US20070212389A1 - Use of a Hydrogel for the Culture of Chondrocytes - Google Patents

Use of a Hydrogel for the Culture of Chondrocytes Download PDF

Info

Publication number
US20070212389A1
US20070212389A1 US10/577,942 US57794204A US2007212389A1 US 20070212389 A1 US20070212389 A1 US 20070212389A1 US 57794204 A US57794204 A US 57794204A US 2007212389 A1 US2007212389 A1 US 2007212389A1
Authority
US
United States
Prior art keywords
hydrogel
chondrocytes
hpmc
hec
cells
Prior art date
Legal status (The legal status 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 status listed.)
Abandoned
Application number
US10/577,942
Other languages
English (en)
Inventor
Pierre Weiss
Jerome Guicheux
Guy Daculsi
Gael Grimandi
Claire Vinatier
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Universite de Nantes
Institut National de la Sante et de la Recherche Medicale INSERM
Original Assignee
Universite de Nantes
Institut National de la Sante et de la Recherche Medicale INSERM
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 Universite de Nantes, Institut National de la Sante et de la Recherche Medicale INSERM filed Critical Universite de Nantes
Assigned to UNIVERSITE DE NANTES, INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE (INSERM) reassignment UNIVERSITE DE NANTES ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: DACULSI, GUY, GRIMANDI, GAEL, GUICHEUX, JEROME, VINATIER, CLAIRE, WEISS, PIERRE
Publication of US20070212389A1 publication Critical patent/US20070212389A1/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • 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
    • 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
    • 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
    • A61L27/3839—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 the site of application in the body
    • A61L27/3843—Connective tissue
    • A61L27/3852—Cartilage, e.g. meniscus
    • 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
    • A61L27/3895—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 using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/30—Synthetic polymers

Definitions

  • the invention relates to the use of a cellulose hydrogel for the three-dimensional culture of chondrocytes, for the implantation thereof in a cartilaginous site.
  • Cartilage is a specialized non-vascularized, non-innervated strong and resilient connective support tissue. It is present in the ribs, the sternum, the nose and ears, but also in the joints. Cartilage contains specialized cells known as chondrocytes and an extracellular matrix (ECM) basically consisting of collagen fibers and proteoglycans.
  • ECM extracellular matrix
  • the articular cartilage may be the seat of numerous changes of inflammatory origin (rheumatoid arthritis, osteoarthritis), of traumatological origin or related to ageing (arthrosis).
  • the establishment of these cartilage pathologies results, over the more or less long term, in degradation of the extracellular matrix and a decrease in cellularity.
  • the absence of vascularization and chondrocyte proliferation imparts to this tissue low repair capacities, which render these catabolic processes irreversible.
  • these degenerative pathologies now affect a significant proportion of the population and are therefore a major public health issue.
  • the scientific community has for a number of years been interested in means for regenerating a functional cartilaginous tissue.
  • Brittberg et al. were the first to propose a repair method based on the transplantation of autologous chondrocytes (Brittberg et al., 1994). This method takes place over three stages: firstly, a cartilage fragment is removed from a non-load-bearing zone in order to isolate chondrocytes, which are then multiplied in vitro as a monolayer, then reimplanted below a periosteal flap of the lesion. The results revealed repair of the cartilaginous defect, but the patients have to undergo two intensive surgical procedures. Furthermore, the phenotype of the cells after the monolayer amplification thereof still has to be determined.
  • Polymers, such as polylacetic acid, polyglycolic acid, carbon fibers, polyester urethane, Dacron and Teflon, have been proposed and used in vitro for the 3D culture of chondrocytes or mesenchyme cells. Some of these synthetic polymers have caused immune responses or inflammation after the implantation thereof in a cartilaginous site (Cancedda et al., 2003).
  • Injectable hydrogel compositions are described, in particular, in U.S. Pat. No. 6,129,761.
  • the inventors have now demonstrated the benefit, for implantation in a cartilaginous site, of the three-dimensional cultivation of chondrocytes within a hydrogel self-crosslinking as a function of pH.
  • the hydrogel used is a hydrogel which self-crosslinks as a function of pH and consists of an aqueous hydroxyethylcellulose (HEC) or hydroxypropylmethylcellulose (HPMC) solution onto which are grafted silane groups which allow the formation of covalent bonds between the HEC or HPMC chains.
  • HEC hydroxyethylcellulose
  • HPMC hydroxypropylmethylcellulose
  • This material preferably consists of a polymer of simplified formula: (HEC or HPMC)-O—X—Si(OZ) 3 which may be obtained by the reaction of hydroxyethylcellulose (HEC) or hydroxypropylmethylcellulose with a compound of formula (I): XSi(OZ) 3 (1) wherein X represents a halogen atom or a hydrocarbon group having an epoxy function, in particular C 2-20 , and Z is selected from among a hydrogen atom, an alkali metal and an alkyl group, in particular C 1-5 .
  • the compound of formula (I) may be, for example, (3-glycidoxypropyl)trimethoxysilane:
  • the organosilicon compound grafts onto the HEC or the HPMC, opening the epoxide, and the methoxysilane groups are hydrolized to produce a polymer of simplified formula:
  • the polymer is preferably a silanized HPMC polymer.
  • This polymer is stable in aqueous solution at a pH greater than or equal to approximately 12.4. Acidification of the solution causes a gradual increase in viscosity and the formation of a hydrogel.
  • This physical phenomenon corresponds to the crosslinking of the polymer by (i) transformation of the silanolate groups into silanol groups: SiO—Na + ⁇ SiOH then formation of a three-dimensional network by (ii) condensation of one silanol on another silanol: (HEC ou HPMC) SiOH+(HEC+HPMC) SiOH ⁇ (HEC ou HPMC) SiOSi (HEC ou HPMC) and/or (iii) condensation of a silanol on a hydroxyl group of the cycles of the cellulose ethers or the substituents: SiOH+(HEC or HPMC)-ROH ⁇ (HEC or HPMC) SiOR-(HEC or HPMC)
  • the synthesized polymer may be in the form of a powder which may be dissolved in an alkaline sodium hydroxide solution.
  • the gelation pH is between 7 and 12, depending on the desired crosslinking rate.
  • the gel obtained may be sterilized by autoclaving (for example, at 121° C. for 20 minutes).
  • the polymer in basic aqueous solution, before crosslinking, may also be mixed, prior to its biological use, with a physiological buffer solution.
  • the pH of the final mixture is thus adapted to the desired handling time prior to crosslinking of the overall mixture.
  • This crosslinking time may be measured by oscillatory rheometry and may vary from 5 minutes to 5 days as a function of the parameters of this mixture—mainly the pH and the temperature (Bourges et al., 2002a; Bourges et al., 2002b).
  • the side silicon carrier groups, of the silanolate or alkali metal or ammonium silanolate precursor type represent from 0.5 to 5% of the total dry weight of the polymer.
  • the invention relates to the use of a silanized HEC or silanized HPMC hydrogel of this type, self-crosslinking as a function of pH, for the three-dimensional ex vivo culture of chondrocytes.
  • the hydrogel used has, in particular, the following advantages:
  • the cells cultured in the hydrogel are preferably human or animal chondrocytes, preferably autologous chondrocytes, of patients having a lesion of the cartilage. These cells may, for example, be removed from a cartilaginous tissue, for example from a portion of the articular cartilage or from the nasal cartilage.
  • the cells cultured in the hydrogel may also be undifferentiated cells capable of chondrogenic differentiation.
  • Mesenchymate stem cells or stem cells originating from fatty tissue are thus included.
  • the former may, in particular, be obtained by removing bone marrow (typically by suction, using a syringe, from the trabecular bones such as long bones, iliac crest), the latter by liposuction (typically by suction under vacuum of fatty masses).
  • chondrocyte phenotype preferably a culture in conventional culture media (DMEM or MEM), to which are added glutamine and antibiotics, if required, and, preferably autologous, animal or human serum, or serum substitutes, and also chondrogenic additives.
  • DMEM conventional culture media
  • glutamine and antibiotics if required
  • autologous, animal or human serum, or serum substitutes if required
  • chondrogenic additives include, in particular, insulin, transferrin, sodium selenite, ascorbic acid, TGF-beta, IGF-1, BMP (bone morphogenetic protein), dexamethasone, linoleic acid, BSA (bovine serum albumin) or pyruvate.
  • the culture is preferably produced under moist conditions at 37° C. in the presence of oxygen concentrations which may vary as required. Hypoxia has been found to be an important factor for the development of a chondrocyte phenotype.
  • the invention also relates to an ex vivo process for the preparation of a complex of cells integrated in a hydrogel, the complex being intended to be injected into a cartilaginous site, wherein said process includes the ex vivo mixing of chondrocytes or of undifferentiated cells, capable of chondrogenic differentiation with a silanized HEC or HPMC hydrogel, crosslinking as a function of pH, in a biological buffer at an appropriate pH for the crosslinking of the hydrogel, under appropriate conditions and for an appropriate period for the integration and the three-dimensional culture of the chondrocytes, optionally derived from the differentiation of said undifferentiated cells, in the hydrogel.
  • a person skilled in the art may use any biological buffer. Examples include the phosphate buffer (PBS, phosphate buffered saline), HEPES or TRIS buffer. Any biological medium known to a person skilled in the art, for example DMEM medium or alpha-MEM medium (alpha minimum essential medium), may also be used.
  • PBS phosphate buffer
  • HEPES phosphate buffered saline
  • TRIS buffer TRIS buffer
  • Any biological medium known to a person skilled in the art for example DMEM medium or alpha-MEM medium (alpha minimum essential medium), may also be used.
  • the process includes the following ex vivo steps:
  • the rheological properties of the hydrogel consisting of silanized HEC or HPMC allow it to be injected at the implantation site, thus promoting the vectorization of the chondrocytes by non-invasive surgery.
  • the hydrogel within which the chondrocytes were cultivated may be implanted in a site of the lesion normally occupied by cartilage.
  • the targeted lesions may be losses of focal cartilaginous substances related to traumatological sequelae or, more generally, any osteoarticular or plastic cartilaginous pathologies or losses.
  • the invention may be applied to the engineering of all of the cartilaginous tissues, i.e. of the following types:
  • the intervertebral disc is a fibrocartilaginous structure consisting of four concentric tissues disposed between the adjacent vertebral bodies.
  • the outer layer is the outer fibrous ring, which consists of markedly oriented collagen fibers.
  • the inner fibrous ring has a lower collagen density and is less organized than the outer ring.
  • the transition zone is a thin zone of fibrous tissue separating the inner ring from the nucleus pulposus, a gelatinous central zone forming a nucleus.
  • the intervertebral discs are hypovascularized and have limited innervation.
  • the discs are fed by the diffusion of nutriments and metabolites.
  • the discs contain relatively few cells embedded in an abundant extracellular matrix consisting mainly of water, proteoglycans, collagen and non-collagenic proteins.
  • the cells of the disc synthesize these macromolecules and maintain and repair this extracellular matrix.
  • the chondrocyte cells are the only cells of the nucleus.
  • the inner fibrous ring, the transition zone and the cartilaginous plate of the adjacent vertebral bodies also contain chondrocytes, whereas the outer ring contains mostly fibroblast-type cells.
  • a specific degree of disc degeneration occurs in all individuals. A clear association has been revealed between the age and the degeneration of the discs. Degeneration has been observed from the second decade of life onward. Persons having symptomatic discs experience acceleration of the normal ageing process, owing to both acquired and genetic factors.
  • the invention therefore also relates to a process for the treatment of the human or animal body, including the administration by injection of hydrogel, as defined above and previously colonized ex vivo by chondrocytes, for example using the process as described above.
  • the injection may be carried out using a system comprising a sterilizable syringe and connection pieces provided with single-use plungers, for example the system sold by HAWE NEOS DENTAL, comprising a syringe sterilized by autoclaving (ref. No. 440, Syringe Hawe-Centrix C-R R , Mark III) and connection pieces (ref. No. 445).
  • FIG. 1A to 1 C illustrate the MTS activity of the chondrocytes cultivated for 24, 48 and 72 hours under control conditions (absence of hydrogel), in contact with the si-HPMC hydrogel, or in the presence of actinomycin D (5 ⁇ g/ml).
  • SW1353 FIG. 1A
  • C28/12 FIG. 1B
  • human nasal chondrocytes FIG. 1C . *p ⁇ 0.001 compared to the control at each stage.
  • FIG. 2A to 2 C illustrate the count of the chondrocytes cultivated for 24, 48 and 72 hours under control conditions (absence of hydrogel), in contact with the si-HPMC hydrogel, or in the presence of actinomycin D (5 ⁇ g/ml).
  • SW1353 FIG. 2A
  • C28/12 FIG. 2B
  • human nasal chondrocytes FIG. 2C . *p ⁇ 0.001 compared to the control at each stage.
  • the syntheses were carried out on quantities of 240 grams of HPMC.
  • the selected polysaccharide was E4M®.
  • the synthesis was carried out using glycidoxypropyltrimethoxysilane (GPTMS) in a 6-litre flask in a heterogeneous medium in an organic solvent (Bourges et al., 2002). The synthesis was carried out while boiling for 3 hours.
  • the silanized HPMC powder was firstly oven-dried for 1 night, then lyophilized (Christ Alpha 1-4 ST).
  • Si-HPMC powder 6 grams were solubilized in 200 ml of a 0.2 M NaOH solution. This mixture was stirred for 48 hours. The Si-HPMC solution was then dialyzed for 16 hours in a 0.09 M NaOH solution. A second dialysis was then carried out in a 0.09 M NaOH solution for 2 hours. This 3% Si-HPMC solution was then aliquoted and steam-sterilized (121° C., 30 minutes).
  • the hydrogel used for the cell culture was prepared extemporaneously by mixing, under a laminar flow hood, 7.5 ml of Si-HPMC solution with 7.5 ml of HEPES buffer, with 10% of FCS being added.
  • the HEPES buffer was prepared by dissolving 3.1 g of HEPES and 0.8 g of NaCl in a 0.03 M HCl solution. This solution was then aliquoted and steam-sterilized (121° C., 30 minutes).
  • This HEPES/Si-HPMC mixture was vortex-stirred for 15 seconds, then centrifuged at 1200 rpm for 2 minutes. This mixture was transferred to the culture plate. The culture medium was added 1 hour later.
  • the inventors measured two rheological parameters: the dispersive module G′′ (characteristic of the liquids) and the conservative module G′ (characteristic of the solids).
  • This measurement was carried out using a rheometer (Rhéostress 300, Thermo Haake, Düsseldorf, Germany) provided with a planar cone having a diameter of 60 mm and an angle of 1° (C60/1 titan, planar cone, Thermo Haake).
  • the gap between the truncation and the plate was 0.053 mm.
  • the measurement was carried out in oscillatory mode with 3 oscillation frequencies (1 Hz, 3.2 Hz and 10 Hz) and an imposed stress of 1 pascal, at a temperature of 37° C. and for 1,200,000 seconds (13.8 days). The results are expressed in Pa.
  • the hydrogel has the characteristics of a viscous liquid during the preparation thereof. During its crosslinking, its physical characteristics develop toward those of a gel.
  • the hydrogel prepared according to the preferred embodiment, presented in Example 1, consists, once the crosslinking process has been completed (10 days), of 1.5% of dry polymer and 98.5% of water—a water content comparable to that of cartilage.
  • the calculated mesh size of the hydrogel (0.22 ⁇ m) appears to be less than the size of a cell.
  • the inventors used the human chondrocyte lines SW1353 and C28/I2, derived from a human chondrosarcoma (Mengshol et al., 2000) and from human costal cartilage immortalized by the T antigen of the simian virus 40 (Goldring et al., 1994). These cells were cultivated in a DMEM and Ham's F12 volume/volume mixture, to which were added 10% of FCS, 1% of penicillin/streptomycin and 1% of L-glutamine (complete DMEM/F12), under a moist atmosphere at 37° C. and containing 5% CO 2 .
  • the culture medium was renewed in its entirety every other day. Once the cells had reached 80 to 90% of the confluence, they were treated with 2 ml of trypsin (0.025%)/EDTA (0.01%). Following incubation for 3 minutes at 37° C., the trypsin/EDTA solution was collected and centrifuged (8 min, 1200 rpm) in the presence of 8 ml of complete DMEM/F12 medium. Following elimination of the supernatant, the cell pellet was reconstituted with 10 ml of complete DMEM/F12. The cells were then counted and distributed over 25 cm 2 flasks having a density of 10,000 cells/cm 2 .
  • Human nasal cartilage was removed, having obtained the informed consent of patients undergoing reconstructive rhinoplasty.
  • the nasal cartilage washed five times in Hank's balanced salts (HBSS) and cut up into chips under a laminar flow hood.
  • the cartilage chips were then incubated for 30 minutes at 37° C. in HBSS containing 1 mg/ml of protease.
  • the chips were then rinsed three times in HBSS, then incubated for 4 hours, with stirring, in complete DMEM (10% FCS, 1% penicillin/streptomycin and 1% L-glutamine) containing 0.625 mg/ml of collagenase.
  • complete DMEM (10% FCS, 1% penicillin/streptomycin and 1% L-glutamine
  • the pellet washed once in HBSS, centrifuged (8 min, 1200 rpm) and re-suspended in 10 ml of complete DMEM.
  • the cells were counted and transferred into a 25 cm 2 flask with a cell density of 10,000 cells/cm 2 .
  • the cells were cultured in a moist atmosphere at 37° C. with 5% CO 2 .
  • the culture media were renewed every other day. Once the cells had reached 80 to 90% confluence, they were treated using trypsin/EDTA as described above.
  • the chips underwent a final step of digestion by incubation for 15 hours in a complete DMEM solution enriched with 0.03% of collagenase.
  • the digestion product was filtered through a cell screen, collected and centrifuged (8 min, 1200 rpm).
  • the cell pellet was re-suspended in 20 ml of complete DMEM.
  • the cells were counted and transferred into a 25 cm 2 flask with a cell density of 10,000 cells/cm 2 .
  • the cells were cultured in a moist atmosphere at 37° C. with 5% CO 2 .
  • the culture media were renewed every other day. Once the cells had reached 80 to 90% of the confluence, they were treated with trypsin/EDTA as described above.
  • the human primary chondrocytes the SW1353 and C28/I2 cells
  • the cells were distributed over 24-well culture plates with a density of 10,000 cells per cm 2 .
  • the cells were cultivated in the presence (500 ⁇ l of Si HPMC per well) or in the absence (500 ⁇ l of complete medium) of hydrogel for 24, 48 and 72 hours.
  • Cells cultivated in the absence of hydrogel were also treated with actinomycin D (5 ⁇ g/ml) or the excipient thereof (DMSO) to provide a positive cytotoxicity control.
  • This colorimetric test measures the capacity of the mitochondria of the living cells to oxidize the tetrazolium salt MTS in formazan.
  • the colored product formed is proportional to the dehydrogenase activity of the mitochondria.
  • the absorbance measurement therefore allows the cell viability to be quantified.
  • the cells were washed with complete medium and incubated for 1 hour at 37° C. in the presence of 100 ⁇ l of MTS reagent containing 48 ⁇ g/ml of PMS and 2 mg/ml of MTS.
  • the absorbance measurement at 490 nm was carried out by a microplate reader (MRX, Dynatech Laboratories, VWR International). The results are expressed as a percentage of MTS activity relative to the control conditions (cells cultivated in the absence of hydrogel).
  • the culture medium with or without hydrogel was drawn up and replaced by 200 ⁇ l of trypsin/EDTA.
  • the cells were incubated for 2 minutes in a moist atmosphere at 37° C. containing 5% CO 2 .
  • the cells were then collected and centrifuged (8 min, 1200 rpm) in the presence of 2 ml of complete culture medium.
  • the pellet was re-suspended in 2 ml of complete culture medium and the cells were counted after vital staining with a trypan blue solution (0.04% in PBS) on a Malassez cell. The results are expressed in the total number of cells per well.
  • the inventors studied by RT/PCR the expression of specific markers of the chondrocyte phenotype after isolation and during a monolayer culture. This study revealed that the freshly isolated nasal chondrocytes (P0) expressed collagen II, aggrecan and collagen X, but also collagen I. During the various passes, the inventors observed a modification of the expression of these various genes. The expression of type II collagen decreased between P0 and P3. The expression of aggrecan dropped between P0 and P1. Collagen X was slightly expressed by the freshly isolated chondrocytes, its expression was no longer detectable at P3. The expression of type I collagen, conversely, increased markedly between P2 and P3.
  • the inventors In order to determine the cytotoxic effect of the hydrogel on the chondrocytes, the inventors analyzed the MTS activity on the SW1353 and C28/I2 cell lines and on the human nasal chondrocytes after 24 hours, 48 hours and 72 hours of culture in contact with the Si-HPMC hydrogel.
  • the inventors carried out a cell count by means of trypan blue staining. This staining allowed the living cells (white) to be distinguished from the dead cells (blue). The count was carried out after 24 hours, 48 hours and 72 hours of culture on contact with the hydrogel for the SW1353 and C28/I2 cell lines and for the human nasal chondrocytes ( FIG. 2A to 2 C).
  • CTG cell tracker green
  • EthD-1 ethidium homodimer 1
  • CTG produces a green fluorescence in living cells.
  • EthD-1 is a DNA intercalating agent, the fluorescence of which increases by 40 times after binding to nucleic acids. It penetrates only those cells having membrane damage. The combined use of these two dyes allows colocalization of the living cells (green staining) and the dead cells (red staining) (Magne et al., 2003).
  • the culture medium was drawn up and replaced by 400 ⁇ l of a 5 ⁇ M CTG solution in complete culture medium. The cells were incubated for 1 hour at 37° C.
  • the CTG solution was then replaced by complete medium and the cells were incubated again for 30 minutes at 37° C. After rinsing with PBS, the cells were then treated for 1 hour, at ambient temperature and in the dark, with 400 ⁇ l of a 1 ⁇ M EthD-1 solution in complete medium without FCS. The medium was finally removed, the hydrogel was drawn up and placed between a slide and cover glass. The images were obtained by epifluorescent microscopy (Axioplan, Zeiss, Iena, Germany) and were recorded using a DC30 digital camera (Kappa opto-electronics Gmbh, Gleichen, Germany).
  • the cells in the gel were also displayed by laser scanning-inverted confocal microscopy (IFR 26, Caroline Vignes-Colombeix) after 24, 48 and 72 hours of culture as specified above.
  • the confocal microscope produces a clear image by eliminating the signals extraneous to the focal plane.
  • This microscope has an object stage able to move along the Z axis, allowing the focal plane to be varied and various planes of the sample thus to be displayed.
  • the inventors carried out staining (CTG and EthD-1) on the SW1353 and C28/I2 chondrocytes and on rabbit articular chondrocytes. They then observed the chondrocytes by fluorescent microscopy. Within the hydrogel, the SW1353 and the C28/I2 chondrocytes or the rabbit articular chondrocytes had a rounded shape and were stained markedly green by the cell tracker green. Rare cells appeared stained red. The SW1353, C28/I2 and rabbit articular chondrocytes developed three-dimensionally in the hydrogel, forming nodules, the number and the size of which increased as a function of the culture time.
  • CTG and EthD-1 staining
  • chondrocyte phenotype In order to analyze the chondrocyte phenotype, the expression of the messengers coding for specific chondrocyte markers was investigated by RT-PCR.
  • the chondrocytes freshly isolated from rabbit articular cartilage were distributed in a proportion of 1 million cells/ml of hydrogel, and 2 ml of this mixture were deposited in wells of six-well culture plates. The cells were then cultivated for 3 weeks prior to freezing in the presence of Trizol®
  • the cells were thawed over ice and scraped: the Trizol® solution was then collected and centrifuged for 10 minutes at 12,000 rpm and at 4° C. The upper phase was removed; 200 ⁇ l of chloroform were added and the mixture was vortexed for 15 seconds. After 10 minutes of incubation at ambient temperature, the samples were centrifuged for 15 minutes at 12,000 RPM and at 4° C. The aqueous phase was removed and the total RNAs were precipitated by centrifugation (12,000 rpm, 15 minutes, 4° C.) in the presence of 500 ⁇ l of isopropanol. The supernatant was removed and the pellet washed with 75% ethanol then dried.
  • RNAs were taken up in 20 ⁇ l of water. The isolated amount of RNAs was then assessed by measuring absorbance at 260 nm. The RNA concentrations were finally adjusted to 1 ⁇ g/ ⁇ l. In order to check the integrity of the extracted RNAs, 500 ng of total RNAs were separated by electrophoresis over 1% agarose gel in a TBE buffer containing EtBr. Migration took place at 100 V for 30 minutes. The total RNAs were displayed using a UV transilluminator.
  • RNA samples 2 ⁇ g were removed; 1 ⁇ l of DNAse 110 ⁇ buffer and 1 ⁇ l of DNAse 1 (5 U/ ⁇ l) were added, and the volume was adjusted to 10 ⁇ l using sterile water. The samples were then incubated for 15 minutes at ambient temperature. The reaction was stopped by adding 1 ⁇ l of 25 mM EDTA and the reaction product was placed at 65° C. for 10 minutes.
  • the PCR was carried out using 2 ⁇ l of cDNA solution, to which were added 0.5 ⁇ l of sense primers and 0.5 ⁇ l of antisense primers at 100 ng/ ⁇ l, 5 ⁇ l of Taq polymerase buffer, 1.5 ⁇ l of MgCl 2 , 0.5 ⁇ l of Taq polymerase and 40 ⁇ l of water.
  • the PCR reactions were carried out using a thermocycler (Eppendorf Mastercycler) under the following conditions: 3 minutes of denaturation at 94° C., followed by 30 cycles of 20 seconds at 94° C. (denaturation), 20 seconds at 60° C. (hybridization) and 20 seconds of elongation at 72° C.
  • the PCR amplification products were electrophoretically separated over 2% agarose gel in tris borate EDTA buffer (TBE) 1 ⁇ .
  • TBE tris borate EDTA buffer
  • the bands were revealed by ethidium bromide using a UV transilluminator.
  • the size of the amplicons is indicated in Table 2.
  • the intensity of the bands obtained over the 2% agarose gels was estimated using Leica Q500 software (Leica Imagine Systems, Cambridge, UK) allowing the semi-quantitative densitometric analysis of the amplified transcripts under various experimental conditions. The results were expressed in an arbitrary unit as the relevant gene/reference gene ratio.
  • type II collagen During a monolayer culture, the inventors observed a reduction in the expression of type II collagen, aggrecan and type X collagen. After 3 weeks of culture, the type II collagen was still slightly expressed, whereas the type X collagen and aggrecan were no longer detectable. At the same time, our results revealed that the expression of type I collagen had increased after 3 weeks of monolayer culture.
  • RNAs were extracted on each pass during monolayer cultures and after 2, 3 and 4 weeks for the three-dimensional cultures.
  • the expression of these markers was modified during monolayer culture; the expression of collagen II and aggrecan decreased during the various passes until it disappeared at the end of three passes, whereas the expression of type I collagen increased markedly from the first pass onward and remained markedly expressed during the various passes.
  • the chondrocytes therefore appeared to be dedifferentiated at the end of three passes in monolayer culture.
  • Hydrogels were prepared as described in the preceding examples, integrating human chondrocytes derived from nasal cartilage (HNC). They were then implanted subcutaneously in nude mice and left for 3 weeks.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Cell Biology (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • Rheumatology (AREA)
  • Botany (AREA)
  • Veterinary Medicine (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Epidemiology (AREA)
  • Transplantation (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Public Health (AREA)
  • Medicinal Chemistry (AREA)
  • Dermatology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Urology & Nephrology (AREA)
  • General Engineering & Computer Science (AREA)
  • Vascular Medicine (AREA)
  • Developmental Biology & Embryology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Materials For Medical Uses (AREA)
  • Compositions Of Macromolecular Compounds (AREA)
  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
US10/577,942 2003-11-04 2004-11-04 Use of a Hydrogel for the Culture of Chondrocytes Abandoned US20070212389A1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
EP03292759A EP1529543A1 (fr) 2003-11-04 2003-11-04 Utilisation d'un hydrogel pour la culture de chondrocytes
EP03292759.2 2003-11-04
PCT/FR2004/002844 WO2005044326A1 (fr) 2003-11-04 2004-11-04 Utilisation d'un hydrogel pour la culture de chondrocytes.

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
PCT/FR2004/002844 A-371-Of-International WO2005044326A1 (fr) 2003-11-04 2004-11-04 Utilisation d'un hydrogel pour la culture de chondrocytes.

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US12/785,874 Division US8440462B2 (en) 2003-11-04 2010-05-24 Use of a hydrogel for the culture of chondrocytes

Publications (1)

Publication Number Publication Date
US20070212389A1 true US20070212389A1 (en) 2007-09-13

Family

ID=34429548

Family Applications (2)

Application Number Title Priority Date Filing Date
US10/577,942 Abandoned US20070212389A1 (en) 2003-11-04 2004-11-04 Use of a Hydrogel for the Culture of Chondrocytes
US12/785,874 Active 2025-01-13 US8440462B2 (en) 2003-11-04 2010-05-24 Use of a hydrogel for the culture of chondrocytes

Family Applications After (1)

Application Number Title Priority Date Filing Date
US12/785,874 Active 2025-01-13 US8440462B2 (en) 2003-11-04 2010-05-24 Use of a hydrogel for the culture of chondrocytes

Country Status (12)

Country Link
US (2) US20070212389A1 (fr)
EP (2) EP1529543A1 (fr)
JP (1) JP4351258B2 (fr)
CN (1) CN100509064C (fr)
AT (1) ATE361108T1 (fr)
AU (1) AU2004287234B2 (fr)
CA (1) CA2543917A1 (fr)
DE (1) DE602004006299T2 (fr)
DK (1) DK1689461T5 (fr)
ES (1) ES2286709T3 (fr)
PL (1) PL1689461T3 (fr)
WO (1) WO2005044326A1 (fr)

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080269895A1 (en) * 2005-09-20 2008-10-30 Steinwachs Matthias R Implant for the Repair of a Cartilage Defect and Method for Manufacturing the Implant
US20100080836A1 (en) * 2007-05-04 2010-04-01 Ascendia Ab Method and means for culturing osteoblastic cells
US20120101577A1 (en) * 2010-10-26 2012-04-26 Kyphon Sarl Activatable devices containing a chemonucleolysis agent
WO2012164101A1 (fr) 2011-06-03 2012-12-06 Institut National De La Sante Et De La Recherche Medicale (Inserm) Hydrogel à base de biomolécule silylée pour la culture de cardiomyocytes et de cellules souches et utilisation de l'hydrogel pour le traitement de l'insuffisance cardiaque
US8480757B2 (en) 2005-08-26 2013-07-09 Zimmer, Inc. Implants and methods for repair, replacement and treatment of disease
US8497121B2 (en) 2006-12-20 2013-07-30 Zimmer Orthobiologics, Inc. Method of obtaining viable small tissue particles and use for tissue repair
US8518433B2 (en) 2003-12-11 2013-08-27 Zimmer, Inc. Method of treating an osteochondral defect
US8829166B2 (en) 2002-06-26 2014-09-09 Zimmer Orthobiologics, Inc. Rapid isolation of osteoinductive protein mixtures from mammalian bone tissue
US9138318B2 (en) 2007-04-12 2015-09-22 Zimmer, Inc. Apparatus for forming an implant
WO2016081742A1 (fr) * 2014-11-19 2016-05-26 The Trustees Of Columbia University In The City Of New York Systèmes, méthodes et dispositifs pour la synchronisation du cycle cellulaire de cellules souches
US10167447B2 (en) 2012-12-21 2019-01-01 Zimmer, Inc. Supports and methods for promoting integration of cartilage tissue explants

Families Citing this family (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009057755A1 (fr) * 2007-11-02 2009-05-07 National Institute Of Advanced Industrial Science And Technology Substrat matriciel d'une chaîne de sucre et procédé de détection d'une molécule de liaison à une chaîne de sucre l'utilisant
JP5544635B2 (ja) * 2009-09-30 2014-07-09 国立大学法人北海道大学 軟骨細胞再分化誘導用基材およびこれを用いた軟骨細胞の製造方法
CN102775831B (zh) * 2012-07-31 2014-11-12 华南理工大学 在材料表面紫外表固化接枝两性离子凝胶涂层的方法
EP3076902A4 (fr) * 2013-12-03 2017-08-16 Cornell University Procédé pour réparer un espace annulaire, et composition de gel de collagène
EP3167911A1 (fr) 2015-11-10 2017-05-17 Oniris Cellules produisant de l'insuline encapsulée dans du si-hpmc- pour le traitement de diabètes de type1
CN108102129B (zh) * 2016-11-25 2021-01-01 山西加乐医疗科技有限责任公司 一种纤维素海绵的制备方法
CN108042567A (zh) * 2017-12-15 2018-05-18 广州金塞迩中西医结合医院有限公司 用于软骨修复的生物组合制剂及其应用
CN109172604A (zh) * 2018-08-08 2019-01-11 浙江大学 一种msc水凝胶复合物及应用
US20230092155A1 (en) * 2019-04-08 2023-03-23 Gn Corporation Co., Ltd. Chondrocyte culture with high tissue regeneration ability

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6001394A (en) * 1995-08-07 1999-12-14 Centre National De La Recherche Scientifique (C.N.R.S.) Biomaterial composition and method for preparing same
US6129761A (en) * 1995-06-07 2000-10-10 Reprogenesis, Inc. Injectable hydrogel compositions

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5902741A (en) * 1986-04-18 1999-05-11 Advanced Tissue Sciences, Inc. Three-dimensional cartilage cultures
US5709854A (en) * 1993-04-30 1998-01-20 Massachusetts Institute Of Technology Tissue formation by injecting a cell-polymeric solution that gels in vivo
FR2798671A1 (fr) * 1999-09-16 2001-03-23 Univ Paris Curie Compositions de chondrocytes, preparation et utilisations

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6129761A (en) * 1995-06-07 2000-10-10 Reprogenesis, Inc. Injectable hydrogel compositions
US6001394A (en) * 1995-08-07 1999-12-14 Centre National De La Recherche Scientifique (C.N.R.S.) Biomaterial composition and method for preparing same

Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8829166B2 (en) 2002-06-26 2014-09-09 Zimmer Orthobiologics, Inc. Rapid isolation of osteoinductive protein mixtures from mammalian bone tissue
US8834914B2 (en) 2003-12-11 2014-09-16 Zimmer, Inc. Treatment methods using a particulate cadaveric allogenic juvenile cartilage particles
US8784863B2 (en) 2003-12-11 2014-07-22 Zimmer, Inc. Particulate cadaveric allogenic cartilage system
US8765165B2 (en) 2003-12-11 2014-07-01 Zimmer, Inc. Particulate cartilage system
US8518433B2 (en) 2003-12-11 2013-08-27 Zimmer, Inc. Method of treating an osteochondral defect
US8524268B2 (en) 2003-12-11 2013-09-03 Zimmer, Inc. Cadaveric allogenic human juvenile cartilage implant
US8652507B2 (en) 2003-12-11 2014-02-18 Zimmer, Inc. Juvenile cartilage composition
US8480757B2 (en) 2005-08-26 2013-07-09 Zimmer, Inc. Implants and methods for repair, replacement and treatment of disease
US8945535B2 (en) 2005-09-20 2015-02-03 Zimmer Orthobiologics, Inc. Implant for the repair of a cartilage defect and method for manufacturing the implant
US20080269895A1 (en) * 2005-09-20 2008-10-30 Steinwachs Matthias R Implant for the Repair of a Cartilage Defect and Method for Manufacturing the Implant
US8497121B2 (en) 2006-12-20 2013-07-30 Zimmer Orthobiologics, Inc. Method of obtaining viable small tissue particles and use for tissue repair
US9138318B2 (en) 2007-04-12 2015-09-22 Zimmer, Inc. Apparatus for forming an implant
US10010649B2 (en) * 2007-05-04 2018-07-03 Ascendia Ab Method and means for culturing osteoblastic cells
US20100080836A1 (en) * 2007-05-04 2010-04-01 Ascendia Ab Method and means for culturing osteoblastic cells
US20120101577A1 (en) * 2010-10-26 2012-04-26 Kyphon Sarl Activatable devices containing a chemonucleolysis agent
US9414930B2 (en) * 2010-10-26 2016-08-16 Kyphon SÀRL Activatable devices containing a chemonucleolysis agent
WO2012164101A1 (fr) 2011-06-03 2012-12-06 Institut National De La Sante Et De La Recherche Medicale (Inserm) Hydrogel à base de biomolécule silylée pour la culture de cardiomyocytes et de cellules souches et utilisation de l'hydrogel pour le traitement de l'insuffisance cardiaque
US10167447B2 (en) 2012-12-21 2019-01-01 Zimmer, Inc. Supports and methods for promoting integration of cartilage tissue explants
WO2016081742A1 (fr) * 2014-11-19 2016-05-26 The Trustees Of Columbia University In The City Of New York Systèmes, méthodes et dispositifs pour la synchronisation du cycle cellulaire de cellules souches

Also Published As

Publication number Publication date
EP1689461A1 (fr) 2006-08-16
EP1689461B1 (fr) 2007-05-02
ES2286709T3 (es) 2007-12-01
CA2543917A1 (fr) 2005-05-19
AU2004287234A1 (en) 2005-05-19
DK1689461T5 (da) 2007-10-15
EP1529543A1 (fr) 2005-05-11
DE602004006299D1 (de) 2007-06-14
PL1689461T3 (pl) 2007-10-31
US20100297764A1 (en) 2010-11-25
DE602004006299T2 (de) 2008-04-10
CN100509064C (zh) 2009-07-08
WO2005044326A1 (fr) 2005-05-19
EP1689461B9 (fr) 2007-08-29
US8440462B2 (en) 2013-05-14
ATE361108T1 (de) 2007-05-15
CN1897989A (zh) 2007-01-17
AU2004287234B2 (en) 2010-08-05
JP4351258B2 (ja) 2009-10-28
DK1689461T3 (da) 2007-09-10
JP2007510410A (ja) 2007-04-26

Similar Documents

Publication Publication Date Title
US8440462B2 (en) Use of a hydrogel for the culture of chondrocytes
RU2435846C2 (ru) Применение стромальных стволовых клеток жировой ткани для лечения свищей
AU2004231498B2 (en) Materials and methods for augmenting and/or repairing intervertebral discs
KR101091084B1 (ko) 연골 손상의 치료를 위한 세포군집체-하이드로겔-고분자 지지체 복합체, 이의 제조방법 및 이를 유효성분으로 함유하는 연골 손상 치료용 조성물
CN105308176B (zh) 从哺乳动物组织分离的椎间盘细胞、使用方法及其制备方法
JP2011041472A (ja) 細胞混合物からなる細胞移植治療用スフェロイド及びその作製方法
KR102479530B1 (ko) 인간 유도 만능 줄기세포로부터 연골세포의 펠렛을 제조하는 방법 및 이의 용도
CA2410944A1 (fr) Cartilage artificiel et procede permettant de le produire
KR101098073B1 (ko) 이식용 연골세포의 제법
CN114306732B (zh) 一种促软骨修复水凝胶材料及其制备方法和应用
KR20040106438A (ko) 주입성 연골 세포 이식물
KR20190124817A (ko) 포자를 닮은 세포들의 계군 및 그 용도
Zeiter et al. The fate of bovine bone marrow stromal cells in hydrogels: a comparison to nucleus pulposus cells and articular chondrocytes
CN103874763A (zh) 从胎儿组织制备亲代细胞库
Ghiasi et al. Use of mesenchymal adult stem cell for cartilage regeneration by hydrogel
KR101649375B1 (ko) 세포이식술을 위한 혼합세포복합체인 세포스페로이드의 제조방법 및 이의 이용방법
JP5373427B2 (ja) 滑膜細胞および細切軟骨片の軟骨修復における使用
Ujam Cartilage Tissue Engineering for Rhinoplasty
MX2008000001A (en) Use of adipose tissue-derived stromal stem cells in treating fistula

Legal Events

Date Code Title Description
AS Assignment

Owner name: INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE M

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:WEISS, PIERRE;DACULSI, GUY;VINATIER, CLAIRE;AND OTHERS;REEL/FRAME:018675/0604

Effective date: 20060505

Owner name: UNIVERSITE DE NANTES, FRANCE

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:WEISS, PIERRE;DACULSI, GUY;VINATIER, CLAIRE;AND OTHERS;REEL/FRAME:018675/0604

Effective date: 20060505

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION