou à proximité de, la transition du trop-plein de métabolites de la voie de fermentation à la voie acide tricarboxylique. Un tissu cartilagineux produit par les méthodes selon l'invention, et ses utilisations pour réparer des défauts de cartilage sont en outre décrits.
PCT/CA2018/051647 2017-12-21 2018-12-21 Méthodes permettant d'améliorer la formation du tissu cartilagineux WO2019119149A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005105992A1 (fr) * 2004-04-21 2005-11-10 New York Eye & Ear Infirmary Formulations de culture de chondrocytes
CA2729917A1 (fr) * 2008-07-06 2010-01-14 The Curators Of The University Of Missouri Implants osteo-cartilagineux, procedes d'arthroplastie, dispositifs et systemes associes

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005105992A1 (fr) * 2004-04-21 2005-11-10 New York Eye & Ear Infirmary Formulations de culture de chondrocytes
CA2729917A1 (fr) * 2008-07-06 2010-01-14 The Curators Of The University Of Missouri Implants osteo-cartilagineux, procedes d'arthroplastie, dispositifs et systemes associes

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
HEYWOOD ET AL.: "Glucose Concentration and Medium Volume Influence Cell Viability and Glycosaminoglycan Synthesis in Chondrocyte-Seeded Alginate Constructs", TISSUE ENGINEERING, vol. 12, no. 12, 2006, pages 3487 - 3496, XP055620435 *

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