Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/14—Macromolecular materials
  • A61L27/20—Polysaccharides
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  • 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
  • A61K35/32—Bones; Osteocytes; Osteoblasts; Tendons; Tenocytes; Teeth; Odontoblasts; Cartilage; Chondrocytes; Synovial membrane
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/14—Macromolecular materials
  • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
  • A61L27/225—Fibrin; Fibrinogen
• • 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
  • A61L27/3604—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 characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
  • A61L27/3612—Cartilage, synovial fluid
• • 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
  • A61L27/3641—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 characterised by the site of application in the body
  • A61L27/3645—Connective tissue
  • A61L27/3654—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
  • A61L27/3804—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
  • A61L27/3895—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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
• • 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
  • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L2430/00—Materials or treatment for tissue regeneration
  • A61L2430/06—Materials or treatment for tissue regeneration for cartilage reconstruction, e.g. meniscus
• • C—CHEMISTRY; METALLURGY
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  • C12N2533/00—Supports or coatings for cell culture, characterised by material
  • C12N2533/50—Proteins
  • C12N2533/56—Fibrin; Thrombin
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  • C12N2533/00—Supports or coatings for cell culture, characterised by material
  • C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue

Definitions

• Cartilage gel for cartilage repair including chitosan and chondrocytes.
• the present invention relates to compositions especially for the reconstruction of cartilage, and to a method for obtaining such compositions. More particularly, the present application relates to an environment or structure extremely favorable not only to the proliferation of cells capable of forming hyaline cartilage, but also to the synthesis of extracellular matrix of cartilage by these cells; the cells in this environment or structure constitute an implantable composition that can be grafted to cartilage lesions in humans or animals. The structure also constitutes a favorable environment during the implementation.
• Cartilage, or cartilage tissue consists of specific cells, namely chondrocytes, distributed within an extracellular matrix, comprising at least 80% of water. Chondrocytes are able to synthesize or degrade the components of the cartilage extracellular matrix, composed of glycosaminoglycans and collagen fibers, essentially type II for hyaline cartilage. Chondrocytes are therefore responsible not only for the synthesis but also for the maintenance of the cartilaginous tissue. Cartilage, and particularly articular cartilage in adulthood, has a very low capacity for self-repair, in particular because of its avascular character and the fact that mature chondrocytes do not proliferate.
• Cartilaginous lesions are therefore essentially irreversible and are therefore an important cause of pain and disability, particularly following trauma, mechanical wear or degenerative joint disease such as osteoarthritis. At present, there is no entirely satisfactory therapeutic solution for treating cartilaginous lesions, and especially large lesions.
• chondrocytes are dedifferentiated and lose their chondrocyte phenotype, expressing then type I collagen rather than type II collagen, so that once reimplanted, they do not lead to the formation tissue with satisfactory properties, but rather with scar tissue or, as part of a cartilaginous repair, the formation of a non-functional fibrocartilage, essentially composed of type I collagen.
• proposed culture media for redifferentiating the chondrocytes which have dedifferentiated during the proliferation stage in monolayers require relatively long times, of the order of 4 weeks for the proliferation step (Liu et al, 2007) during which the chondrocytes must undergo several passes in order to reach a sufficient number.
• redifferentiation is more complicated after two passages in particular (Hautier et al, 2008).
• Seeding in a 3D structure can also offer the advantage of facilitating in vivo implantation of chondrocytes, limiting cell leakage and treating large lesions.
• this biopolymer is known to be biodegradable, biocompatible, non-toxic, hemocompatible, cytocompatible, and in addition, bioactive, hemostatic, healing, bacteriostatic and fungistatic.
• chitosan is known to promote cell adhesion. He is also appreciated for its ability to maintain the chondrocyte phenotype and stimulate the process of extracellular matrix synthesis during chondrocyte culture. In addition, there is no reported infection or allergy related to chitosan, so it is non-immunogenic. As for its resorption time, it is possible to vary it by playing on its physicochemical properties. Chitosan is relatively easy to use and can be produced in a variety of physical forms, including solutions, films (Lahiji, et al, 2000), fibers, sponges, beads, hydrogels or microparticles. . It has therefore been used in extremely varied structures.
• the polymer is crosslinked in situ, once injected with the chondrocytes at the lesion (Hoemann et al, 2005).
• this approach has the disadvantage that the injected chondrocytes tend not to remain at the injection site because of the polymerization time.
• other authors have tested compositions, from a crosslinked polymer in situ and allowing the realization of a graft-capable tissue (Hao et al, 2010). These methods may nevertheless present difficulties during implantation.
• some authors have tested the hydrogel form and more particularly the physical hydrogel, synthesized without the addition of crosslinking agent and thus promoting the biocompatibility and bioresorbability of the structure.
• the three-dimensional structure is largely resorbed during the in vitro step of synthesis of the extracellular matrix, said structure is no longer part of the graft (see in particular WO02078760, Laboratoires Genévrier et al).
• Such a method is particularly long to implement, from 4 to 6 weeks, before obtaining a neo-tissue capable of being grafted, and said neo-tissue is no longer supported by the 3D structure at the time of implantation. .
• hyaluronic acid Another polymer that has been frequently tested in the field of chondrocyte implantation is hyaluronic acid. It is a natural polysaccharide, also biocompatible and biodegradable. Moreover, it is a major component of the liquid synovial and glycosaminoglycans (GAGs) present in articular cartilage.
• GAGs glycosaminoglycans
• Hyaluronic acid helps protect the joints by increasing the viscosity of the synovial fluid and making the cartilage more elastic. It has also been shown that hyaluronic acid promotes the expression of the chondrocyte phenotype.
• hyaluronic acid has sometimes been combined with chitosan compositions in the chondrocyte culture.
• the present inventors have developed a method for obtaining chondrocytes sufficient number, within a structure ensuring both their perfect multiplication, their redifferentiation and the production of cartilaginous matrix.
• This structure is also suitable for implantation at the level of the lesion.
• chitosan in the form of physical hydrogel particles, is an excellent environment for chondrocytes, not only for the synthesis of extracellular matrix of hyaline cartilage, but also with a view to their multiplication. Indeed, against all odds, the inventors were able to produce in the same structure of chitosan, a proliferation step making it possible to obtain a proliferation rate similar to or even greater than that observed by the ordinary technique of monolayers (on plastic or in sponges ) and then easily allowing the re-differentiation of the cells.
• the inventors have therefore developed a method, successively allowing the multiplication of chondrocytes, directly after extraction in the primary state, then to induce the re-differentiation and the synthesis of specific extracellular matrix, within the same structure. , thus avoiding the stages of trypsination and structural change.
• this structure is compatible with in vivo implantation without the need for further modification.
• chondrocytes distributed homogeneously within the structure and with a density allowing their reimplantation, in a reduced time compared to what is described up to to date, under conditions very favorable to the repair of the cartilaginous tissue, in a structure directly compatible with implantation.
• chitosan is meant a polysaccharide composed of D-glucosamine units (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit) linked to ⁇ - (1 -4). It can be produced by chemical or enzymatic deacetylation of chitin; it also exists in the natural state.
• the polysaccharide is generally associated with negligible amounts of beta-glucan.
• beta-glucan in amounts consistent with those found naturally in chitosan, is without influence.
• a chitosan will be said to be “pure” even in the presence of beta-glucan, as long as the presence of this latter remains less than 5% by mass, relative to the polysaccharide.
• chitosan derivative denotes any polymer of chitosan which has undergone a reaction intended to modify the chemical groups of chitosan to change the functionalities thereof, for example methylation, halogenation, etc.
• a derivative of chitosan does not have more than three types of derivation, preferably only two types of derivation, and more preferably only one type of derivation.
• Chitosan derivatives particularly contemplated in the context of the present invention are glycol-chitosan, N-succinyl chitosan, N- or O-Carboxymethyl Chitosan, without this list being exhaustive.
• the degree of acetylation is the percentage of acetylated units relative to the number of total units (acetylated and deacetylated units), it can be determined by Fourier transform infrared spectrometry (I TF) or by proton NMR .
• chitosan hydrogel it is meant that the very major constituent, therefore more than 80% or even more than 90% or even 95%, of the hydrogel (by mass) is chitosan, apart from water.
• a hydrogel of chitosan derivative refers to a hydrogel of which the very major constituent, more than 80% or even more than 90% or 95% of the hydrogel is the derivative of chitosan, apart from the water.
• a hydrogel of chitosan or of chitosan derivative means a hydrogel preferably comprising at least 70% of water, or even at least 80% of water.
• physical hydrogel is meant a hydrogel obtained by a gelling process in an aqueous medium or in an aqueous-alcoholic medium, not requiring the addition of crosslinking agent.
• chondrocyte phenotype we designate chondrocyte type cells, preferentially expressing collagen type II compared to type I collagen.
• hyaluronic acid is meant a polysaccharide composed of D-glucuronic acid units and D-N-acetylglucosamine, linked together by glycosidic bonds.
• Hyaluronic acid derivative denotes any hyaluronic acid polymer that has undergone a reaction aimed at modifying the chemical moieties of hyaluronic acid in order to change its functionalities, for example by esterification.
• the present invention thus relates, according to a first aspect, to an in vitro method for obtaining a composition, which can be implanted by arthroscopy for cartilage repair, comprising particles or fragments of physical chitosan hydrogel or a chitosan derivative. , and cartilage-forming cells, preferably hyaline cartilage.
• the composition obtained at the end of the implementation of the process can be described as a cartilage gel.
• a cartilage gel indeed comprises cells, synthesizing cartilage matrix, all distributed homogeneously without cell gradient, in a three-dimensional structure based on chitosan particles.
• Such a method according to the invention comprises in particular a step of amplification of primary cells, in a three-dimensional structure (3D structure) and then a step of redifferentiation and induction of extracellular matrix synthesis, within this same three-dimensional structure, that is, without changing the environment cells.
• chitosan is indeed a biocompatible biomaterial, bioabsorbable with non-toxic, non-immunogenic, cytocompatible and bioactive degradation products. It is also fully compatible with the pharmaceutical requirements as an implantable device.
• the chitosan or chitosan derivative hydrogel is a physical hydrogel obtained without the addition of a crosslinking agent.
• step (ii.) Induction of differentiation, or re-differentiation, and extracellular matrix synthesis by said amplified cells, within the three-dimensional structure of step (i).
• the cells that are amplified in the first step are seeded in this 3D structure.
• They can be either chondrocytes or chondrocyte precursor cells obtained from stem cells, for example mesenchymal stem cells, or induced pluripotent cells (IPS). It can be any cell differentiated into chondrocytes. Preferably, it is chondrocytes, and more preferably articular chondrocytes.
• Chondrocytes or stem cells can be obtained by any method known to those skilled in the art for recovering these cells from a biological sample that may contain them. Such methods are described in particular in the document FR2965278 (University of Caen Basse-Normandie, et al).
• It is preferably human or animal cells, especially equine or canine. It can be articular cells, ear or even from the nasal septum.
• Particularly preferred cells are human cells, for example human chondrocytes, and more particularly human articular chondrocytes for a human patient. It is the same for an animal, especially for a horse or a dog.
• the primary cells seeded in the structure may be allogeneic, xenogeneic, heterologous, or autologous cells, vis-à-vis the organism to be treated.
• these are autologous cells, that is to say that come from the patient, human or animal, which must be treated. Most preferably, it is therefore autologous human chondrocytes, which can be reimplanted in the donor at the end of the method of the invention.
• chondrocytes for example for race horses or dogs, which may also benefit from chondrocyte transplantation. It can also be xenogeneic or allogeneic cells, because certain measures well known to those skilled in the art can be implemented to avoid rejection during implantation.
• the cells are seeded in a three-dimensional structure or scaffold, or biomaterial, comprising fragments or physical hydrogel particles of pure chitosan, or derivative of chitosan. Said particles thus form a scaffold or structure compatible and favorable to the formation of a three-dimensional tissue, until the cells produce enough extracellular matrix to maintain the structure mechanically.
• the cells are added after the gelation and formation phase of the chitosan hydrogel particles. The cells are therefore located outside the hydrogel fragments or particles; they remain on the surface of the fragments or particles, they are neither trapped nor encapsulated in the hydrogel, they do not infiltrate the pores of the hydrogel either. They can therefore circulate freely around the particles, as well as nutrients and waste, during the different steps of the process of the invention.
• the mixture made at the beginning of culture between the chitosan particles and the cells thus makes it possible to promote a good distribution of the cells within the three-dimensional structure at the end of this process.
• the chitosan used for the design of the device or three-dimensional structure is obtained for example by deacetylation of chitin which can come from the exoskeleton of arthropods (shrimps, insects, crab ...), the endoskeleton of cephalopods (squid) or the cell wall of fungi.
• chitosan may be conformation a (cell wall of mushrooms, shrimps, crabs) or ⁇ (squid) or ⁇ (insects), which strongly influences its biological properties.
• the chitosan used can come from these different sources, but it will preferably be used a chitosan of non-animal origin, for reasons of biocompatibility, low endotoxin content, batch reproducibility and compliance. to pharmaceutical standards.
• the chitosan used in the context of the present invention is chitosan extracted from the cell wall of fungi, more particularly Paris fungi, Agaricus bisporus.
• Paris mushroom extract is in accordance with pharmaceutical requirements in terms of levels of endotoxins, microbiological residues and heavy metals. Succession of the steps:
• the method of the invention is characterized in particular by the succession of two steps, a first step of amplification of primary cells in a three-dimensional structure and a second step of induction of differentiation and extracellular matrix synthesis (ECM), within this same three-dimensional structure.
• the major advantage of this method lies in the fact that it is not necessary to change the structural cells, between the amplification step and the re-differentiation step with MEC synthesis, or elsewhere. between the step of re-differentiation and that of implantation. It follows that
• the primary cells are seeded in the structure of the invention, without prior proliferation step, and therefore without the need to take off, particularly by trypsinization, or by any other means likely to damage the wall cellular. They then proliferate within the structure and are then induced to redifferentiate and produce
• the cartilaginous matrix again without the need to take off, trypsinize, or make them undergo any other treatment likely to damage the cell wall.
• the method of the invention thus makes it possible to obtain cells which, after the extraction, have not been damaged and have not undergone stress conditions, which makes it possible to guarantee no
• the viability rate of the cells is greater than
• the two major steps of the process of the invention that is to say on the one hand the amplification and on the other hand the re-differentiation and the synthesis of MEK, are distinct stages. .
• the inventors have indeed observed that the ability to proliferate and the ability to produce ECM were preferentially successive steps for cells such as
• the two steps as described are therefore not only successive but also distinct, the second step
• the amplification step is distinct from that of the cartilage matrix synthesis when the number of chondrocytes increases in the first step and remains relatively stable in the second step, with little or no amplification. It is considered that the amplification step is distinct from that of the cartilage matrix synthesis as a function of the viscosity of the medium: with the observation of a low viscosity during the amplification step and an increased viscosity during the second step, confirming the production of extracellular matrix.
• extracellular matrix synthesis is low.
• Collagen type II (COLII) is a characteristic marker of hyaline cartilage; it is a homo-trimer of three chains to 1 (II), encoded by the gene Col2a1. The analysis of this type of collagen is conventionally performed to identify differentiated chondrocytes.
• Collagen type 1 (COLI), a heterotrimeric ⁇ 1 2 ⁇ 2- ⁇ produced from the Coll ai and Col1 a2 genes, is conventionally considered as a marker of the de-differentiation of chondrocytes.
• the method according to the invention is characterized by maintaining the capacity of the cells which have been cultured within the structure to redifferentiate into chondrocytes.
• This capacity it is meant that the majority of the cells, at the end of the second step, have a chondrocyte phenotype, preferably at least 60%, preferably at least 70%.
• the inventors have indeed observed that the majority of the cells seeded in the structure as described, based on chitosan hydrogel particles or a derivative, exhibit a stable chondrocyte phenotype during the second step of the extracellular matrix synthesis process. .
• the cells within the composition exhibit little or no expression of type I collagen and / or do not produce it at the protein level but express a COLII / COLI differentiation index greater than 1.
• the cells present in this composition synthesize type II collagen proteins with a COLII / COLI protein ratio greater than 1, preferably greater than 1.5; and / or the level of messenger RNA of type II collagen is significantly higher than the level of type I messenger RNA.
• the method according to the invention as described above preferably makes it possible to obtain the composition or cartilage gel ready for implantation in less than 40 days, preferably in less than 36 days, or even in less than thirty days. days, for example in less than 28 days, or less than 21 days. It thus makes it possible, starting from a biopsy of chondrocytes or primary stem cells differentiated into chondrocytes, to obtain a composition ready for implantation, having a sufficient number of chondrocytes to be able to repair the articular lesion, and this in one month and a half, or less than a month, or less than three weeks.
• the amplification step is carried out in one to three weeks, preferably in about two weeks, from 12 to 16 days, or even in less than two weeks.
• the multiplication of the number of living cells, relative to the number of cells initially seeded in the structure is at least 4, or even at least 6, or even at least 7 or greater than 7.
• the multiplication step lasts between 1 and 3 weeks and must make it possible to multiply the cells within the 3D structure by a factor of at least 4, or even at least 6, or even at least 7.
• a biopsy of 300 to 500 mg of cartilage comprising approximately 10 6 to 1.5 ⁇ 10 6 cells makes it possible to obtain, at the end of the amplification step of 4 ⁇ 10 6 cells at 10, 5 x 10 6 cells because of the amplification factor observed by the inventors, and this in 1 to 3 weeks.
• Such a number of cells is considered suitable for implantation of a chondrocyte transplant.
• the second step related to re-differentiation accompanied by extracellular matrix synthesis, may have a variable duration.
• such a step lasts between 2 and 4 weeks, preferably about three weeks, or less.
• the duration of this second step may possibly be adjusted according to the chosen mode of reimplantation.
• composition or cartilage gel comprising chondrocyte cells, distributed in a newly synthesized cartilage matrix, and which are capable of continuing tissue synthesis.
• cartilaginous within a 3D structure composed of particles of physical chitosan hydrogel or a derivative of chitosan, such that said composition or cartilage gel is directly implantable in a human being or an animal, in particular to fill a lesion articular cartilaginous, for example following traumatic and limited injuries of the articular cartilage, or lesions of the type beginning superficial osteoarthritis, or deeper lesions of the osteochondral type.
• kits or cartilage gel ready for injection or implantation in vivo in a human being or an animal is thus obtained.
• the 3D structure comprising the chitosan hydrogel particles or the chitosan derivative may be partly biodegraded during the process, it is preferably partially biodegraded, preferably less than 50% of the 3 D structure. based on chitosan hydrogel particles prior to seeding.
• the composition of the invention or cartilage gel may be injectable in a human being or an animal.
• the duration of the second stage of synthesis of MEK will be reduced by a few days, to ensure that the composition remains injectable.
• the MEC synthesis step will be adjusted in order to obtain the desired consistency so that the shape of the composition can be adjusted to that desired.
• composition as described above comprises hyaline cartilage, synthesized by the cells during the second stage of the process, hence its name cartilage gel.
• a major advantage of the present invention resides indeed in the directly implantable nature of the composition obtained at the end of the process.
• it is not necessary to change the cells of the 3D structure, so it is not necessary to subject them to any treatment. There is no need either to ensure the disappearance of the structure, nor to wait for such degradation.
• the chitosan-based structure is still present at the end of the MEC synthesis step and is part of the composition, or neo-tissue, intended to be implanted.
• such a structure serves as a scaffold ensuring the maintenance of the cells in a suitable environment such that it allows, after implantation, the continuation of the synthesis of MEC, in order to perfectly fit at the lesion, for example articular.
• the structure of chitosan is not only intended to promote the synthesis of MEC in vitro, but also, after reimplantation, to support the implanted cells; it therefore participates in the structure of the re-implanted neo-tissue.
• the structure also ensures an optimal spatial distribution of the seeded cells, it therefore allows a harmonious distribution of the synthesized MEC during the process of the invention, and also after reimplantation.
• the number of cells implanted in the structure of the invention is variable depending on the size of the lesion that it is desired to fill, and also as a function of the number of cells that it is possible to take for seeding. .
• at least about 10 5 elementary cells are seeded, in particular at least about 6 ⁇ May 10 primary cells, or at least 10 6 primary cells, preferably human primary chondrocytes, canine or equine;
• the final composition preferably comprises at least 3 ⁇ 10 5 chondrocytes, or at least 6 ⁇ 10 6 chondrocytes, or even more.
• it will preferably be seeded 10 6 to 1.5 x 10 6 cells to obtain, at the end of the amplification step 4 to 10.5 x 10 6 cells.
• the chondrocytes within the composition are at a concentration of at least about 10 6 cells / g, preferably at least about 6 ⁇ 10 6 cells / g of 3D structure at the time of placing. in culture.
• two different culture media are used.
• a medium that promotes the proliferation of cells without inducing the synthesis of MEK.
• the proliferation of cells is generally accompanied by a phenomenon of de-differentiation; however, a medium that preserves their ability to redifferentiate into chondrocytes at the end of the proliferation stage will be used.
• the inventors have indeed highlighted the fact that, within a three-dimensional structure, it was preferable to carry out a step of intense amplification, without inducing the synthesis of MEC. Indeed, the three-dimensional structures and chitosan were known before the invention for their ability to promote the chondrocyte phenotype by limiting the dedifferentiation during the extracellular matrix synthesis step. In an entirely unexpected manner, the inventors have shown that it is possible to amplify the chondrocytes without inducing massive extracellular matrix synthesis, within a three-dimensional structure based on chitosan hydrogel or a derivative.
• a medium that is particularly suitable for the proliferation stage is an amplification-inducing medium, in particular a medium comprising fibroblast growth factor (FGF-2), and incidentally also insulin, corresponding to a FI medium shown in the experimental section (Claus et al, 2012).
• FGF-2 is preferably present between 2 and 10 ng / mL and insulin between 2 and 10 ⁇ g / mL.
• other culture media well known to those skilled in the art, can also be used, including all the culture media generally used for this kind of cells, but in monolayers. Because of the three-dimensional structure and chitosan, the cells seeded in the structure of the invention retain mainly their round morphology and their ability to subsequently redifferentiate into chondrocytes.
• a most preferred medium comprises BMP-2 (Bone Morphogenetic Protein 2); preferably, the medium is that used in the experimental part, composed in particular of BMP-2 and insulin, and preferably also triiodothyronine T3 (Liu et al., 2007, Claus et al, 2012), corresponding to a medium says "ILO".
• BMP-2 is preferably at a concentration between 100 and 500 ng / mL, insulin between 2 and 10 ⁇ g / mL and triiodothyronine T3 between 50 and 250 mM.
• BMP-2 will preferably be selected from the same species as the cells used, ie human BMP-2 for human cells. It is the same for animal cells.
• preference will be given to culture media that do not oppose the subsequent reimplantation of the composition, at the end of the MEC synthesis step. In particular, compounds that are likely to generate rejection reactions that are not compatible with the implantable device regulations will be avoided.
• the first and second stages can, independently of one another, take place in normoxia or hypoxia conditions.
• the pure chitosan hydrogel, or chitosan derivative is made from chitosan, preferably extracted from fungi and whose weight average molecular weight (Mw), will preferably be greater than 150 kDa (ie 150,000 g / mol ) to promote the physical gelation process by the presence of long macromolecular chains. It will preferably be between 150 and 220 kDa.
• the process for producing the physical chitosan hydrogel as described in the experimental part may easily be adapted by those skilled in the art , using well known techniques.
• chitosan for the hydrogel, which has a variable degree of acetylation; preferably, however, the degree of acetylation of chitosan is between 5% and 60%, preferably greater than 25%, for example between 25 and 60%, or between 28 and 40%. This degree of acetylation induces indeed a favorable environment for the cells, leading to good adhesion between the hydrogel formed and the cells, and good results in chondrogenesis.
• a solution of chitosan whose concentration is sufficiently high to allow the entanglement of the macromolecular chains to be used is preferably used, and thus to promote physical gelation.
• the hydrogel is indeed obtained in the context of the invention by a completely physical process, without any chemical crosslinking agent.
• the concentration of chitosan in solution may be between 0.5-4% (w / w), preferably greater than 1, 5% or even 2%.
• the chitosan or the chitosan derivative has a concentration in mass in the hydrogel of between 3.4 and 4.2% before neutralization.
• Several methods for gelling chitosan can be used in the context of the present invention. The following particularly suitable methods can be mentioned, such as physical gelling by gaseous means (ammonia) or physical gelation in an aqueous-alcoholic or aqueous medium.
• the hydrogel used in the context of the present invention is obtained by a method of evaporation in an alcoholic medium, as illustrated in Example 1 in the experimental part; such a process is also called hydro-alcoholic gelation.
• the hydrogel obtained is preferably between 3 and 5 mm thick.
• the pore size of the hydrogel obtained must be both smaller than the cell size and both sufficient to allow free diffusion of nutrients and waste disposal.
• the embodiments described above and implemented in the experimental part make it possible to obtain such a pore size.
• the chitosan or chitosan derivative hydrogel is such that the size of its pores does not allow the cells to penetrate inside the hydrogel.
• the cells seeded within the three-dimensional structure according to the invention therefore proliferate without penetrating into the hydrogel. Because the cells do not multiply in the pores of the hydrogel but they can freely circulate around the hydrogel particles, allows to obtain a homogeneous distribution or distribution of the cells in the structure, without the important ones. cell gradients reported in Correia et al, 201 1.
• the skilled person is able to determine the pore size of a hydrogel and adjust the parameters of its realization to ensure that the pore size is small enough to prevent the penetration of cells, including chondrocytes while still allowing the free diffusion of nutrients.
• the hydrogel thus obtained is manipulated by any suitable means, well known to those skilled in the art; in this way, the hydrogel is fragmented into particles.
• the hydrogel particles thus obtained are of irregular shape, but preferably have a relatively homogeneous size distribution, ie 50% of the particles have a size of between -20% and + 20% of the size. average.
• the particles have an average size of between 10 ⁇ and 1,500 ⁇ ; preferably between 200 ⁇ and 1200 ⁇ (1, 2 mm), and more preferably between 400 ⁇ and 700 ⁇ .
• particle size we mean the length of the edge, if the particles are assimilated to rectangles, the length of the largest diameter if the particles are assimilated to ellipses.
• the particles are preferably assimilable to ellipses.
• the inventors have shown better results for particles of a size average greater than a hundred microns, especially beyond 60 ⁇ , or even beyond 400 ⁇ and less than 1, 2 mm.
• the hydrogel is first obtained by gelation hydroalcoholic, and then fragmented by any appropriate means.
• the three-dimensional structure according to the invention thus comprises physical hydrogel particles of chitosan or of one of its derivatives, as detailed above, constituting a three-dimensional structure in which the cells are seeded, or migrate naturally.
• the cells are mixed with the hydrogel particles.
• the physical hydrogel of pure chitosan or chitosan derivative is preferably solely composed of chitosan or a derivative, and water with a content preferably of at least 70%, preferably at least 80%. In particular, it does not enter into the composition of this hydrogel, chemical crosslinking agent, or other polymer, especially polysaccharide or derivative, apart from ⁇ -glucan naturally associated with chitosan.
• the large percentage of water ensures that it is indeed a chitosan hydrogel and not a sponge-like structure or other, obtained by lyophilization of a chitosan solution. It should be noted that the large percentage of water in the hydrogel can best mimic the natural environment of chondrocytes, the cartilaginous tissue comprising about 80% water.
• an anionic molecule is added to the chitosan or chitosan derivative hydrogel particles making it possible to reinforce the mechanical and biological properties of the three-dimensional structure.
• This molecule does not enter the composition of the hydrogel, it is a component added after gelation of the chitosan hydrogel or chitosan derivative, and preferably after completion of the hydrogel particles.
• the anionic molecule therefore only interacts with the surface of the hydrogel particles according to the invention, thus promoting the formation of hairy chitosan particles by the linear chains of this anionic molecule.
• the anionic molecule associated with the surface of the chitosan particles is preferably in the form of a polymer, it is for example hyaluronic acid, or chondroitin sulfate.
• the chitosan chains having positive charges, due to amino groups in protonated form NH 3+ , and the chains of this anionic molecule interact by electrostatic bonds, thus forming a stable complex in a physiological medium (pH of between 5 and 8, and more generally between 6 and 7).
• the anionic molecule thus makes it possible to electrostatically cross-link the hydrogel fragments or particles by interacting with the cationic chains of the chitosan at the periphery of the particles, thus reinforcing the mechanical properties of the three-dimensional structure or 'scaffold' in which the cells are seeded.
• the anionic molecule can be added to the three-dimensional structure before seeding with the primary cells, and therefore be present during the proliferation and synthesis step of MEC, and therefore also during the subsequent implantation of the composition.
• it is also envisaged to seed the primary cells in a three-dimensional structure devoid of the anionic molecule and to add the latter either during the first multiplication step or at the end. the first step at the start of the second redifferentiation step, or at the end of the second step of re-differentiation and synthesis of MEC, before implantation.
• the anionic molecule will preferably be hyaluronic acid, one of the constituents of the synovial fluid, known for its chondroprotection properties and favorable to chondrogenesis. It is thus added the amount of hyaluronic acid needed to modify the viscoelastic characteristics of the three-dimensional structure. Electrostatic interactions occur between the protonated NH 3 + amino groups of chitosan and the carboxylic groups of hyaluronic acid.
• the relative proportion of the anionic molecule vis-à-vis the chitosan hydrogel is preferably between 1 and 10%, preferably between 1 and 3%.
• Hyaluronic acid may be of animal origin, for example by rooster or non-animal extraction, obtained by bacterial fermentation.
• hyaluronic acid will most preferably be selected from bacterial origin. Indeed, hyaluronic acid obtained by bacterial fermentation is known for its better biocompatibility properties, thus avoiding allergies and rejections, reproducibility of batches and compliance with pharmaceutical standards.
• the hyaluronic acid used in the context of the present invention is in accordance with pharmaceutical standards.
• Its weight-average molecular weight is preferably from 50 kDa to 4 MDa, it will preferably be greater than 500 kDa, preferably between 500 kDa and 2 MDa, for example between 1 MDa and 2MDa.
• the constituents of the three-dimensional structure which are particles of chitosan hydrogel or of chitosan derivatives, with or without the addition of anionic compound chains, must be resorbable in vivo. . In order to obtain such a property, it is important that none of the constituents of the three-dimensional structure oppose its resorbable nature.
• the combination of the chitosan hydrogel particles or chitosan derivatives, with or without the anionic compound chains will be resorbed after several weeks once implanted, for example after at least two weeks, preferably at less than 4 weeks. It is generally preferred that the resorption time does not exceed 6 months, preferably does not exceed 4 months. Depending on the type of application envisaged, the resorption time may be adjusted by those skilled in the art.
• compositions according to the present invention can be adapted in terms of shape, diameter, concentration, content according to the different applications envisaged.
• the three-dimensional structure formed of chitosan hydrogel particles or of chitosan derivative, with or without the chains of anionic compound can be elaborated so that its shape corresponds to that of the observed lesion in which it will be reimplanted to the resulting from the process according to the invention.
• the method makes it possible to obtain a composition or gel of cartilage ready for implantation or injection in vivo, in particular in a human being, or an animal such as a dog or a horse.
• the present invention relates to a three-dimensional structure formed of particles of physical chitosan hydrogel or of a chitosan derivative, and of an anionic molecule associated with these particles.
• a matrix may advantageously be used to seed chondrocytes by making them proliferate and then to synthesize the extracellular matrix, more particularly the matrix characteristic of hyaline cartilage, in particular for use according to the present invention, before being implanted or injected, under form of cartilage gel, especially within an articular lesion.
• the invention also relates to an implantable or injectable composition comprising this three-dimensional structure and differentiated chondrocytes capable of synthesizing cartilage tissue.
• the composition, or cartilage gel also comprises the cartilaginous matrix, synthesized by the chondrocytes it contains.
• the various elements mentioned are as described for the first aspect of the invention, in particular the three-dimensional structure, the chitosan or its derivative, the hydrogel, the particles, the anionic molecule, with regard to the method of the invention.
• chitosan is preferably a chitosan obtained from fungi, and more particularly extracted from the cell wall of Paris mushrooms, Agaricus bisporus.
• the particles have the sizes specified above, ie between 10 ⁇ and 1200 ⁇ , preferably between 400 and 700 ⁇ on average.
• the structure is therefore preferably a 3D structure formed of particles of physical chitosan hydrogel, of average size between 400 ⁇ and
• anionic molecule as specified for the process of the invention, it is preferably a polymeric molecule and most preferably hyaluronic acid or a hyaluronic acid derivative or a hyaluronic acid complex, and more specifically hyaluronic acid from bacterial fermentation.
• the differentiated chondrocytes present in the composition of the invention are, for example, articular chondrocytes. Most preferably, it is human or animal chondrocytes, in particular canines or equines. Chondrocytes are differentiated chondrocytes presenting a chondrocyte phenotype, in particular having a COLII / COLI differentiation index greater than 1. Preferably, the chondrocytes present in this
• the composition mainly synthesizes type II collagen proteins with a COLII / COLI protein ratio greater than 1, preferably greater than 1.5; and / or the level of messenger RNA of type II collagen is significantly greater than the level of type I messenger RNA, with a ratio of the COLII / COLI transcriptional level of the cells greater than 1, for example greater than 100, or greater at 1000.
• the relative proportion of chondrocytes in the composition corresponds to a concentration of between 10 6 and 10 7 cells / g of 3D hydrogel structure, at the time of culturing.
• composition of the invention may also comprise other compounds or molecules, and
• ECM extracellular matrix
• a composition as described, or a cartilage gel can be obtained by implementing the method of the invention, in particular by culturing the cells within the three-dimensional structure, then their proliferation, followed by their re-differentiation accompanied by MEC synthesis.
• the three-dimensional structure as described is advantageously absorbable, in particular bioabsorbable in vivo.
• the properties of the chitosan hydrogel or the chitosan derivative will be chosen according to the desired time scale until the integral resorption of the three-dimensional structure once the composition is implanted.
• the resorption time will be adapted so that such resorption is carried out concomitantly with the synthesis of the cartilaginous matrix by the chondrocytes present in the composition; preferably, the resorption time will be adjusted so that the cartilaginous matrix formed by the chondrocytes completely replaces the three-dimensional structure of chitosan hydrogel or chitosan derivative.
• the composition as obtained at the end of the process of the invention, or as described according to the second aspect of the invention is for a therapeutic and / or surgical use, in particular for use as an implant or graft in the repair or reconstruction of cartilaginous tissue, or in the treatment of osteoarthritis, and more generally in the treatment of any pathology characterized by degradation or disappearance of the cartilaginous tissue, in particular cartilaginous lesion, for example, following traumatic and limited injuries of the articular cartilage, or deeper lesions of the osteochondral type.
• Such a composition for use in vivo is especially envisaged in surgery, in rheumatology, or as a carrier of active principle.
• the composition, or cartilage gel is implantable by arthroscopy.
• chondrocyte transplantation is chondrocyte transplantation.
• the chondrocytes present in the composition to be grafted are autologous or allogeneic cells, preferably they are human, canine or equine chondrocytes.
• the present invention also relates to a three-dimensional structure formed of particles or fragments of physical hydrogel of pure chitosan or a derivative of chitosan, and its use for inoculating cells in vitro, in particular for the purpose of proliferation and synthesis of extracellular matrix, in particular for the implementation of the method according to the first aspect of the present invention.
• the three-dimensional structure is as previously described; the same goes for the chitosan hydrogel or chitosan derivative.
• the latter is preferably extracted from Paris fungi as explained above, having a weight average molecular weight of preferably between 150 and 220 kDa.
• the hydrogel particles preferably have an average size of between 200 ⁇ and 1.2 mm, and most preferably between 400 and 700 ⁇ .
• An anionic molecule is preferably added to the hydrogel particles of the three-dimensional structure; it is preferentially an anionic polymer, especially hyaluronic acid or a hyaluronic acid derivative or a hyaluronic acid complex, especially obtained by bacterial fermentation.
• the hydrogel particles preferably have a size of between 200 ⁇ and 1 , 2 mm, preferably between 400 and 700 ⁇ , and / or the chitosan has a weight average molecular weight of preferably greater than 50 kDa, preferably between 150 and 220 kDa, and / or chitosan has a degree of acetylation between 5 and 60%, preferably between 28 and 40%, and / or hyaluronic acid has a weight average molecular weight of between 50 kDa and 4MDa, preferably between 1 and 2 MDa.
• the proportion of hyaluronic acid with respect to chitosan hydrogel is preferably between 1 and 10%, preferably between 1 and 3%.
• this three-dimensional structure is advantageously used for seeding or culturing primary cells, in particular primary chondrocytes, or primary stem cells differentiated into chondrocytes, notably mesenchymal stem cells.
• primary cells in particular primary chondrocytes, or primary stem cells differentiated into chondrocytes, notably mesenchymal stem cells.
• other types of cells can be cultured in this three-dimensional structure, including bone cells, fibroblasts, keratinocytes, or combinations of some of these cells, without this list being limiting.
• the present inventors have in fact demonstrated that this three-dimensional structure provided a three-dimensional architecture particularly favorable to cells, whether in proliferation or multiplication phases, or in extracellular matrix synthesis phases.
• this three-dimensional structure is biodegradable and bioabsorbable, and can therefore be implanted in vivo,
• a three-dimensional structure of the invention that is to say a three-dimensional structure comprising fragments or particles of physical hydrogel of chitosan or derivative of chitosan, electrostatically crosslinked by an anionic molecule, preferably a polymer, in particular hyaluronic acid or a hyaluronic acid derivative or a hyaluronic acid complex, said structure being devoid of cells, so that the structure 3D is precisely colonized, in vivo, by cells.
• an anionic molecule preferably a polymer, in particular hyaluronic acid or a hyaluronic acid derivative or a hyaluronic acid complex
• the three-dimensional structure as described in the context of the various aspects of this invention is preferably sterilized before any seeding.
• FIGURES are a diagrammatic representation of FIGURES.
• Fig.1 shows a physical hydrogel photograph of pure chitosan, obtained by Cryo-Scanning electron microscopy.
• Fig.2 shows a physical hydrogel photograph of pure dehydrated chitosan, obtained by Scanning Electron Microscopy.
• FIG. 3 shows a photograph of pure physical chitosan hydrogel particles, after eosin treatment, obtained by optical microscopy.
• Fig.4 shows the survival rate of the cells seeded in a 3D M1-type structure, as a function of the initial chondrocyte density, measured with the Live and Dead kit on 7-day culture fractions (undergoing amplification , in FI medium).
• the enumeration of dead cells (in black) and living cells (shaded) is performed with ImageJ software from x-ray fluorescence microscopy. The percentage of dead cells is calculated for each condition by the ratio of dead cells to total cells.
• Fig.5 shows the evolution as a function of time of the chondrocyte population in 3D structures based on particles of physical chitosan hydrogel extracted from mushrooms, supplemented or not by hyaluronic acid, with particle sizes different, or within 3D structures based on physical hydrogel particles of chitosan extracted from squid, or even chondrocytes grown in monolayers, under the conditions of culture "FI".
• the number of cells is brought to the ordinate, the culture time in days is plotted on the abscissa
• Fig.6 shows the evolution of the chondrocyte population as a function of time for different initial densities of chondrocytes grown in monolayers, under the "FI" culture conditions, confirming that the cell population obtained is identical from 7 days the initial density.
• Fig.7 shows optical micrographs versus time, chondrocytes cultured within three-dimensional structures of pure hydrogel particles (M1).
• FIG. 7A represents the cells at the end of the amplification step, in F1 medium, 14 days after seeding.
• Fig.7B represents cells during the MEC synthesis step, in BIT medium, 24 days after seeding, ie 10 days after induction of re-differentiation and synthesis of MEK.
• the magnification factor is x 20.
• Fig.8 shows the amount of messenger RNAs of type I collagen and type II collagen relative to the GAPDH gene, measured by quantitative RT-PCR, for chondrocytes cultured within a 3D structure (M1) with several densities initial cell counts compared with the single-layer technique after 35 days of culture.
• Fig.9 shows the ratio of messenger RNAs of COLII / COLI genes, obtained by quantitative RTC-PCR, chondrocytes seeded within a 3D structure (M1) with several initial cell densities compared with the single-layer technique, at after 35 days of culture.
• Fig. 10 shows the Western Blot analysis, protein levels of type I collagen and type II collagen, for chondrocytes cultured in three-dimensional structure M1 compared to chondrocytes grown in monolayers, after 35 days.
• the level of actin expression serves as a control.
• Fig.1 1 shows the immunohistochemical analysis of chondrocytes grown in 3D M1 and M2 structure, compared to those grown in monolayers (x20) (MC) after 35 days, for the same initial cell density (6.10 5 cells) by HES and SO stains as well as immunolabelings of collagen type I and collagen type IL
• the chitosan used is of non-animal origin, it is extracted from the cell wall of Paris mushrooms, Agaricus bisporus. Its weight average molar mass (Mw) is 170 g / mol; and its degree of acetylation (DA) of 32%. It is used in powder form.
• the pure chitosan is dissolved in an acid solution of acetic acid (1% in water) in stoichiometric amount with the amine groups of chitosan.
• the solution is stirred at ambient temperature until complete dissolution of the chitosan, ie for at least 3 hours, preferably at least 6 hours.
• the 1,2-propanediol is then added in an amount identical to that of acetic acid and the mixture is stirred for at least 30 minutes, preferably for 1 hour at room temperature.
• the mixture can then be degassed at room temperature, or under vacuum if necessary, if the solution has a lot of air bubbles.
• the solution is then poured into containers, of the multi-well plate type or petri dishes of 3 cm in diameter, and then left to stand, preferably overnight.
• the solution is then placed in a vacuum oven, preferably at 50 ° C., for the time necessary for forming the gel, preferably at least 20 hours.
• the gelation step can also be performed at room temperature, but then requires longer times (5-8 days depending on the intrinsic characteristics of chitosan).
• the thickness of the solution before gelling may be between 2 and 7 mm, preferably between 3-6 mm, to promote evaporation and hydrophobic type interactions for good gel setting.
• the physical hydrogel obtained is then neutralized in a basic medium, with a 0.1N NaOH sodium hydroxide solution, preferably for 1 hour. This is followed by several washes with water, preferably sterile. The washings preferably last about 1 hour each to remove excess alcohol and return the hydrogel to a neutral pH. It has generally been carried out at least 6 washes.
• the gel thus obtained has a water content of about 80% by weight.
• the final mass concentration of chitosan in the hydrogel is between 1% and 4.5% before neutralization, preferably between 3.4 and 4.2% before neutralization.
• the hydrogel obtained at the end of these different steps has a thickness of 3 to 6 mm, preferably between 4 and 5 mm thick, it is translucent white in color, and its surfaces are smooth and regular. However, its appearance may vary depending on the intrinsic properties of the basic chitosan, including the degree of acetylation, the molar mass and the concentration. It is in the form of a viscoelastic block, the mechanical properties of which depend on the intrinsic characteristics of the starting chitosan, in particular again the degree of acetylation, the molar mass and the concentration.
• the hydrogel obtained is easy to handle, it is detached without difficulty and without tearing the flat surface on which it was made.
• Example 2 Synthesis of chitosan hydrogel particles / fragments in order to develop the 3D structure (M 1 structure and M2 structure).
• the chitosan hydrogel obtained at the end of Example 1 is cut into small squares of 1 mm side and then placed in 10 ml of water, preferably sterile.
• the hydrogel is then milled using an ultra-turrax, at 6000 to 17000 rpm, for 10 seconds at 2-4 times.
• grinding is preferably carried out at 6000 rpm for 10 seconds on 3 occasions, making it possible to reach particle sizes of: 400-700 ⁇ (50% ), or 250-900 ⁇ (> 80%), with an average of the order of 650 microns.
• the solution obtained is centrifuged, preferably at 1375 g for 7 minutes, in order to recover the pellet consisting of particles of chitosan hydrogel.
• Figure 3 illustrates an example of chitosan particles obtained.
• a mini-spoon is used to measure the amount of chitosan particles that will be put in contact with the chondrogenic cells. Preliminary tests validated the reproducibility of the measurement.
• the inventors also made a second structure (M2), by adding an anionic component, interacting with the cationic functions of chitosan.
• the polymer chosen is hyaluronic acid, preferably of bacterial origin because such a component is known for its better biocompatibility properties, in order to avoid allergies or possible rejections.
• the weight-average molecular weight of the hyaluronic acid used in producing the M2 structure is about 2 MDa.
• Hyaluronic acid is added after the preparation of the chitosan hydrogel particles.
• Example 3 Culture of cells within the 3D structure.
• the cells used in the context of this example are human chondrocytes obtained from human samples and treated according to the protocol described in FR2965278 (University of Caen Basse-Normandie, et al).
• the hydrogel particles obtained at the end of Example 2, with hyaluronic acid (3D M2 structure) or without (3D M1 structure), are sterilized, for example at 121 ° C. for 15 min, before contacting the cells.
• a few mini-spoons of hydrogel particles, preferably 2 mini-spoons corresponding to 80 to 84 particles, are taken and introduced into the well of a 24-well culture plate previously covered with an insert (pore size 8). ⁇ ).
• the cells are added between 10 5 and 10 7 , preferably of the order of 6.10 5 cells / well, for 80-84 hydrogel particles, which are gently mixed with the chitosan hydrogel particles. This proportion of cells relative to the 3D structure corresponds to approximately 6.7 x 10 6 cells per gram of 3D structure at the time of seeding.
• the culture is carried out in a controlled atmosphere in an oven at 37 ° C with a CO 2 content of 5% under normoxic conditions.
• the cells adhere spontaneously to the chitosan hydrogel particles.
• the amount of cells falling to the bottom of the well is considered negligible.
• the culture medium chosen is favorable to the multiplication of the cells.
• the medium chosen is a solution of 50/50 DMEM-HAM F12 + 1% AB (streptomycin / penicillin) + SVF supplemented with a solution called "IF" comprising FGF-2 at 5ng / ml + insulin at 5 mg / ml. (Claus et al, 2012).
• the mixture described in the previous step is covered, after a short time, of this culture medium known to be favorable to the proliferation of cells.
• the culture medium is renewed 3 times a week, for cultures in 3D structures and for monolayer cultures.
• the proliferation period lasts between one and two weeks to obtain a sufficient number of cells.
• the inventors observed that the amplification phase lasted about two weeks, when the cells were seeded in a structure consisting of pure hydrogel particles (3D M1 structure) and could be shortened to 1 week in the presence of supplemented hydrogel particles. by linear chains of an anionic molecule such as hyaluronic acid (3D M2 structure).
• the initial amount of cells of 6.10 5 cells / insert can be quite increased, if the condition of the cell number / hydrogel mass or cell number / hydrogel volume, or number of cells / number of cells. hydrogel particles is respected. For example, it is quite possible to seed 1 to 1.5 ⁇ 10 6 cells / insert, provided to add the necessary amount in 3D structure, in order to obtain more than 4 ⁇ 10 6 cells / insert at the end of the process, or even 10.5 x 10 6 cells / insert, or more.
• Reverse phase optical microscopy was performed. It validated that the cells adhere well to the chitosan hydrogel particles and that this environment is favorable to their culture.
• the culture conditions three-dimensional structure M1 or M2, and FI culture medium) promote the proliferation and division of chondrocytes.
• the cells observed can proliferate either in isolation or in clusters.
• the 3D structures of hydrogel particles show predominantly round cells.
• the elongated shape, characteristic of fibroblasts is not observed in the 3D structures M1 and M2, except sometimes at the periphery, that is to say at the interface between the structure and the external environment.
• the inventors have performed in parallel monolayer cultures. After 24 hours of culture, in the same FI medium as the cells seeded in the M1 and M2 structures, the chondrocytes adopt an elongated morphology characteristic of fibroblastic cells. Viability of the cells:
• the viability of the chondrocytes seeded in the three-dimensional structures was measured with the Live and Dead kit on 7-day culture fractions (during amplification, in FI medium). The enumeration of dead cells (red) and living cells (green) is performed with ImageJ software from x-ray fluorescence microscopy. The percentage of dead cells is calculated for each condition by the ratio of dead cells to total cells. The viability is greater than 93%, or even greater than 97% which shows a good biocompatibility of the 3D structure.
• Proliferation assays were performed by counting T4 Cellometer total cells after trypsin detachment of cells and staining of trypan blue dead cells. The measurements were carried out after 1 day (D1), 14 days (D14) and 21 days (D21) of culture after inoculation of primary chondrocytes at D0.
• Figure 5 illustrates the evolution of the cell population.
• the increase in the number of cells is clearly observed.
• the cells survive and proliferate very well in 3D hydrogel structure as in monolayers (MC).
• M1 or M2 the cells remain round during the multiplication stage while they adopt an elongated form, of fibroblastic type, in monolayers.
• FIG. 6 illustrates that, in monolayers, the cell population is identical from 7 days, regardless of the initial density of cells seeded.
• hyaluronic acid to the three-dimensional structure of chitosan hydrogel particles (M2 structure) causes a very strong acceleration of cell proliferation, much greater than the M1 structure or the monolayer culture, especially of a factor 2.
• the multiplication and differentiation steps are preferably distinct: in a first step, the cells multiply and in a second step, they differentiate and produce extracellular matrix.
• the culture medium used for the preceding multiplication step is modified after 15 days. We replaces the "FI" culture medium with a "BIT" medium in order to promote the step of cell differentiation and extracellular matrix production.
• the culture medium is then preferably composed of: 50/50 DMEM-HAM F12 + 1% AB (streptomycin / penicillin) + SVF, to which is added a BIT solution composed of: BMP-2 at 200ng / ml + Insulin at ⁇ s / ⁇ + triiodothyronine T3 at 100mM (Claus et al, 2012).
• This new culture medium is renewed every two or three days.
• the redifferentiation and chondrogenesis period preferably lasts 3 weeks.
• hydrogels structures M1 and M2
• the cells are predominantly round at the end of the amplification phase and continue to be during the entire extracellular matrix production phase. This point is illustrated in particular in FIG.
• the cells are perfectly round in hydrogels (structures M1 and M2), less in monolayers.
• the cells can agglutinate the hydrogel particles and form a kind of "ball" more or less compact. This observation is carried out under most conditions containing the hydrogels, not in the monolayers, however, which serve as control. This observation is evidence of the high production of extracellular matrix that has accumulated around cells. Cells make more extracellular matrix in three-dimensional environment than in monolayers.
• composition consisting of said beads remains, under certain conditions, injectable, despite the synthesis of a large amount of extracellular matrix.
• the composition is implantable.
• the transcription rate of the following proteins was quantified.
• the transcription rate serves as a reference for comparing the COLI and COLII transcription levels. Indeed, it is well known that the chondrocyte phenotype and the extracellular matrix production is accompanied by a strong synthesis of COLII transcripts, whereas the COLI transcripts generally accompany the de-differentiation process, in particular towards a fibroblastic phenotype.
• the results are shown in Figure 8.
• the COLII / GAPDH results obtained at the end of the MEC synthesis step show that there are more COLII transcripts for the hydrogel cultures than for the monolayer cultures.
• the COLI / GAPDH results show that, on the contrary, there are more COLI transcripts in monolayer cultures than in cultures within three-dimensional structures.
• the result of the calculation of the COLII / COLI ratio is shown in FIG. It shows that there is a much better ratio, and therefore a much better re-differentiation after de-differentiation, within 3D structures consisting of chitosan hydrogel particles compared to monolayers.
• the three-dimensional environment tested thus favors the re-differentiation of de-differentiated chondrocytes, following an intense prior multiplication, by a factor of at least 6. This 3D structure promotes the expression of the chondrocyte phenotype.
• the culture conditions (three-dimensional structure based on chitosan hydrogel, and BIT culture medium) thus favor the re-differentiation of chondrocytes and the production of cartilage matrix, compared to a monolayer culture.
• compositions obtained were then observed in immunohistochemistry, in order to compare the implementation of the new method, in three-dimensional structure, and the traditional culture in monolayers, at the level of the synthesis of MEK, proteoglycans, type I and IL collagen.
• the results are illustrated by the photos in Fig.1.1.
• proteoglycans in the three-dimensional structures are observed more clearly than in monolayers (MC in FIG. 11) thanks to the Safranine O (SO) staining, which evidence the presence of GAG.
• SO Safranine O
• the coloring of the cells within the matrix by immuno-tagging Collagen I also confirms the virtual absence of collagen I, when the chondrocytes are cultured within three-dimensional structures.
• the cells and 3D structure constitutes the end of culture. , or between 3 and 6 weeks, a cartilaginous neo-tissue that can be injected or implanted by arthroscopy.
• chondrocytes For example, for a sample of 0.3g-0.5g of human cartilage, it can be extracted 1- 1, 5x10 6 cells (chondrocytes). Insofar as the inventors have shown, in the preceding examples, that from 6.10 5 initial cells per insert, it is possible to obtain 3.6 ⁇ 10 6 cells / insert in 0.09 g of corresponding hydrogel structure. at 80-84 hydrogel particles, the following concentration data are obtained:
• the succession of steps, in the same three-dimensional medium, comprising particles of chitosan hydrogel with / without structuring molecule, amplification and then of differentiation / chondrogenesis is very favorable for obtaining a cartilaginous neo-tissue injectable or implantable having excellent mechanical and biological properties.
• hyaluronic acid improves the system by accelerating the process of amplification of the cells and making it possible to increase the number of cells to be implanted or to gain a week on the global protocol.
• Example 7 Comparison of the different 3D structures.
• the inventors have reproduced the 3D structures described in the preceding examples, in particular in Example 2, by varying the type of chitosan constituting the hydrogel particles, the size of the hydrogel particles, and the presence and concentration of acid. hyaluronic.
• the proliferation rates were compared and the results obtained are shown in Table 1.
• the value 1 was assigned to the M1 structure corresponding to particles of several hundred microns, obtained from chitosan fungi.
• the proliferation rate obtained with structure M2 is 2 times higher compared to structure M1 (mushroom extract not supplemented with hyaluronic acid), which itself makes it possible to obtain a proliferation rate 1, 5 times higher with respect to chitosan extracted from squid, or with particles of the order of tens of ⁇ .

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