WO2011025367A1 - Procédé de culture de chondrocytes et utilisations pour le traitement d'arthrite et de lésions chondrales - Google Patents

Procédé de culture de chondrocytes et utilisations pour le traitement d'arthrite et de lésions chondrales Download PDF

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WO2011025367A1
WO2011025367A1 PCT/NL2010/050505 NL2010050505W WO2011025367A1 WO 2011025367 A1 WO2011025367 A1 WO 2011025367A1 NL 2010050505 W NL2010050505 W NL 2010050505W WO 2011025367 A1 WO2011025367 A1 WO 2011025367A1
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medium
cells
chondrocytes
mosm
tonicity
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Jahr Holger
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Erasmus University Medical Center Rotterdam
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    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • 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
    • C12N2500/60—Buffer, e.g. pH regulation, osmotic pressure
    • 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
    • C12N2501/02—Compounds of the arachidonic acid pathway, e.g. prostaglandins, leukotrienes
    • 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
    • C12N2501/04—Immunosuppressors, e.g. cyclosporin, tacrolimus

Definitions

  • the present invention relates to the field of tissue engineering, more specifically to culturing of chondrocytes for use in the treatment of chondral lesions, osteoarthritis and rheumatoid arthritis.
  • Cartilage or more precisely, hyaline articular cartilage (HAC) is a connective tissue covering the ends of bones in joints and is composed of specialized cells, chondrocytes, that produce a large amount of extracellular matrix. This matrix is crucial for the unique biomechanical properties of this tissue and it is composed of a collagen fiber network, providing tensile strength and flexibility, and abundant ground matrix, rich in proteoglycans.
  • HAC hyaline articular cartilage
  • glycosaminoglycan (GAG) side chains of the proteoglycans are sulphated and responsible for a characteristic high fixed negative charge density (Lesperance, L.M. et al., 1992, J. Orthop. Res. 10:1-13), which binds mobile cations, mainly sodium cations. This binding determines the
  • RA rheumatoid arthritis
  • RA rheumatoid arthritis
  • chondrocyte implnatation is currently the most developed hyaline repair technique for the knee (Brittberg, M et al., 1994, N. Eng. J. Med. 331:889-895).
  • Characterized chondrocyte implantation employing phenotypical pre- screening prior to implantation, has recently been proven to improve structural repair (Saris, D.B. et al., 2008, Am. J. Sports Med. 36:235-246).
  • chondrocyte dedifferentiation during in vitro expansion for autologous chondrocyte implantation.
  • spherical chondrocytes will gradually convert into fibroblast-like cells (Von der Mark, K. et al., 1977,
  • Chondrocyte dedifferentiation may also play a role in the pathogenesis of OA and RA, as the ability of aging chondrocytes to produce, maintain and repair the extracellular matrix is compromised (Loeser, R. F., 2009, Osteoarthritis Cartilage 17:971-979) and as COLl is shown to be present in chondrocyte clusters in fibrillated areas of late- stage OA cartilage while it is absent in healthy cartilage (Gouttenoire, J. et al., 2004, Biorheology 41:535- 542).
  • SOX9 mRNA expression was significantly upregulated in freshly isolated and passaged human articular chondrocytes after exposure to hypertonic solutions of 550 mOsm, this overexpression indicating prevention of dedifferentiation.
  • the current inventor now have found that proliferation and prevention of dedifferentiation of chondrocytes, can advantageously be performed, when culturing in vitro, by a method comprising culturing said cells in a medium having a physiological level of tonicity.
  • said medium has a tonicity of 350 - 480 mOsm, more preferably a tonicity of about 380 mOsm.
  • said physiological level of tonicity is also applied during isolation of the cells.
  • the proliferation of the cells in such a method comprises passaging of the cells.
  • the chondrocytes are isolated from a subject having osteoarthritis or rheumatoid arthritis.
  • a calcineurin inhibitor is added to the culture medium, and preferably said calcineurin inhibitor is chosen from the group consisting of FK506 (tacrolimus/Prograft®), FK520, cyclosporine, tacrolimus, ascomycin and pimecrolimus.
  • a COX-2 inhibitor is added to the culture medium, preferably chosen from the group consisting of celecoxib, rofecoxib, valdecoxib, lumiracoxib, meloxicam, tramadol, etoricoxib, parecoxib, valdecoxib, licofelone, NS-398 and sulphonanilides such as nimesulide.
  • the presence of a COX-2 inhibitor in the medium causes reduction and not total inhibition of the COX-2 activity of the cells in the medium.
  • Another embodiment of the present invention is a method for autologous chondrocyte implementation (ACI) or matrix-assisted ACI (MACI) in which chondrocytes are proliferated with a method according to the invention.
  • a further embodiment of the present invention is a method for ameliorating osteoarthritis (OA) or rheumatoid arthritis (RA) by intra- articular washing a joint of a subject suffering from said OA or RA with a medium as defined above, preferably wherein said medium also comprises chondrocytes or (osteo-chondro) progenitors.
  • the invention comprises the use of a medium as defined above for treatment of OA or RA.
  • a further embodiment is the use of a medium as defined above for intra-articular injection in a joint after surgery of said joint.
  • said medium further comprises chondrocytes, preferably autologous chondrocytes.
  • a further embodiment of the present invention is a method for isolating chondrocytes from cartilage tissue comprising use of a medium as defined above 1 as buffer. Accordingly, also part of the invention is the use of a medium as defined above as a buffer medium for isolation of chondrocytes from cartilage tissue.
  • the invention comprises the use of a medium as defined above for the culture of mesenchymal stem cells or other chondrocyte progenitor cells.
  • FIG. 3 Hypertonic conditions activate nuclear factor of activated T-cells 5 in osteoarthritis human articular chondrocytes.
  • NFAT5 nuclear factor of activated T-cells 5
  • S100A4 and SLC6A12 target genes S100A4 and SLC6A12 in transduced chondrocytes either expressing (NFAT5 shRNA) or not expressing (control) ⁇ 7K4T"5-specific shRNAs, 24 hours after increasing tonicity to 380 mOsm.
  • NFAT5 shRNA nuclear factor of activated T-cells 5
  • control ⁇ 7K4T"5-specific shRNAs
  • chondrocytes, (a), gene expression of collagen type I and type II, respectively under standard culture tonicity (280 mOsm, 280) and physiological, hypertonic conditions (380 mOsm, 380) in the presence or absence of FK506 (values in ng/niL). n 12, *p ⁇ 0.05 compared to 280, #p ⁇ 0.05 compared to 380.
  • Figure 6 Combined effects of calcineurin inhibition and physiological tonicity (hypertonicity) on chondrogenic and hypertrophic marker gene expression in osteoarthritic human chondrocytes, (a, b), gene expression; (c), enzyme activity. Chondrogenic markers: aggrecan, Sox9 (); hypertrophic markers: collagen type X (COLlO) alkaline phosphatase (ALPL) and matrix
  • MMP13 metalloproteinase-13
  • FIG. 7 Combined effects of calcineurin inhibition and physiological tonicity (hypertonicity) on ECM marker gene expression in osteoarthritic human chondrocytes.
  • Matrix metalloproteinases MMP-I, -3; i.e. interstitial collagenase or fibroblast collagenase and stromelysin-1, respectively
  • TIMP-I and -2 their inhibitors
  • ADAMTS-4 and -5 aggrecanases 1 and 2
  • Figure 8 Combined effects of calcineurin inhibition and physiological tonicity (hypertonicity) on collagen protein expression of P2 osteoarthritic human chondrocytes.
  • FIG. 10 Relative expression of ECM components in cartilage explants cultured in vitro at 380mOsm without or with addition of calcineurin inhibitor FK506 (+, 50ng/mL).
  • A chondrogenic markers COL2, AGCl, and Sox-9 next to unwanted COLl;
  • B hypertrophic marker expression (COLlO, ALPL and MMP- 13).
  • n 6.
  • ATDC5 cells specifically up-regulate COL2, but not COLl or
  • COLlO in response to culture medium of 380 mOsm.
  • Western Blot analysis 14 days post exposure to either maintaining cells at 280 mOsm or changing to 380 mOsm culture medium. Representative images of three independent blots are shown. TUB, ⁇ - tubulin (loading control).
  • Proteoglycan content of ATDC5 at different culture tonicities The figure shows Alcian Blue staining for proteoglycans (PG, e.g. aggrecan etc.), next to relative DNA amounts (Crystal- Violet cell proliferation assay). Note that PG, e.g. aggrecan etc., next to relative DNA amounts (Crystal- Violet cell proliferation assay). Note that PG
  • Osteoarthritis is characterized by an imbalance between matrix synthesis and degradation: increased synthesis of catabolic enzymes (e.g. MMPs and ADAMTS') by hyaline chondrocytes causes collagen (i.e. mainly collagen type II, COL2) and proteoglycan (i.e. aggrecan, AGCl) depletion in the extracellular matrix (ECM). Both molecules are major structural components of cartilage's ECM: COL2 networks provide biomechanical strength, while sulfated proteoglycans ensure the characteristic high fixed negative charge density (FCD) of this tissue. The latter binds mobile cations (e.g.
  • FCD fixed negative charge density
  • FCD determines the physiological extracellular tonicity of healthy hyaline articular chondrocytes in vivo to between 350 and 480 mOsm (milliosmoles per kilogram of water), while OA is associated with a severity- depending drop in tonicity to between 280 and 350 mOsm.
  • ADAMTS' are generally considered to be rather catabolic markers, other molecules, such as COL2, AGCl, SOX9, and TIMPs can be used as anabolic markers for testing the 'health' and usefulness of (cultured) chondrocytes.
  • NFAT5 can be used as a positive marker for chondrocyte health and utility.
  • COLl which is a marker for the unwanted fibrocartilage formation, can be used. Quantitative PCR assays for COL2, SOX9, AGCl and COLl have been reported earlier (Mandl, E.W. et al., 2004, Matrix Biol. 23:231-243).
  • NFAT5 nuclear factor of activated T- cells 5
  • HsNFAT5_Fw 5'-gggtcaaacgacgagattgtg-3'
  • Assays and primer/probe sets for testing the expression levels of MMPs, and TIMPs are described by Nutall, R.K. et al. (2003, MoI Cancer Res. 1:333-345) and assays for ADAMTS' are commercially available (e.g. from Anaspec, Fremont, CA or Qiagen, Venlo, The Netherlands).
  • chondrocytes tend to rapidly loose their most important marker, COL2, the expression of which decreases with within the first two passages (expansion rounds) by about 1000-fold.
  • the expression of COL2 (and further important markers such as SOX9 and aggrecan) is preserved in medium with 380 and 480 mOsm in a tonicity- dependent manner.
  • moderately elevated, physiological tonicity (380 mOsm) did not affect chondrocyte proliferation, while higher tonicities dose-dependently inhibited proliferation and diminished cell viability.
  • Physiological tonicity improved expression of chondrogenic markers and NFAT5 and its target genes, while suppressing dedifferentiation marker collagen type I and improving type II/type I expression ratios >100-fold. Effects of physiological tonicity were similar in osteoarthritic and normal (non-osteoarthritic) chondrocytes, indicating a disease-independent mechanism. NFAT5 RNA interference abolished tonicity- mediated effects and revealed that NFAT5 positively regulates collagen type II expression, while suppressing type I.
  • a tonicity of about 380 mOsm yields an optimum for the expansion of chondrocytes in vitro.
  • the chondrocytes do not or only occasionally dedifferentiate, while they are not inhibited in their expansion (see Table 1). It follows from Table 1 that for 4 doublings of the cell count, a culture will have to be cultured on average for about 11-12 days under the currently used regimen of a tonicity of 280 mOsm.
  • the culturing time for the same amount of doubling is— on average— only slightly larger, i.e. about 13 days.
  • the slightly hypertonic medium of the invention having a hypertonicity of 350-480 mOsm, preferably 380-480 mOsm, more preferably 350-450 mOsm, more preferably 360-400 mOsm and most preferably about 380 mOsm, provides an ideal medium for proliferation of chondrocytes, whereby
  • the medium can thus be used as a culturing medium for the ex vivo culturing of autologous chondrocytes (to be used for any form of treatment, like ACI or MACI, of OA, RA and other arthritic conditions), but it can also be used as medium in the isolation of such autologous chondrocytes from the subject which is to be treated.
  • This beneficial effect of the medium of the invention is specifically of use in proliferation of chondrocytes which involves multiple passaging or expansion rounds.
  • chondrocytes that are isolated from subjects suffering from osteoarthritis, whose cells are deemed to be in a pathological state, react similarly to the same order of tonicity with respect to the marker genes tested. This may imply that physiological tonicity, postulated to be around 380 mOsm for chondrocytes, is sensed by OA cells and normal cells in a similar fashion. However, late stage OA cells, isolated from fibrillated areas, are dedifferentiated, flattened cells that have lost their spherical shape as an integral part of the chondrocyte phenotype because of cytoskeletal changes.
  • dedifferentiation of chondrocytes can yet be improved by addition of one or more calcineurin inhibitors to the medium. It is shown in the present application that addition of the FDA-approved calcineurin inhibitors FK506 or FK520 dose-dependently affected the expression of the tested markers.
  • Aggrecan expression was improved by 80% by application of the 380 mOsm medium and 110% by application of the medium + 50 ng/ ⁇ l FK506; SOX9 expression was improved by more than 250% in both occasions and COL2 expression increased 10-fold in the medium of the invention, but >20-fold in the medium to which FK506 was added (see Fig. 5).
  • TGF ⁇ has been the most commonly used chondrogenic factor to induce redifferentiation (Mandl, 2002, supra; van Osch, G.J. et al, 2001, Plast. Reconstr. Surg. 107:433).
  • TGF ⁇ is added to the medium and will further potentiate the beneficial effects on the proliferation and the inhibition of dedifferentiation of chondrocytes.
  • Addition of TGF ⁇ may be specially be useful when isolating and culturing cells from subjects suffering from OA or RA.
  • calcineurin inhibitors have an additional effect on the effects of physiologic tonicity when culturing chondrocytes also opens further possibilities for treatment. Since at least some calcineurin inhibitors, such as FK506, also have anti- inflammatory effects, they have been suggested for treatment of arthritis (e.g. Magari, K. et al., 2003, Br. J. Pharmacol.
  • calcineurin inhibitors are used to block or decreasing an immune response that can be caused by a scaffold seeded with chondrocytes, or by non-autologous chondrocytes when introduced in a joint for repair of chondric lesions, such as in ACI, MACI or other treatments for OA and RA.
  • the calcineurin inhibitor(s) may be chosen from the group of FK506 (also known as tacrolimus or Prograft®), FK520, cyclosporine A, voclosporin, ascomycin and pimecrolimus. Further, (genetically engineered) cell-permeablp peptides derived froro the family of naturaJJy occurring regulators of
  • calcineurin activity so-called RCANs (Davies et al. FASEB Journal 2007, 21: 3023-28) or those described by Roehrl et al. PNAS 2004, 101 no. 20: 7554-7559) may be used.
  • RCANs Rosehrl et al. PNAS 2004, 101 no. 20: 7554-7559
  • Other synthetic peptides such as the IBP import blocking peptide (IBP) described by Hallhuber et al. (Future Cardiol. 2007, 3(l):91-8) preventing the binding of calcineurin to importin and inhibiting its nuclear shuttling may also be included.
  • IBP IBP import blocking peptide
  • oleanane triterpenoids such as liquidambaric acid, oleanolic acid, 3a-acetoxy-25-hydroxy-olean-12-en- 28-oic acid or lantanolic acid and more preferably 3a-acetoxy-25-hydroxy- olean-12-en-28-oic acid (IC50: 4.63mM) and lantanolic acid (IC50: 12.62mM) as the latter exhibit the strongest inhibitory activity against NFAT
  • transcription factors (Dat et al. Biol. Pharm. Bull. 2004, 27(3) 426—428). Also members of the pyrazolopyrimidine family such as NCI3 (Sieber et al., Eur, J. Immunol. 2007. 37: 2617-2626) or related compounds which bind to
  • calcineurin and cause an allosteric change interfering with NFAT activation or NFAT nuclear import inhibitors (Venkatesh et al., 2004, PNAS 101:8969- 8974).
  • the specificity of the treatment may be increased by releasing cell-permeable NFAT-specific inhibitors like 1 IR-VIVIT (H-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Gly-Gly-Gly-Met-Ala-Gly-Pro-His-Pro-Val-Ile-Val-Ile-Thr- Gly-Pro-His-Glu-Glu-NH 2 ; Noguchi et al. Nature Medicine 2004 10, 305 - 309; doi:10.1038/nm994; Luoma et al. J Neuroscience, 2008, 28(12):3159 -31;
  • the method of culturing chondrocytes comprises the use of a medium to which one or more COX-2 inhibitors are added.
  • COX-2 inhibitors are known to be useful in the treatment of OA and RA (see e.g. Ehrlich, G. E., 1999, Inflammopharmacol. 7:265-267) and several of them are actually marketed for these indications.
  • COX-2 inhibitors have a biphasic effect, where completely blocking COX-2 also would give undesired effects on growth and differentiation of the cells. It thus is proposed that only small doses of COX-2 inhibitors should be given, in order to leave at least some residual COX-2 action. It is thus preferred to consider COX-2 reduction and not total inhibition. It has been established that COX activity is involved in normal bone metabolism and that NSAIDS and COX-2 inhibitors have a negative impact on bone repair (Zhang, X. et al., 2002, J. Clin. Invest. 109:1405-1415).
  • a COX- 2 reducing compound according to the invention may be chosen from the group consisting of celecoxib, rofecoxib, valdecoxib, lumiracoxib, meloxicam, tramadol, etoricoxib, parecoxib, valdecoxib, licofelone, NS398 and sulphonanilides such as nimesulide. Since complete inhibition is not preferred, the concentration in the medium of the invention should not be excessive; and a person skilled in the art is able to choose the appropriate concentration by checking the COL2 expression and general health of the chondrocytes.
  • the medium is also to be used therapeutically (see below), then careful dosing is even more warranted, since it has been established in the mean time that COX- 2 inhibitors have negative effects on the cardiovascular system. If the medium is only applied locally (e.g. intra- articularly) the same considerations would be needed as for in vitro applications: a dose that is sufficient to stimulate COL2 expression, but does not have detrimental effects on the chondrocytes (or any other cells that are present in the treated area). If the medium also is able to reach other parts of the body, then cardiovascular effects should be prevented by minimizing the dose further.
  • the method for proliferation and prevention of dedifferentiation of chondrocytes according to the invention i.e. with a medium as defined above, can be advantageously be applied in methods for autologous chondrocyte implementation (ACI) or matrix-assisted ACI (MACI) such as the CARTICEL® method of GenZyme Corp.
  • ACI autologous chondrocyte implementation
  • MMI
  • the medium as defined above can be used for ameliorating OA or RA, or any other form of arthritis, by intra-articular washing or rinsing.
  • Intra-articular washing of a joint of a subject suffering from arthritis is a commonly used procedure to alleviate arthritic complaints (e.g. Tanaka, N. et al., 2005, Clin. Rheumatol. 25:65-69). It is also possible in such a washing or rinsing protocol to use medium to which (autologous) chondrocytes have been added.
  • the medium of the invention itself, thus, can be used for treatment of arthritic complaints.
  • the medium also comprises chondrocytes, which may be autologous or not, and which may be seeded on a scaffold or not.
  • the medium preferably is injected into the affected joint(s) of a subject, but it may also be applied during surgery for washing or rinsing the joint.
  • Also part of the invention is a method for isolating chondrocytes from cartilage tissue using the medium according to the invention.
  • This isolation is advantageously performed during surgery (e.g. Akens, K. and Hurtig, M. B., 2005, BMC Musculoskeletal Disorders 6:23; Stoop, R. et al., 2007, Arthritis Res. Ther. 9:R60).
  • the invention further comprises the use of a medium according to the invention for the isolation of chondrocytes from cartilage tissue of a subject.
  • chondrocytes may also be derived from progenitor cells, such as mesenchymal stem cells.
  • progenitor cells such as mesenchymal stem cells.
  • osteo-progenitor cells Other cell types that would benefit comprise mammalian bone marrow-derived stromal cells or MSCs when aiming at chondrogenic differentiation (see above) or
  • mesenchymal stem cells or other progenitor cells may be autologous or heterologous. It is also possible to derive suitable cells from yet less frequently used sources such as the ear (elastic) cartilage or rib (costal) or .
  • a subject according to the invention can be a mammal, but said subject preferably is a race horse, a pet animal or, most preferably, a human being. Said subject can be a healthy subject, but it can also be a subject having an arthritic condition such as OA or RAor suffering from osteochondritis dissecans, ..
  • a method for treatment may also apply for the following surgical interventions: (stem) cell-based knee or hip or spine
  • the invention may also improve chondrectomy and debridement or brasion and microfracture surgery.
  • Osteoarthritis as the second leading cause of disability in the elderly population, can be also dealt with by several non- surgical treatments like e.g. using braces or drug therapies, such as anti- inflammatory substances (eg. diclofenac, ibuprofen, and naproxen— which is not very effective) or COX-2 selective inhibitors (see above) which mainly alleviate the pain.
  • anti- inflammatory substances eg. diclofenac, ibuprofen, and naproxen— which is not very effective
  • COX-2 selective inhibitors see above
  • Genzyme Corporation provides the only FDA approved ACI treatment: Carticel— but this treatment is designated for young, healthy patients with medium to large sized (non-OA) damage to cartilage (thus cartilaginous lesions) and not (yet) applicable to OA patients. The reason is that the patient's own chondrocytes (e.g.
  • 1OK— IOOK cells are removed arthroscopically from a non load-bearing area from either the intercondylar notch or the superior ridge of the medial or lateral femoral condyles. This is still difficult to accomplish, but the invention provides for an enahncement in obtaining cells that would be suitable for such a treatment..
  • cells were then grown in vitro at Genzyme for approximately six weeks until reaching 10-12M cells to be implanted in a second surgery under a periosteal flap to hold the cells in place. Approximately 1,500-3,000 Carticel procedures are currently being performed annually (over 10,000 in total) with costs of $20,000-$35,000 ea.
  • CARTICEL II is a second generation procedure using a "fleece matrix" (i.e. a scaffold) into which the grown harvested chondrocyte cells are planted (i.e. MACI).
  • CARTICEL II procedure is about to undergo clinical trials under the supervision of the FDA in the United States.
  • BioTissue Technologies GmbH (Freiburg, Germany) moved the CARTICEL technology forward using a patient's hyaline biopsy to be grown in a 3D resorbable matrix prior to implantation via open or arthroscopic procedures (a modified, simpler MACI, without using a periosteal patch).
  • FAB Fi Advanced Biopolymers
  • Biomaterials and Arthro Kinetics or co.don AG (URL http://www.codon.de/).
  • CHONDROSPHERE a third generation liquid product 3D matrix, which may be optimally benefit from the claimed invention as cell spheroids (lmm diameter) are implanted through a syringe.
  • Autologous mesenchymal stem cell transplantation protocols are currently successfully used in human pilot studies in human knees (about 400 patients, costs > $7,000 ). All aforementioned methods would benefit from the present invention, either by applicability of the medium to isolate cells, to grow cells, or to use as medium in which the matrix is being introduced into the body, as well as for washing and rinsing the area in which surgery is taking place.
  • osteochondral autografting is a technique that requires that the surgeon transplants sections of bone and cartilage. If this employs a bi- phasic scaffold, such a procedure may benefit from the present invention with respect to cartilage (re-)generation. Basically, all three methods of grafting cartilage defects (periosteal grafting, osteochondral grafting (mosaicplasty), and articular cartilage paste grafting) may benefit as the invention has been shown to work on cartilage explants as well [see Fig. 10].
  • spinal disc herniation prolapsus disci intervertebralis
  • fibrocartilagenous, load- distributing annulus fibrosus the nucleus pulposus regenerative surgeries may benefit from the present invention.
  • stem cell therapy may include stem cell therapy and the present invention would also be useful therein.
  • Using injections of cell-suspensions with the medium of the invention may actually work better for all these spinal surgeries than for knee or hip surgeries due to anatomic differences and better concealments.
  • autologous mesenchymal stem cells in animal models were successful in arresting intervertebral disc degeneration.
  • high-end physiological tonicities i.e. 450mOsmo
  • the therapeutic compounds that are optionally present in the medium of the invention can be given in a formulation in which they are normally administered to diseased subjects.
  • it is possible to administer slow release formulations of said compounds e.g. employing injectable microspheres such as
  • TnnoCorc ' s proprietary SynBiosysTM biodegradable multi-block co-polymer platform. This platform forms the basis for the development of long-acting, injectable microparticles for systemic and local delivery of active pharmaceutical ingredients.
  • injectable microspheres with a size of 25-100 ⁇ m, more preferably 50-100 ⁇ m. most preferably 50-75 ⁇ m are to be used, because spheres ⁇ 20um are quickly cleared from the synovia (i.e. joint space fluid) and thus compromising drug release dynamics and very small particles (i.e. 0.5-10 ⁇ m) are known to stimulate unwanted macrophage activation (i.e. inflammation).
  • SynBiosys m polymer platform allows encapsulating small and medium-sized pharmaceutical actives with a wide range of release profiles to create tailored drug-loaded microparticles.
  • Said microparticles (or any other comparable slow-release pharmaceutical carrier) is advantageously used to release calcineurin inhibitors (chosen from the above-mentioned list) and Cox-2 specific inhibitors.
  • the 280 mOsm and 380 mOsm isolations were also performed with cartilage obtained from the femoral condyles and tibial plateau of two non-OA donors (further referred to as normal donors) undergoing above-knee amputation surgery after trauma.
  • HACs Primary (PO), passage 1 (Pl), passage 2 (P2) and passage 3 (P3) HACs were monolayer expanded in medium corresponding to their isolation tonicity (280 mOsm, 380 mOsm, 480 mOsm or 580 mOsm), with an initial seeding density of 6,000 cells/cm2.
  • HACs Primary HACs were cultured for expansion in monolayers at a seeding density of 7,500 cells/cm2 in medium corresponding to their isolation tonicity (280 mOsm, 380 mOsm, 480 mOsm or 580 mOsm).
  • PO cells to P3 cells were seeded in high-density monolayers (20,000 cells/ cm2) and were cultured for an additional 5 days and 7 days before analysis of mRNA (quantitative RT-PCR) and protein expression (Western blotting), respectively.
  • NMDG-Cl iV-methyl-d-glucamine chloride
  • lentiviral vectors for nontransient shRNA-mediated gene silencing in primary chondrocytes.
  • Bam ⁇ I/MunI restriction fragments of the parental pLKO.l-puro vector - each containing the U 6 promotor and one out of five different, sequence-verified antihuman NFAT5 shDNAs (MISSION shRNA library) - were subcloned into corresponding restriction sites of recipient vector pRRL.PPT.PGK.GFPpre.
  • Lentiviral particles were produced in HEK293T cells by transient transfection using a calcium
  • TRCN0000020019 to 23 for humans and TRCN0000085644, TRCN0000085647 for mouse both from Sigma-Aldrich, Mission shRNA library; http://www.sigmaaldrich.com/life-science/functional- genomics-and-rnai/shrna/shrna-search-and-order.html was identified as the best performing anti-NFAT5 shRNA clone by quantitative PCR-based knockdown efficiency determination, and was used in subsequent experiments.
  • Pl OA HACs from two donors were seeded (15,000 cells/cm2) and cultured for 4 days in control medium (280 mOsm). Three hours prior to transduction, cells were deprived of antibiotics, and then were transduced for ⁇ 18 hours, refreshed with control medium with antibiotics and cultured for an additional 4 days before harvesting for fluorescence-activated cell sorting (FACS) analyses. Cells were resuspended in PBS with 10% FCS and antibiotics, and were washed. Cells were collected and stained with Hoechst 33258 (1 mg/ml; Molecular Probes/Invitrogen Corp., Carlsbad, CA, USA) to discriminate between dead cells and live cells.
  • FACS fluorescence-activated cell sorting
  • FACS was performed on the FACSAria (Becton Dickinson BV, Breda, The Nederlands), and eGFP-expressing cells were collected (>50%, multiplicity of infection ⁇ 1) and reanalyzed for purity (>95%) using Cell Quest Pro Software (Becton Dickinson Biosciences BV, Breda, The Nederlands).
  • the eGFP-expressing populations were seeded (10,000 cells/cm2) and cultured in control medium up to 80% confluency. Cells were then switched to medium of 380 mOsm or were kept on control medium for 24 hours prior to RNA analysis.
  • RNA isolation, purification, quantification and cDNA synthesis are described elsewhere (Uitterlinden, A.J. et al.,2006, Osteoarthritis Cartilage 14:250-257).
  • Expression levels of AGCl, SOX9 and COL2 were studied as chondrogenic markers, while COLl was studied as a dedifferentiation marker.
  • Quantitative PCR assays for COL2, SOX9, AGCl and COLl have been reported earlier (Mandl, E.W., supra).
  • concentration was quantified by the bicinchoninic acid assay according to the manufacturer's protocol (#23225; Thermo Fisher ScL, Rockford, IL, USA). Aliquots (10 ⁇ g) were subjected to 10% SDS-PAGE prior to electroblotting onto nitrocellulose membranes (Protran BA83; Schleicher & Schuell BV, s- Hertogenbosch, The Netherlands).
  • Blots were blocked in 5% low-fat dry milk in Ix PBS, 0.05% v/v NP-40, were incubated with primary antibodies - anti-type II collagen and anti-type I collagen, both 1:100 (SouthernBiotech, Birmingham, Alabama, USA), or 1:10,000 anti- ⁇ -Tubulin (Sigma) - were washed, were incubated with secondary antibodies (both 1:1,000; Dako Cytomation,
  • OA HACs isolated at 480 mOsm showed severely inhibited proliferation compared with cells at 280 mOsm and 380 mOsm (Table 1).
  • COL2/COL1 ratio during chondrocyte expansion (Figure Id), from sevenfold in PO cells to 100-fold in expanded P3 cells.
  • Physiological tonicity also upregulated COL2 protein expression (Figure Ie): levels significantly increased (between 1.5-fold and 2.2-fold) in PO, Pl and P2 chondrocytes.
  • Physiological tonicity also significantly increased AGCl (Figure 2a) and SOX9 (Figure 2b) mRNA levels in nonosteoarthritic human articular chondrocytes (NHACs).
  • NHACs nonosteoarthritic human articular chondrocytes
  • COL2 mRNA levels were significantly upregulated, from 5.8-fold in PO cells to 270-fold in expanded P3 NHACs ( Figure 2c).
  • hypertonicity also downregulated COLl expression with increasing passage number in NHACs: the COL2/COL1 ratios increased during expansion (Figure 2d), from 6.8-fold in PO cells to 355-fold in expanded P3 cells.
  • Hypertonicity activates NFAT5 in human articular chondrocytes
  • NFAT5 knockdown inhibits hypertonicity -induced chondrogenic marker expression
  • NFAT5 RNAi also downregulated chondrogenic markers: AGCl by 80%, SOX9 by 32% and COL2 by 84%, as compared with non-RNAi controls ( Figure 4b).
  • Human articular cartilage was explanted from macroscopically normal areas of the femoral condyles and tibial plateau of 4 patients undergoing total knee replacement surgery for OA.
  • Human articular chondrocytes were isolated under standard culture conditions (280 mOsm) and elevated medium tonicity (380 m ⁇ m).
  • Primary HACs were culture expanded in monolayer (7,500 cells/cm 2 ) at both isolation tonicities (280 or 380 mOsm).
  • Alkaline phosphatase activity in human chondrocytes was determined using the Oolorimetrie Assay Kit (ab$3369; Abeam pic, 330 Cambridge Science Park, Cambridge, CB4 OFL, UK) according to the manufacturer's instructions.
  • ALP activity of the test samples were then calculated as relative activity and corrected for the amount of DNA per culture well. For reference see: Bessej', O.A., Lowry O. H. and Brock M.J.:(1946) J.Biol. Chem. 164 321).
  • MMP-13 activity MMP- 13 activity in chondrocytes was determined using the SensoLyte® Plus 520 MMP-13 Assay Kit from AnaSpec (AnaSpec, 34801 Campus Drive, Fremont, CA 94555) according to the manufacturer's guidelines.
  • the kit is designed to specifically detect MMP-13 activity in culture medium, from which MMP-13 is captured by immobilized MMP-13 antibody, and its proteolytic activity is measured by 5-FAM/QXLTM520 FRET peptide.
  • the fluorescence of 5-FAM fluorophore
  • QXLTM520 quencher
  • Cell culture media were collected and centrifuged for 15 min at 1000xg, at 4°C and stored at -70 0 C until use.
  • MMP-13 standard (10 ⁇ g/mL, Component B) was diluted 1:50 in MMP dilution buffer (Component C) to get 200 ng/mL and six 1:2 serial dilutions were then prepared in MMP dilution buffer, next to a blank control. Hundred ⁇ L sample/well, MMP-13 standards, and blank control were transferred to the anti human MMP-13-coated microplate, covered and incubated on a shaker (at room temperature for 2h.
  • physiological tonicity significantly increased mRNA levels of important anabolic markers such as AGCl, SOX9 and COL 2 compared to standard culture.
  • important anabolic markers such as AGCl, SOX9 and COL 2 compared to standard culture.
  • FK506 dose-dependently increased this expression even further: SOX9 was induced 6-times, COL2 increased to 50-fold in Pl chondrocytes, COL2 protein 14-fold and AGCl up to 2.6-times.
  • unwanted COLl mRNA expression was suppressed to 50% under these conditions.
  • expression of anti-catabolic genes such as TIMP-I and -2 significantly increased 4-times by 380 mOsm and FK506.
  • Physiological tonicity provides a simple, yet effective, means to improve marker expression during cytokine-free isolation and in vitro expansion of human articular chondrocytes. Compared to FK506 alone, elevated tonicity significantly improved chondrogenic marker expression. Combining FK506 and elevated tonicity significantly improved chondrogenic marker expression even further, while suppressing the few tonicity-induced catabolic and hypertrophic marker genes and their activities where measured As a major component of cartilage's extracellular matrix, the proteoglycan aggrecan imparts compressive resistance to the tissue. Although aggrecan theoretically can be cleaved by several members of ADAMTS family of metalloproteases (i.e.
  • explants were taken from human osteoarthritic articular cartilage from macroscopically normal areas of the femoral condyles and tibial plateau using a 6 mm diameter dermal biopsy punch, and separated from the underlying bone by dissection with a scalpel. After dissection, the explants were all pooled. For each condition, two explants were randomly taken and cultured for 3 days in a 24-well plate with 330 ⁇ l medium per explant. The explants were cultured in control medium (280 mOsm) or hypertonic medium (380 mOsm), with or without addition of 50 ng/mL FK506 for 24 hours, 7 and 14 days. After ex vivo culture, the cartilage explants were snap-frozen in liquid nitrogen and then processed in a Mikro- Dismembrator S (B. Braun Biotech International GmbH, Melsoder.
  • Ex vivo cultured cartilage tissue explants basically showed the same trends in marker gene regulation as isolated chondrocytes under the same conditions (see Fig. 9). Also here, explants which were cultured for 3 days in medium of physiological tonicity (380 mOsm) showed only a modest down-regulation in COLl and mild up-regulation of Sox- 9 and COL2 when compared to control medium (Fig. 10). Interestingly, physiological tonicity slightly increased mRNA levels of hypertrophic markers COLlO and MMP- 13, but not alkaline phosphatase (ALPL). However, in combination with FK506 (50ng/uL) all unwanted markers (COLl; ALPL, COLlO and MMP-13) were down-regulated to or below control levels. In addition, chondrogenic markers (Sox-9, AGCl and COL2) were profoundly up-regulated after addition of FK506 (Fig. 10).
  • Physiological tonicity especially in combination with FK506 provides a simple, yet effective, means to also improve marker gene expression during ex vivo culture of human articular cartilage explants.
  • Combining FK506 and elevated tonicity significantly improved chondrogenic marker expression, while suppressing hypertrophic marker genes.
  • ADCo chondroger ⁇ e cells
  • teratocarcinoma cells mature m vitro to become hypertrophic and deposit a mineralized extracellular matrix and represent a robust model for
  • the c ⁇ lls are a good model to study chondrogenic differentiation in comparison to clinically relevant human articular
  • chondrocytes or adult mesenchymal stem cells from human bone marrow (Tare et. al. 2005 (European Cells Materials 10 suppl. 2, ISSN 1473-2262).
  • ATDC5 differentiate into chondrocytes and eventually hypertrophic
  • chondrocytes in the presence of insulin, transferring (iron transporter) and sodium selenite (antioxidant).
  • Cells were cultured as described by Atsumi et al. 1990, Cell Differentiation Development vol. 30 (2): 109-116). In brief, cells were seeded at 6,400 cells/cm 2 and propagated in proliferation medium
  • ⁇ DMEM F12+ L-glutamine (GIBCO 32500-043), 5% FCS (PAA A15-101), 1% antibiotic/antimycotic solution (Invitrogenl5240-062), 1% non-essential amino acids (Invitrogen 11140-035) until starting differentiation by adding 1:500 insulin (lO ⁇ g/ml, Sigma T2643), 1:3000 transferrin (lO ⁇ g/ml, Roche
  • 10652202001 and 1:33333 sodium selenite (30 nM, Sigma S9133).
  • Cells were cultured in standard medium ( ⁇ 280mOsm) and tonicity was increased by adding sterile NaCl as described for human HACs.
  • GAG staining and measurements ATDC5 cells (e.g. IxIO 5 cells/well in 12 multi-well plate) were rinsed twice with ice-cold 0.9% (w/v) NaCl and then fixed 10 min in 4 % formaldehyde, prior to washing in demineralized water.
  • the dye was then extracted with 1 ml of 10 % (v/v) acetic acid/water and incubated for 15 min at room temperature with gentle agitation, transferred into in 96 well plates (200 ⁇ l each) and measured at 590 nm in a spectrophotometer against a blank (10% acetic acid/water).
  • MmNFATc2 (NM_010899) FW, GGAGAACTGGAGAAACTGCG; Rv,
  • MmNFATc4 (NM_023699) Fw, TGGAAGGGACTAAGGGTGTG; Rv,
  • TGTGGCAGATACAGATCAAGC iVIarkiewicz «t a I. 2007, . J, Dermatol. SoL 47:217-226) and MmCol2 (NM_031163) Fw, GATGACATTATCTGTGAAG; Rv ATCTCTGATATCTCCAGG; MmCoIlO (NM_0099255) Fw
  • CTTTGTGTGCCTTTCAATCG 3 Rv GTGAGGTACAGCCTACCAGTT; and MmCOX-2 (NM..01H98) Fw CAGGAAGTCTTTGGTCTGGTGCC, Rv
  • GCTGGTTTGGAATAGTTGCTCATCA All primers are listed according to the convention in ⁇ ' - 3 ' direction. Data were normalized and relative expression calculated as described for human cells, but 62-microglobin (mB2MG) expression was used as an interna] control.
  • Protein analyses were essentially identical to those performed for human cells, using the same protocol, buffers, antibodies and dilutions as above.
  • Results represent the mean ⁇ standard deviation.
  • ATDC5 cells express all NFAT family members with increasing NFAT5 expression towards the end of culture.
  • culturing ATDC ⁇ s at 380mOsm decreased expression of COLl, but further increased that of COL2, COLlO and Cox-2.
  • using 480mOsm culture medium resulted in a decreased expression of all these markers to or below control levels (day 14, 280mOsm).
  • 380mOsm increased the proteoglycan (GAG) production by ATDC ⁇ s (Fig. 14), while 480mOsm suppressed it to below control levels.
  • GAG proteoglycan
  • ATDC ⁇ s are proliferating optimally and equally well at both 280mOsmo and 380mOsmo, respectively.
  • 480mOsmo decreased the proliferation rate by 50% (Fig. 14).
  • ATDC ⁇ s produced more GAGs per cell (ratio GAG staining/proliferation assay) than in any other condition.
  • 480mOsm proofed to be sub-optimal in this respect.
  • collagen production Fig. 12
  • Fig. 13 the same trend was seen as for mRNA synthesis
  • Physiological tonicity (380mOsm) not only improved chondrogenic marker gene expression, while suppressing hypertrophic markers, in human adult chondrocytes but also in murine chondrogenic ATDC5 cells. Again, culturing these cells at 380mOsm proofed to be better than using 480mOsm.
  • ATDCo cells were incubated with a combination of physiological tonicity and a Cox-2 inhibitor (NS39S: N194, Sigma).
  • Physiological tonicity provides an effective means to also improve marker expression in ATDC5 cells. Adding NS398 to 380mOsm improves the most important chondrogenic marker COL2 on mRNA and protein level, while it may also increase COLl mRNA levels. Therefore, addition of FK506 might help to prevent induction of hypetrophic markers (as shown for HACs), while (modest) inhibition of NS398 might improve expression of chondrogenic markers.

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Abstract

L'invention concerne la prolifération et la prévention de la dédifférenciation de chondrocytes, qui peuvent être avantageusement mises en œuvre, en culture in vitro, par un procédé comprenant la culture desdites cellules dans un milieu dont le niveau de tonicité est physiologique. De préférence, ledit milieu a une tonicité de 350 — 480 mOsm, mieux encore une tonicité d'environ 380 mOsm. Dans un autre mode de réalisation, ledit niveau physiologique de tonicité est également appliqué pendant l'isolement des cellules. Avantageusement, la prolifération des cellules dans ce procédé comprend le passage (sous-culture) des cellules. Dans un mode de réalisation préféré du procédé de l'invention, les chondrocytes sont isolés à partir d'un sujet souffrant d'ostéoarthrite ou de polyarthrite rhumatoïde. Mieux encore, des inhibiteurs de calcineurine et/ou des inhibiteurs de COX-2 sont ajoutés audit milieu. Le milieu peut être ensuite utilisé dans tous les cas dans lesquels une réparation chondrocytique in vivo est nécessaire, et donc également avec des protocoles ICA et ICAM.
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CN114540285A (zh) * 2017-06-25 2022-05-27 科.东股份公司 用于制备可移植性软骨组织的方法

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* Cited by examiner, † Cited by third party
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CN106083892A (zh) * 2016-06-13 2016-11-09 杭州中美华东制药有限公司 高纯度他克莫司化合物及其制备方法
CN106083892B (zh) * 2016-06-13 2019-02-19 杭州中美华东制药有限公司 他克莫司化合物及其制备方法
CN114540285A (zh) * 2017-06-25 2022-05-27 科.东股份公司 用于制备可移植性软骨组织的方法

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