THREE-DIMENSIONAL STRUCTURE COMPRISING CHITOSAN HYDROGEL PARTICLES

专利摘要:
The present invention relates to a three-dimensional structure formed of particles of physical chitosan hydrogel or chitosan derivative, associated with an anionic polymer, wherein said hydrogel particles are of average size between 10 microns and 1.5 mm. The anionic polymer is preferably hyaluronic acid or a hyaluronic acid derivative or a hyaluronic acid complex. This structure can be advantageously used to seed cells, especially chondrocytes, for the purpose of proliferation and extracellular matrix synthesis. The structure is also suitable for use in cartilage repair or for use as an implant, or as a graft or as a carrier of active ingredient in humans or animals.
公开号:FR3062064A1
申请号:FR1850498
申请日:2018-01-23
公开日:2018-07-27
发明作者:Pascale HAZOT；Frederic Mallein Gerin
申请人:Advanced Chitosan Solutions Biotech；Centre National de la Recherche Scientifique CNRS；Universite Claude Bernard Lyon 1 UCBL；
IPC主号:

专利说明:

® Agent (s): ERNEST GUTMANN - YVES PLASSERAUD SAS.
FR 3 062 064 - A1 ® THREE-DIMENSIONAL STRUCTURE COMPRISING PARTICLES OF CHITOSAN HYDROGEL. (® The present invention relates to a three-dimensional structure formed by particles of physical hydrogel of chitosan or derivative of chitosan, associated with an anionic polymer, where said particles of hydrogel are of average size ranging between 10 gm and 1.5 mm. The anionic polymer is preferably hyaluronic acid or a derivative of hyaluronic acid or a complex of hyaluronic acid. This structure can advantageously be used to seed cells, in particular chondrocytes, for the purposes of proliferation and synthesis of matrix. The structure is also suitable for use in cartilage repair or for use as an implant, or as a graft or as a vector of active principle in humans or animals.
The present invention relates to compositions allowing in particular the reconstruction of cartilage, and to a process making it possible to obtain such compositions. More particularly, the present application relates to an extremely favorable environment or structure not only for the proliferation of cells able to form hyaline cartilage, but also for the synthesis of extracellular matrix of cartilage by these cells; the cells in this environment or structure constitute an implantable composition which can be grafted to the level of cartilage lesions in humans or animals. The structure also constitutes a favorable environment during the establishment.
Cartilage, or cartilage tissue, is made up of specific cells, namely chondrocytes, distributed within an extracellular matrix, comprising at least 80% water. Chondrocytes are capable of synthesizing or degrading the components of the extracellular matrix of cartilage, composed of glycosaminoglycans and collagen fibers, essentially type II for hyaline cartilage. Chondrocytes are therefore responsible not only for synthesis but also for the maintenance of cartilage tissue.
Cartilage, and particularly articular cartilage in adulthood, has a very low capacity for self-repair, due in particular to its avascular character and to the fact that mature chondrocytes do not proliferate. Cartilage damage is therefore essentially irreversible and therefore constitutes 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 cartilage lesions, and especially large lesions.
Different surgical procedures have been tested, consisting either of filling the lesion with materials aimed at mimicking the elastic and compressible properties of natural cartilage, or of grafting cells, in particular chondrocytes, in the hope that they de novo synthesize the extracellular matrix and thus repair the lesion. A more promising approach is based on the grafting of a cartilage tissue, that is to say a neo-tissue obtained from chondrocytes. Autologous chondrocyte transplantation (“ACI” for autologous chondrocyte implantation) has been used for several decades now to treat patients with cartilage lesions. However, a major difficulty in transplanting chondrocytes, and especially autologous chondrocytes, lies in the number of cells to be implanted. Indeed, it can generally only be taken from a reduced number of chondrocytes from the patient's native cartilage which must be treated; it is therefore necessary to carry out a first stage of expansion of the sampled chondrocytes in order to drastically increase their number. However, the in vitro expansion of chondrocytes until a sufficient number of cells are obtained for implantation proves to be very delicate. During this expansion, it is in fact known that the chondrocytes indifferentiate and lose their chondrocyte phenotype, then expressing type I collagen rather than type II collagen, so that once re-implanted, they do not lead to the formation a tissue with satisfactory properties, but rather a scar-style tissue or, in the context of a cartilage repair, the formation of a non-functional fibrocartilage, essentially composed of type I collagen. Some authors have proposed culture media making it possible to redifferentiate the chondrocytes which have become differentiated, during the proliferation step in monolayers. However, these processes require relatively long times, of the order of 4 weeks for the proliferation stage (Liu et al, 2007) during which the chondrocytes must undergo several passages in order to reach a sufficient number. However, it has been observed that redifferentiation is more complicated after 2 passages in particular (Hautier et al, 2008).
Others have tested the re-differentiation of chondrocytes, after the stage of proliferation in monolayers, by seeding in 3D (three-dimensional) structures, called "scaffolds". These structures have the advantage of mimicking the architecture of the cartilage tissue. Such an environment is favorable to the re-differentiation of chondrocytes and to the synthesis of extracellular matrix as observed by the secretion of proteoglycans and type II collagen (Tallheden et al, 2005). However, the prior multiplication step is voluntarily shortened in order to limit the dedifferentiation of chondrocytes.
Seeding in 3D structure can also offer the advantage of facilitating the in vivo implantation of chondrocytes, limiting cell leakage and treating large lesions.
Many materials have been tested for the seeding of chondrocytes in 3D structure; include alginate, collagen, polyethylene glycol and polylactic acid. The most suitable are those which respond to the following biological properties: cytocompatibility, low immunogenicity, and biodegradability with non-toxic degradation products. Thus natural polymers, which have these biological properties, certainly by their chemical and biological similarities with living tissues, present themselves as candidates of choice. Natural polymers are highly biocompatible, easily bioresorbable and bioassimilable by the body.
A natural polymer often cited for its attractive properties as a basic biomaterial of three-dimensional structures, is chitosan.
Indeed, according to the literature, this biopolymer is known to be biodegradable, biocompatible, non-toxic, hemocompatible, cytocompatible, and in addition, bioactive, hemostatic, healing, bacteriostatic and fungistatic.
In addition, chitosan is known to promote cell adhesion. It is also appreciated for its ability to maintain the chondrocyte phenotype and stimulate the process of synthesis of extracellular matrix during the culture of chondrocytes. In addition, no infection or allergy related to chitosan has been reported, so it is not immunogenic. As for its resorption time, it is possible to vary it by varying its physicochemical properties.
Chitosan is relatively easy to use and can be produced in various physical forms, including solutions, films (Lahiji, et al, 2000), fibers, sponges, beads, hydrogels or microparticles . It has therefore been used in extremely varied structures.
In the context of the seeding of chondrocytes in 3D structure, particular attention is paid to the structures allowing both a good distribution and a good maintenance of the chondrocytes as well as an environment favorable to the stimulation of their chondrogenic potential. To this end, different approaches have been considered. Among the various structures tested, mention may be made of beads or fragments of material, polymers intended to encapsulate cells and hydrogels whose pore size is adjusted so that the chondrocytes can lodge there. Some authors have considered injectable compositions, with a polymer encapsulating the chondrocytes (WO2011 / 104131, Univ. De Liège et al). According to certain variants, the polymer is crosslinked in situ, once injected with the chondrocytes at the level of the lesion (Hoemann et al, 2005). However, this approach has the disadvantage that the injected chondrocytes tend not to remain at the injection site, due to the polymerization time. Conversely, other authors have tested compositions, using a cross-linked polymer in situ and allowing the production of a tissue that can be grafted in the form of a plug (Hao et al, 2010). These methods can nevertheless present difficulties during implantation.
In order to best mimic the structure of the cartilage tissue, some authors have tested the hydrogel form and more particularly the physical hydrogel, synthesized without adding a crosslinking agent and thus promoting the biocompatibility and bioresorability of the structure.
It has therefore been envisaged the development of a neo-tissue by bringing it into contact with a physical hydrogel of chitosan (Montembault et al). However, the prior multiplication step in monolayers is deliberately rapid in order to avoid or limit the dedifferentiation of the chondrocytes before inoculation, consequently implying a relatively low multiplication of the cells.
According to a variant, the three-dimensional structure is largely absorbed during the in vitro step of synthesis of the extracellular matrix, said structure is no longer part of the graft (see in particular W002078760, Laboratoires Genevrier et al). Such a process 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. .
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. In addition, it is a major component of synovial fluid and glycosaminoglycans (GAG) present in articular cartilage. 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.
As a result, hyaluronic acid has sometimes been combined with chitosan compositions, in the culture of chondrocytes.
Authors (Correia et al, 2011) have proposed, for example, three-dimensional sponge-like structures of a mixture of chitosan and hyaluronic acid for seeding chondrocytes. However, these structures do not allow a homogeneous distribution of cells with the observation of an increasing cellular gradient from the outside to the inside of the structure.
The document by Denuzière et al (1998) describes sponges of polyelectrolyte complexes, in particular based on chitosan and hyaluronic acid. The polycationic chains of chitosan interact electrostatically with the polyanionic chains of hyaluronic acid. The conclusion that emerges from this is, however, that chitosan sponges alone are to be preferred, compared to chitosan sponges complexed with GAGs such as hyaluronic acid, for the proliferation of chondrocytes and for other associated biological properties. .
A hydrogel of chitosan and photo-crosslinked hyaluronic acid has also been proposed in which chondrocytes are encapsulated (Park et al, 2013). However, such encapsulation does not sufficiently promote proliferation despite relatively long culture times (proliferation factor 4 in 3 weeks).
Furthermore, although chitosan seems to have, according to the literature, interesting properties for the reconstruction of cartilage, it should be noted that the rate of proliferation and synthesis of extracellular matrix of chondrocytes on chitosan sponges alone strongly depends on the size of the pores of the structure (Griffon et al, 2006).
Some authors have even considered that chitosan is not adapted to the proliferation of chondrocytes (Suh et al, 2000).
Currently, none of these 3-D (three-dimensional) environments solve the problem of proliferation of chondrocytes, in a state compatible with the regeneration of cartilage tissue for transplantation. There is therefore a great need to obtain, according to a rapid process, a satisfactory number of chondrocytes, which are differentiated and capable of generating a cartilage tissue with adequate properties.
In addition, all of the methods propose environmental changes between the multiplication step and the differentiation step, which involves loss of material and time as well as the use of trypsinization, which is harmful to cells.
The present inventors have developed a process for obtaining sufficient chondrocytes, within a structure ensuring at the same time their perfect multiplication, their redifferentiation and the production of cartilage matrix. This structure is also suitable for implantation at the level of the lesion.
In this context, the present inventors have found, quite unexpectedly, that chitosan, in the form of physical hydrogel particles, is an excellent environment for chondrocytes, not only for the synthesis of extracellular matrix, but also in view of their multiplication. Indeed, against all expectations, the inventors were able to carry out in the same chitosan structure, a proliferation step making it possible to obtain a proliferation rate similar or even higher than that observed by the ordinary technique of monolayers (on plastic or in sponges ) and then easily allowing the redifferentiation of cells. The inventors have therefore perfected a process, successively making it possible to multiply chondrocytes, directly after extraction in the primary state, then to induce re-differentiation and synthesis of extracellular matrix, within the same structure, thus avoiding the trypsination and structural change stages. In addition, this structure is compatible with in vivo implantation without requiring any additional modification.
Thanks to the process of the invention, it is thus possible to obtain a composition comprising chondrocytes, at a density allowing their reimplantation, in a reduced time compared to what has been described to date, under very favorable conditions. the repair of cartilage tissue, in a structure directly compatible with implantation.
In the context of this description, the following terms have more particularly the following meaning:
By chitosan is meant a polysaccharide composed of D-glucosamine units (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit) linked in β- (1-4). It can be produced by chemical or enzymatic deacetylation of chitin; it also exists in its natural state. In naturally occurring chitosan, the polysaccharide is generally associated with negligible amounts of beta-glucan. In the context of the present invention, the natural presence of 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 betaglucan, as long as the presence of the latter remains less than 5% by mass, relative to the polysaccharide.
The term “chitosan derivative” denotes any polymer of chitosan which has undergone a reaction aimed at modifying the chemical groups of chitosan to change its functionalities, for example methylation, halogenation, etc. Preferably, a chitosan derivative 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 envisaged in the context of the present invention are glycol-chitosan, / V-succinyl chitosan, N- or 0-Carboxymethyl Chitosan, without this list being limiting.
The degree of acetylation (DA) is the percentage of acetylated units compared to the number of total units (acetylated and deacetylated units), it can be determined by Fourier transform infrared spectrometry (IRTF) or by proton NMR.
By chitosan hydrogel, it is meant that the very majority constituent, therefore more than 80% or even more than 90%, even 95%, of the hydrogel (by mass) is chitosan, apart from water. Similarly, a hydrogel of chitosan derivative means a hydrogel of which the very majority constituent, more than 80%, even more than 90% or 95% of the hydrogel is the derivative of chitosan, apart from the water.
By physical hydrogel is meant a hydrogel obtained by a gelling process in an aqueous medium or in an alcoholic medium, not requiring the addition of crosslinking agent.
By chondrocyte phenotype, we mean cells of the chondrocyte type, preferentially expressing collagen type II over collagen type I.
By hyaluronic acid is meant a polysaccharide composed of units of Dglucuronic acid and D-N-acetylglucosamine, linked together by glycosidic bonds.
The term “hyaluronic acid derivative” denotes any polymer of hyaluronic acid which has undergone a reaction aimed at modifying the chemical groups of hyaluronic acid to change its functionalities, for example by esterification.
The present invention therefore relates, according to a first aspect, to an in vitro process for obtaining a composition, which can be implanted by arthroscopy for cartilage repair, comprising particles or fragments of physical hydrogel of chitosan 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 qualified as cartilage gel. Such a cartilage gel in fact comprises cells, synthesizing the cartilage matrix, all in a three-dimensional structure based on chitosan.
Such a method according to the invention notably comprises a step of amplifying primary cells, in a three-dimensional structure (3D structure) then a step of redifferentiation and of induction of the synthesis of extracellular matrix, within this same three-dimensional structure, that is to say without changing the environment cells.
As indicated above, chitosan is indeed a biocompatible, bioresorbable biomaterial with non-toxic, non-immunogenic, cytocompatible and bioactive degradation products. It is also fully compatible with pharmaceutical requirements as an implantable device. In the context of the invention, the chitosan or chitosan derivative hydrogel is a physical hydrogel obtained without the addition of crosslinking agent.
By this method, it is possible to obtain a composition or neo-tissue comprising chondrocytes, within a matrix structure, which is immediately ready for implantation, with undamaged chondrocytes capable of continuing the synthesis of extracellular matrix. after relocation. Due to the absence of a 3D structure change, the process is easier to implement and makes it possible to obtain a good quality neo-fabric. This process is also faster than those described in the prior art.
The process according to the invention is therefore characterized by the following stages:
(i.) Amplification of primary cells, in a three-dimensional structure comprising physical hydrogel particles of pure chitosan or of chitosan derivative, then (ii.) Induction of differentiation, or re-differentiation, and of matrix synthesis extracellular by said amplified cells, within the three-dimensional structure of step (i.).
These two stages take place successively and not simultaneously in order to optimize the conditions and the yields of each of these stages.
Cells :
The cells which are amplified, in the first step, are seeded in this 3D structure. They can be either chondrocytes or precursor cells of chondrocytes obtained from stem cells, for example mesenchymal stem cells, or induced pluripotent cells (IPS). They can be any cells differentiated into chondrocytes. Preferably, these are chondrocytes, and more preferably articular chondrocytes.
In the context of the invention, it is also possible to use cocultures of differentiated chondrocytes and / or stem cells differentiated into chondrocytes.
The chondrocytes or the stem cells can be obtained by any method known to a person skilled in the art making it possible to recover these cells from a biological sample capable of containing them. Such methods are in particular described in document FR2965278 (Univ. De Caen Basse-Normandie, et al).
They are preferably human or animal cells, in particular equines or canines. These can be joint, ear or even cells 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, in particular for a horse or a dog.
The primary cells seeded in the structure can be allogeneic, xenogenic, heterologous, or autologous cells, vis-à-vis the organism to be treated. According to a preferred implementation, these are autologous cells, that is to say which originate from the patient, human or animal, which must be treated. Most preferably, these are autologous human chondrocytes, which can be reimplanted in the donor at the end of the process of the invention. It is the same for animal chondrocytes, for example for racehorses or dogs, which are also likely to benefit from a chondrocyte transplant. It can also be xenogenic or allogeneic cells, since certain measures well known to those skilled in the art can be implemented to avoid rejection during implantation.
According to one embodiment, these are not human embryonic stem cells.
The three-dimensional structure and the chitosan:
The cells are seeded within a three-dimensional or scaffold structure, or else biomaterial, comprising fragments or particles of physical hydrogel of pure chitosan, or of chitosan derivative. Said particles thus form a scaffold or structure compatible and favorable for the formation of three-dimensional tissue, until the cells produce enough extracellular matrix to maintain the structure mechanically. The chitosan used for the design of the device or three-dimensional structure is obtained for example by deacetylation of the chitin which can come from the exoskeleton of arthropods (shrimps, insects, crab ...), from the endoskeleton of the cephalopods (squid) or the cell wall of fungi. Depending on its origin, chitosan can be of conformation a (cell wall of fungi, shrimps, crabs) or β (squid) or φ (insects), which strongly influences its biological properties.
In the context of the present invention, 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. pharmaceutical standards. Preferably, the chitosan used in the context of the present invention is chitosan extracted from the cell wall of fungi, more particularly button mushrooms, Agaricus bisporus. Indeed, in the context of implantable devices, due to regulatory requirements, it is particularly advantageous to have no material which is of animal origin. The chitosan used in the present invention, extract of button mushrooms, complies with pharmaceutical requirements in terms of endotoxin, microbiological residue and heavy metal levels.
Succession of stages:
The method of the invention is characterized in particular by the succession of two stages, a first stage of amplification of primary cells in a three-dimensional structure and a second stage of induction of differentiation and synthesis of extracellular matrix (ECM), at the within this same three-dimensional structure.
The major advantage of this process lies in the fact that it is not necessary to change the cells of structure, between the amplification step and the re-differentiation step with synthesis of MEC, or for that matter between the re-differentiation stage and the establishment stage. It follows that at no time is it necessary to carry out a step of trypsinization of the cells. In fact, after the extraction, the primary cells are seeded in the structure of the invention, without prior proliferation step, and therefore without the need to take them off, in particular by trypsinization, or by any other means liable to damage the wall cellular. They then proliferate within the structure, then are induced to redifferentiate and produce cartilaginous matrix, once again without it being necessary to take them off, to trypsinize them, or to subject them to any other treatment of nature to damage the cell wall.
The method of the invention therefore makes it possible to obtain cells which, after extraction, have not been damaged and have not undergone stress conditions, which makes it possible to guarantee not only an optimization of the number of cells samples, by reducing cell death, but also guarantees that the cells, at the end of the process, are not in cell death programs and do not express signals which could be harmful to the host organism, after reimplantation .
Preferably, 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 MEC, are understood to be distinct steps . The inventors have in fact observed that the capacity to proliferate and the capacity to produce MEC are preferentially successive stages for cells such as chondrocytes, so that the yields of proliferation and synthesis of cartilage matrix were much better when the two stages were distinct. In addition, there are environments that can favor one or the other of these stages, exclusively, so that it is preferable to carry them out one after the other.
Collagen type II (COLII) is a characteristic marker of hyaline cartilage; it is a homo-trimer of three a1 (ll) chains, encoded by the Col2a1 gene. The analysis of this type of collagen is conventionally carried out to identify differentiated chondrocytes.
The typical collagen (COLI), heterotrimer α1 ₂ a2i produced from the Collai and Col1a2 genes is classically considered as a marker for the de-differentiation of chondrocytes.
It is considered that the amplification stage is distinct from that of the synthesis of cartilage matrix when in particular the levels of messenger RNA transcribed COLII remain very low during the amplification stage and increase sharply during the second stage after redifferentiation.
Furthermore, it is considered that the amplification stage is distinct from that of the synthesis of cartilage matrix according to the viscosity of the medium: with the observation of a low viscosity during the amplification stage and an increased viscosity during of the second step, confirming the production of extracellular matrix.
Preferably, during the first step, the synthesis of extracellular matrix is negligible. It is also possible to observe whether such a matrix is synthesized by using, for example, immunohistochemistry methods, such as those described in the examples below.
Preferably, during the second stage, the number of chondrocytes is stable, there is little or no amplification.
The method according to the invention is characterized by maintaining the capacity of the cells which have been seeded within the structure to redifferentiate into chondrocytes. By maintaining this capacity, it is meant that the majority of cells, at the end of the second step, have a chondrocyte phenotype, preferably at least 60%, preferably at least 70%.
The inventors have in fact observed that the majority of cells seeded in the structure as described, based on chitosan hydrogel or a derivative, exhibit a stable chondrocyte phenotype during the second step of the process of synthesis of extracellular matrix. At the end of the process, the cells within the composition show 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. Preferably, 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 messenger RNA of type I.
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, for example in less than 28 days, or even less than 21 days. It therefore allows, 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.
To this end, 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.
During this stage, the multiplication of the number of living cells, compared to the number of cells initially seeded in the structure, is at least 4, even at least 6, or even at least 7 or more than 7.
Of course, it may be decided to extend the multiplication stage for a sufficient time to obtain a determined number of cells, provided that the confluence is preferably not reached. The density of the cells during seeding in the 3D structure must of course be adapted accordingly.
According to a preferred implementation, the multiplication stage 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.
Consequently, a biopsy of 300 to 500 mg of cartilage, comprising approximately 10 ⁶ to 1.5 x 10 ⁶ cells makes it possible to obtain, at the end of the amplification step, from 4 × 10 ⁶ cells to 10, 5 x 10 ⁶ cells due to the amplification factor observed by the inventors, and this in 1 to 3 weeks. Such a number of cells is considered suitable for the implantation of a chondrocyte transplant.
The second stage, linked to re-differentiation accompanied by synthesis of extracellular matrix, can have a variable duration.
In view of the reimplantation of the cartilage gel, it is however preferable that such a stage lasts between 2 and 4 weeks, preferably about three weeks, or much less. The duration of this second stage can possibly be adjusted according to the chosen mode of reimplantation.
At the end of the implementation of the process of the invention, there is therefore obtained a cartilage composition or gel, comprising chondrocytic cells, distributed in a newly synthesized cartilage matrix, and which are capable of continuing the synthesis of tissue cartilaginous, within a 3D structure composed of particles of physical hydrogel of chitosan 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 cartilaginous joint, for example following traumatic and limited lesions of the articular cartilage, or lesions of the beginning superficial arthritis type, or deeper lesions of the osteochondral type.
At the end of the process, a kit or gel of cartilage ready for injection or in vivo implantation in a human or animal is obtained. If the 3D structure comprising the particles of chitosan hydrogel or the chitosan derivative, can be partially biodegraded during the process, it is preferably very little, preferably less than 50% of the initial 3 D structure based on particles of chitosan hydrogel before sowing.
Depending on the duration of the stages of the process of the invention, in particular of the stage of synthesis of MEC, it is also conceivable that the composition of the invention or cartilage gel can be injectable in a human being or an animal. To this end, the duration of the second stage of synthesis of MEC will be reduced by a few days, in order to ensure that the composition remains injectable.
Conversely, in particular when it is desirable to obtain an implant having a well-defined shape, 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. . It is also possible to produce the structure directly in a container having the desired shape and to carry out the various stages of the process in this container in such a way that, at the end of the stage of synthesis of cartilaginous matrix, the cartilage gel have the shape induced by the container.
The composition as described above comprises hyaline cartilage, synthesized by the cells during the second step of the process, hence its name cartilage gel.
A major advantage of the present invention resides in fact in the directly implantable nature of the composition obtained at the end of the process. In particular, there is no need to change the 3D structure cells, so there is no need to subject them to any treatment. There is also no need to ensure the disappearance of the structure, nor to wait for such degradation. On the contrary, according to the method of the invention, the structure based on chitosan is always present at the end of the stage of synthesis of MEC and is part of the composition, or neo-tissue, intended to be implanted. Indeed, such a structure serves as a scaffold ensuring the maintenance of the cells in an adapted environment such that it allows, after implantation, the continuation of the synthesis of MEC, in order to be perfectly inserted at the level of the lesion, for example articular. The structure of chitosan is therefore not the only vocation 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 reimplanted neo-tissue.
The structure also ensures 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 as a function of the size of the lesion which it is desired to fill, and also as a function of the number of cells which it is conceivable to remove for the purpose of seeding. . Preferably, however, at least about 10 ⁵ primary cells are seeded, in particular at least about 6x10 ⁵ primary cells, or at least 10 ⁶ primary cells, preferably primary human, canine or equine chondrocytes; at the end of the process of the invention, the final composition preferably comprises at least 3 × 10 ⁵ chondrocytes, or at least ⁶ × 10 ⁶ chondrocytes, or even more. With a view to obtaining a composition which can be directly implanted in a cartilage lesion, it will preferably be seeded 10 ⁶ to 1.5 x 10 ⁶ cells making it possible to obtain, at the end of the amplification step, from 4 to 10 , 5 x 10 ⁶ cells.
According to an implementation of the invention, the chondrocytes within the composition are at a concentration of approximately at least 10 ⁶ cells / g, preferably approximately at least 6x10 ⁶ cells / g of 3D structure at the time of seeding.
Culture media:
According to a particularly preferred embodiment, two different culture media are used. One for the first amplification stage and the other for the second stage of induction of re-differentiation and synthesis of MEC; the passage from one to the other of the stages being therefore achieved by the change of medium, the two mediums being distinct.
It is notably envisaged to use, during the first step, a medium which promotes cell proliferation, without inducing the synthesis of MEC. The proliferation of cells is generally accompanied by a phenomenon of de-differentiation; however, a medium will be used which preserves their ability to redifferentiate into chondrocytes at the end of the proliferation stage.
The inventors have in fact highlighted the fact that, within a three-dimensional structure, it was preferable to carry out an intense amplification step, without however inducing the synthesis of MEC.
Indeed, three-dimensional structures and chitosan were known before the invention for their ability to promote the chondrocyte phenotype by limiting dedifferentiation, during the step of synthesis of extracellular matrix. Unexpectedly, the inventors have shown that it is possible to amplify the chondrocytes without inducing the synthesis of extracellular matrix, within a three-dimensional structure based on chitosan hydrogel or a derivative.
A medium particularly suitable for the proliferation step is a medium inducing amplification, in particular a medium comprising fibroblast growth factor (FGF-2, “fibroblast growth factor”), and incidentally also insulin, corresponding to a medium known as “F1” illustrated in the experimental part (Claus et al, 2012). FGF-2 is preferably present between 2 and 10 ng / mL and insulin between 2 and 10 pg / mL. However, other culture media, well known to those skilled in the art, can also be used, in particular all the culture media generally used for this type of cell, but in monolayers. Because of the three-dimensional structure and chitosan, the cells seeded in the structure of the invention retain their round morphology and their ability to subsequently differentiate into chondrocytes.
For the culture medium of the second step, preferably using media conventionally used to promote the differentiation or re-differentiation of dedifferentiated chondrocytes and allowing the synthesis of specific MEC. Such environments are well known to those skilled in the art. A very particularly 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 of triiodothyronine T3 (Liu et al, 2007, Claus et al, 2012), corresponding to a medium says "BIT". BMP-2 is preferably at a concentration between 100 and 500 ng / mL, insulin between 2 and 10 pg / mL and triiodothyronine T3 between 50 and 250 mM.
The BMP-2 will preferably be chosen from the same species as the cells used, ie a human BMP-2 for human cells. The same is true for animal cells.
As well for the culture medium of the first stage as that of the second, culture mediums which will not oppose the subsequent reimplantation of the composition, will be preferred, at the end of the stage of synthesis of MEC. In particular, compounds which risk generating rejection reactions, which are not compatible with the regulations relating to implantable devices, will be avoided.
Incidentally, it is possible to envisage stages of elimination of the culture medium before the injection or the reimplantation at the end of the two stages mentioned above.
The first and second stages can, independently of one another, take place in normoxia or hypoxia conditions.
Realization of chitosan hydroqel:
The hydrogel of pure chitosan, or of chitosan derivative, is produced from chitosan, preferably extracted from fungi and whose average molar mass by weight (Mw), will preferably be greater than 150 kDa (ie 150,000 g / mol ) to promote the physical gelling process by the presence of long macromolecular chains. It will preferably be between 150 and 220 kDa.
If the chitosan has a widely different molar mass, in particular if it is extracted from another source, the process for producing the physical hydrogel of chitosan as described in the experimental part can be easily adapted by a person skilled in the art , using well known techniques.
In addition, it is also possible to use chitosan, for the hydrogel, which has a variable degree of acetylation; preferably, however, the degree of acetylation of the 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 in fact induces a favorable environment for the cells, leading to good adhesion between the hydrogel formed and the cells, and good results in chondrogenesis. Indeed, by increasing the degree of acetylation, one favors the cationic character of the hydrogel, by increasing the cationicity of the residual amine sites, and by this way, one favors indeed the interaction with the cells, via electrostatic interactions .
With a view to the formation of the hydrogel, a solution of chitosan is preferably used, the concentration of which is high enough to allow the entanglement of the macromolecular chains, and thus to promote physical gelling. The hydrogel is in fact obtained within the framework of the invention by an entirely physical process, without any chemical crosslinker. The concentration of chitosan in solution can be between 0.5-4% (w / w), preferably greater than 1.5%, or even 2%. Preferably, the chitosan, or the chitosan derivative has a mass concentration in the hydrogel of between 3.4 and 4.2% before neutralization.
Several gelling methods for chitosan can be used in the context of the present invention. Mention may in particular be made of the following processes, which are particularly well suited, such as physical gelling by gas (ammonia) or physical gelling in an aqueous or alcoholic medium.
Preferably, the hydrogel used in the context of the present invention is obtained by an evaporation process in alcoholic medium, as illustrated in example 1 in the experimental part.
The hydrogel obtained is preferably between 3 and 5 mm thick.
The pore size of the hydrogel obtained must be both smaller than the size of the cells and both sufficient to allow the free diffusion of nutrients and the elimination of waste. The embodiments described above and implemented in the experimental part make it possible to obtain such a pore size. It should be noted that, within the framework of the present invention, the hydrogel of chitosan or of derivative of chitosan is such that the size of its pores does not allow penetration of the cells inside the hydrogel. The cells seeded within the three-dimensional structure according to the invention therefore proliferate without entering the hydrogel. A person skilled in the art is able to determine the size of the pores of a hydrogel and to adjust the parameters of its production in order to ensure that the size of the pores is sufficiently small to prevent penetration of cells, in particular of chondrocytes. , while nevertheless allowing the free diffusion of nutrients.
In order to obtain the hydrogel particles constituting the basis of the three-dimensional structure of the invention, the hydrogel thus obtained is handled everywhere suitable means, well known to those skilled in the art.
The hydrogel particles thus obtained are irregular in shape, but preferably have a relatively homogeneous size distribution, that is to say that 50% of the particles have a size of between -20% and + 20% of the size. average. According to a preferred implementation, the particles have an average size of between 10 μm and 1500 μm; preferably between 200 µm and 1200 µm (1.2 mm), and more preferably between 400 µm and 700 µm. By particle size is meant 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 comparable to ellipses.
The inventors have shown better results for particles of an average size greater than a hundred microns, in particular beyond 60 μm, or even beyond 400 μm and less than 1.2 mm.
A certain variability in the particle sizes of chitosan hydrogel seems to be favorable for the culture of chondrocytes.
Design of the three-dimensional structure:
The three-dimensional structure according to the invention therefore comprises particles of physical hydrogel of chitosan or one of its derivatives, as detailed above, constituting a three-dimensional structure within which the cells are seeded, or migrate naturally. The physical hydrogel of pure chitosan or of chitosan derivative preferably consists only of chitosan or of a derivative, and of water with a content preferably at least 70%, preferably at least 80%. In particular, it does not enter into the composition of this hydrogel, neither chemical crosslinking agent, nor other polymer, in particular polysaccharide or derivative, apart from the β-glucan naturally associated with chitosan.
According to a particularly preferred implementation, an anionic molecule is added to the particles of chitosan hydrogel or of chitosan derivative making it possible to reinforce the mechanical and biological properties of the three-dimensional structure. This molecule is not part of the hydrogel composition, it is a constituent added after gelling of the chitosan hydrogel or chitosan derivative.
The anionic molecule is preferably in the form of a polymer, it is for example hyaluronic acid, or chondroitin sulfate. The chitosan chains exhibiting positive charges, due to the amino groups in protonated form NH ³⁺ , and the chains of this anionic molecule interact by electrostatic bonds, thus forming a stable complex in physiological medium (pH between 5 and 8, and more generally between 6 and 7). The anionic molecule thus allows electrostatic crosslinking of the fragments or particles of hydrogel, thus reinforcing the mechanical properties of the three-dimensional structure or 'scaffold' within which the cells are seeded.
It should be noted that the anionic molecule can be added to the three-dimensional structure before seeding with the primary cells, and therefore be present during the stage of proliferation and synthesis of MEC, and therefore also during the subsequent implantation of the composition. In the context of the invention, 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 from the first step when starting the second step of redifferentiation, 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 synovial fluid, known for its chondroprotection properties and favorable for chondrogenesis. The amount of hyaluronic acid necessary to modify the visco-elastic characteristics of the three-dimensional structure is thus added. Electrostatic interactions occur between the amino groups in NH3 + protonated form of chitosan and the carboxylic groups of hyaluronic acid.
It can also be a derivative of hyaluronic acid or a complex of hyaluronic acid. The relative proportion of the anionic molecule with respect to the chitosan hydrogel is preferably between 1 and 10%, preferably between 1 and 3%.
The hyaluronic acid can be of animal origin, for example by extraction of cock crest or of non-animal origin, obtained by bacterial fermentation. In the context of the present invention, the hyaluronic acid will most preferably be chosen 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 complies with pharmaceutical standards.
Its molecular weight by weight is preferably from 50 kDa to 4 MDa, it will preferably be chosen greater than 500 kDa, preferably between 500 kDa and 2 MDa, for example between 1 MDa and 2MDa.
Very particularly preferably in the context of the present invention, the constituents of the three-dimensional structure, which are particles of chitosan hydrogel or of chitosan derivatives, with or without addition of chains of anionic compound, 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 absorbable nature.
Preferably, the association of the particles of chitosan hydrogel or of chitosan derivatives, with or without the chains of anionic compound, will be absorbed after several weeks once implanted, for example after at least two weeks, preferably at minus 4 weeks. It is generally preferred that the absorption time does not exceed 6 months, preferably even does not exceed 4 months. Depending on the type of application envisaged, the absorption time can be adjusted by a person skilled in the art.
It is important to note that the compositions according to the present invention can be adapted in terms of shape, diameter, concentration, content depending on the different applications envisaged. In particular, the three-dimensional structure formed of particles of chitosan hydrogel or of chitosan derivative, with or without the chains of anionic compound, can be worked out so that its shape corresponds to that of the lesion observed in which it will be reimplanted at the resulting from the process according to the invention.
As specified above, 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 the dog or the horse.
However, before its implantation, it can be envisaged to change the medium, or else to add additional compounds, in particular soluble within the three-dimensional structure. For example, it is conceivable to add pharmaceutical compounds such as anti-inflammatory agents, anesthetics, analgesics, corticosteroids, vitamins, minerals, compounds aimed at reducing the immune response, and / or promoting transplantation, all or part of these compounds or a combination of these compounds, without this list being exhaustive.
According to a second aspect, the present invention relates to a three-dimensional structure formed of particles of physical hydrogel of chitosan or of a derivative of chitosan, and of an anionic molecule associated with these particles. Such a matrix can advantageously be used to seed chondrocytes by making them proliferate and then synthesize extracellular matrix, before being implanted or injected, in the form of cartilage gel, in particular 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 cartilaginous tissue. It should be noted that the composition, or cartilage gel, also comprises cartilage matrix, synthesized by the chondrocytes which 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 process of the invention. The preferred implementations within the framework of this first aspect are also preferred within the framework of this second aspect. In particular, the chitosan is preferably a chitosan obtained from mushrooms, and more particularly extracted from the cell wall of button mushrooms, Agaricus bisporus. The particles have the sizes specified above, ie between 10 μm and 1200 μm, preferably between 400 and 700 μm on average. The structure is therefore preferably a 3D structure formed of particles of physical hydrogel of chitosan, of average size between 400 μm and 700 μm, where said chitosan is extracted from button mushrooms.
As regards the anionic molecule, as specified for the process of the invention, it is preferably a polymeric molecule and most preferably hyaluronic acid or a derivative of hyaluronic acid or a complex of hyaluronic acid, and more specifically hyaluronic acid from bacterial fermentation.
The differentiated chondrocytes, present within the composition of the invention, are for example articular chondrocytes. Most preferably, these are human or animal chondrocytes, in particular canine or equine. The chondrocytes are differentiated chondrocytes, having a chondrocyte phenotype, in particular having a COLII / COLI differentiation index greater than 1. Preferably, the chondrocytes present in this composition mainly 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 collagen type II is significantly higher than the level of messenger RNA of type I, with a ratio of the COLII / COLI transcription level of the cells greater than 1, for example greater than 100, or greater at 1000.
According to an implementation of the invention, the relative proportion of chondrocytes within the composition corresponds to a concentration of between 10 ⁶ and 10 ⁷ cells / g of 3D hydrogel structure, at the time of seeding.
The composition of the invention may also include other compounds or molecules, and in particular the extracellular matrix (ECM).
Preferably, a composition as described, or cartilage gel, is capable of being obtained by implementing the method of the invention, in particular by seeding the three-dimensional structure with cells, then their proliferation, followed by their re-differentiation accompanied by synthesis of MEC.
It may also be envisaged to add additional compounds, in particular soluble compounds, for example chosen from anti-inflammatory agents, anesthetics, analgesics, corticosteroids, vitamins, minerals, compounds intended to reduce the immune response, and / or to promote the transplant, all or part of these compounds or a combination of these compounds without this list being exhaustive.
As described in the context of the method of the invention, the three-dimensional structure as described is advantageously absorbable, in particular bioresorbable in vivo. The properties of the chitosan hydrogel or the chitosan derivative will be chosen according to the desired time scale until the complete absorption of the three-dimensional structure, once the composition is implanted. Preferably, the resorption time will be adapted so that such resorption is carried out concomitantly with the synthesis of the cartilage matrix by the chondrocytes present in the composition; preferably, the resorption time will be adjusted so that the cartilage matrix formed by the chondrocytes completely replaces the three-dimensional structure of chitosan hydrogel or chitosan derivative.
According to another aspect of the invention, 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 therapeutic and / or surgical use, in particular for use as an implant or graft in the repair or reconstruction of cartilage tissue, or in the treatment of osteoarthritis, and more generally in the treatment of any pathology characterized by degradation or disappearance of cartilage tissue, in particular cartilage lesion, for example following traumatic and limited lesions of the articular cartilage, or deeper lesions of osteochondral type. Such a composition for use in vivo is especially envisaged in surgery, in rheumatology, or as a vector of active principle. The composition, or cartilage gel, is implantable by arthroscopy.
A use especially envisaged is the transplant of chondrocytes. Preferably, the chondrocytes present in the composition to be grafted are autologous or allogenic cells, preferably it is human, canine or equine chondrocytes.
According to yet another aspect, the present invention also relates to a three-dimensional structure formed of particles or fragments of physical hydrogel of pure chitosan or of a chitosan derivative, and its use for the seeding of cells in vitro, in particular for the purposes of proliferation and synthesis of extracellular matrix. The three-dimensional structure is as described above; the same is true for chitosan or chitosan derivative hydrogel. The latter is preferably extracted from button mushrooms as explained above, having a weight-average molar mass preferably between 150 and 220 kDa. The hydrogel particles preferably have an average size between 200 µm and 1.2 mm, and most preferably between 400 and 700 µm. An anionic molecule is preferably added to the hydrogel particles of the three-dimensional structure; it is preferably an anionic polymer, most particularly hyaluronic acid or a derivative of hyaluronic acid or a complex of hyaluronic acid, in particular obtained by bacterial fermentation.
All the preferred embodiments detailed above concerning the elements of the three-dimensional structure are also applicable to this aspect of the invention, and more particularly the following characteristics: the hydrogel particles preferably have a size of between 200 μm and 1 , 2 mm, preferably between 400 and 700 μm, and / or the chitosan has a weight-average molar mass preferably greater than 50 kDa, preferably between 150 and 220 kDa, and / or the chitosan has a degree of acetylation of between 5 and 60%, preferably between 28 and 40%, and / or hyaluronic acid has a weight-average molar mass 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%. As described above, this three-dimensional structure is advantageously used for seeding primary cells, in particular primary chondrocytes, or primary stem cells differentiated into chondrocytes, in particular mesenchymal stem cells. However, other types of cells can be inserted into this three-dimensional structure, in particular bone cells, fibroblasts, keratinocytes, or combinations of some of these cells, without this list being exhaustive. The present inventors have in fact demonstrated that this three-dimensional structure provides a three-dimensional architecture which is particularly favorable to cells, whether in phases of proliferation or multiplication, than in phases of synthesis of extracellular matrix. Furthermore, as described above, this three-dimensional structure is biodegradable and bioresorbable, and can therefore be implanted in vivo, in humans or animals, once seeded with cells.
In the context of the present invention, it is also envisaged to implant in vivo such 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 with an anionic molecule, preferably a polymer, in particular hyaluronic acid or derivative of hyaluronic acid or a complex of hyaluronic acid, said structure being devoid of cells, such that the structure 3D is precisely colonized, in vivo, by cells.
The three-dimensional structure as described in the context of the various aspects of this invention is preferably sterilized before any seeding.
The present invention relates in particular to the following additional aspects:
According to a first aspect, the invention relates to an in vitro process for obtaining an implantable composition for tissue repair of cartilage comprising particles of chitosan hydrogel and cells capable of forming hyaline cartilage, said process comprising the steps following:
(i.) Amplification of primary cells, in a three-dimensional structure comprising particles of physical hydrogel of chitosan or of chitosan derivative, then (ii.) Induction of the synthesis of extracellular matrix by said amplified cells, within the structure three-dimensional in step (i.), wherein said cells are primary articular chondrocytes and / or primary mesenchymal stem cells differentiated into chondrocytes
According to a second aspect, the invention also relates to a method according to aspect 1, wherein said method does not include any step of trypsinization of the cells.
According to a third aspect, the invention also relates to a method according to aspect 1 or 2, in which the induction step (ii.) Of the synthesis of extracellular matrix is carried out by changing the culture medium.
According to a ^4th aspect, the invention relates to a method according to any one of aspects 1 to 3, wherein said composition is carried out in less than 45 days, preferably less than 35 days, in particular less than 28 days.
According to a ^5th aspect, the invention relates to a method according to any of the preceding aspects, wherein the amplification step lasts between 1-3 weeks, preferably less than two weeks, and allows the multiplication of the number of cells by at least 4, preferably by
6.
According to a ^6th aspect, the invention relates to a method according to any one of aspects 1 to 5 wherein the extracellular matrix synthesis step lasts between 2 and 4 weeks, preferably less than three weeks.
According to a ^7th aspect, the invention relates to a method according to any one of aspects 1 to 6 wherein the hydrogel particles are of mean size of between 10 microns and 1.5 mm, preferably between 400 pm and 700 pm .
According to an 8 ^th aspect, the invention relates to a process according to any one of aspects 1 to 7 in which the chitosan has a weight-average molar mass greater than 50 kDa, preferably between 150 and 220 kDa.
In a ^ninth aspect, the invention relates to a method according to any one of aspects 1 to 8 in which the chitosan is extracted from fungi, preferably from Agaricus bisporus.
According to a io ^th aspect, the invention relates to a method according to any one of aspects 1 to 9 in which the three-dimensional structure also comprises an anionic polymer, preferably hyaluronic acid, which is present during the two stages or else added at the end of step (i.) or (ii.).
In a n ^th aspect, the invention relates to a process according to any one of aspects 1 to where hyaluronic acid is extracted from bacterial fermentation, with a weight-average molar mass greater than 1MDa, preferably greater than 2 MDa.
I2 in a ^third aspect, the invention relates to a method according to any one of aspects 1 to where the amplification step is performed with a medium comprising FGF-2 (fibroblast growth factor) and insulin.
I3 according to a ^third aspect, the invention relates to a method according to any one of aspects 1 to 11 wherein the synthesis of extracellular matrix induction step is performed with a medium comprising the BMP-2 (bone morphogenic Protein - 2), insulin and triiodothyronine T3.
According to I4 ^th aspect, the invention relates to a method according to any one of aspects 1 to 13, wherein said cells are articular chondrocytes or from the nasal septum or ear, of human, canine or equine.
According to a ^15th aspect, the invention relates to an implantable composition comprising a three-dimensional structure formed of physical hydrogel particles of chitosan or chitosan derivative, together with an anionic polymer, preferably hyaluronic acid and differentiated chondrocytes or all cells differentiated into chondrocytes.
According to I6 ^th aspect, the invention relates to an implantable composition according to the ^15th aspect, wherein the chondrocytes synthesize preferably of type II collagen protein and type II collagen mRNA, expressing a ratio COLII / COLI greater than 1 .
In a I7 ^th aspect, the invention relates to an implantable composition according to any one of aspects 15 and 16, obtainable by the process according to any one of aspects 1-14.
According to an ^18th aspect, the invention relates to a composition implantable by arthroscopy according to any one of aspects 15 to 17, for use in cartilage repair.
I9 according to a ^third aspect, the invention relates to a formed three-dimensional structure of physical hydrogel particles of chitosan or chitosan derivative, associated with a hyaluronic acid polymer, wherein said chitosan is extracted from fungi, and wherein said hydrogel particles are of average size between 400 µm and 700 µm.
According to a ^20th aspect, the invention relates to the use of a three-dimensional structure formed of physical hydrogel particles of chitosan or chitosan derivative, together with an anionic polymer, preferably hyaluronic acid, for the inoculation of chondrocytes for the purpose of proliferation and synthesis of extracellular matrix of said chondrocytes.
FIGURES:
Fig. 1: shows a photograph of a physical hydrogel of pure chitosan, obtained by scanning electron-microscopy.
Fig. 2: shows a photograph of a physical hydrogel of dehydrated pure chitosan, obtained by scanning electron microscopy.
Fig. 3: shows a photograph of physical hydrogel particles of pure chitosan, after treatment with eosin, obtained by optical microscopy.
Fig. 4: shows the survival rate of cells seeded in a 3D M1-type structure, as a function of the initial density of chondrocytes, measured with the Live and Dead kit on culture fractions at 7 days (in the process of amplification , in middle Fl). The enumeration of dead cells (in black) and of living cells (grayed) is carried out with the ImageJ software from snapshots taken under fluorescence microscopy at x20. The percentage of dead cells is calculated for each condition by making the ratio of dead cells / total cells.
Fig. 5: shows the evolution over time of the population of chondrocytes in 3D structures based on physical hydrogel particles of chitosan extracted from fungi, whether or not supplemented with hyaluronic acid, with particle sizes different, or within 3D structures based on particles of physical hydrogel of chitosan extracted from squid, or of chondrocytes cultivated in monolayers, under the culture conditions “F1”. The number of cells is plotted, the culture time in days is plotted on the abscissa
Fig. 6: shows the evolution of the population of chondrocytes as a function of time for different initial densities of chondrocytes cultivated in monolayers, under the culture conditions “F1”, confirming that the cell population obtained is identical from 7 days onwards or the initial density.
Fig. 7: shows optical microscopy as a function of time, of 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 inoculation.
FIG. 7B represents the cells during the stage of synthesis of MEC, in BIT medium, 24 days after seeding, that is to say 10 days after the induction of re-differentiation and synthesis of MEC. The magnification factor is x 20.
Fig. 8: shows the quantity 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 cells in comparison with the monolayer technique, after 35 days of culture.
Fig. 9: shows the ratio of the messenger RNAs of the COLII / COLI genes, obtained by quantitative RTC-PCR, of chondrocytes seeded within a 3D structure (M1) with several initial cell densities in comparison with the monolayer technique, after 35 days of culture.
Fig. 10: shows the Western Blot analysis of the protein levels of type I collagen and type II collagen, for chondrocytes cultured in three-dimensional structure M1 compared to chondrocytes cultured in monolayers, after 35 days. The level of actin expression serves as a control.
Fig. 11: shows the immunohistochemistry analysis of chondrocytes cultivated in 3D structure M1 and M2, compared to those cultivated 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 immunolabelling of type I collagen and type II collagen.
EXAMPLES:
Example 1: Synthesis of physical hydrogel based on chitosan.
The chitosan used is of non-animal origin, it is extracted from the cell wall of button mushrooms, Agaricus bisporus. Its weight average molar mass (Mw) is 170 g / mol; and its acetylation degree (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 a stoichiometric amount with the amine groups of the chitosan. The solution is stirred at room temperature until the chitosan has completely dissolved, ie for at least 3 hours, preferably at least 6 hours.
Then 1.2 propanediol is added in an amount identical to that of acetic acid and the mixture is left to stir for at least 30 min, preferably 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 or petri dish type 3 cm in diameter, then is left to stand, preferably overnight. The solution is then placed in a vacuum oven, preferably at 50 ° C, for the time necessary for the formation of the gel, preferably at least 20 hours.
The gelation step can also be carried out at room temperature, but then requires longer times (5-8 days depending on the intrinsic characteristics of the chitosan).
The thickness of the solution before gelation can be between 2 and 7 mm, preferably between 3-6 mm, to promote evaporation and hydrophobic interactions for good gel setting.
The physical hydrogel obtained is then neutralized in basic medium, with a 0.1 N NaOH soda solution, preferably for 1 hour. It is then washed several times with water, preferably sterile. The washes preferably last about 1 hour each to remove excess alcohol and bring the hydrogel back to neutral pH. It was generally carried out at least 6 washes.
The gel thus obtained has a water content of about 80% by mass. 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.
It is important to control the temperature and humidity conditions during the synthesis of chitosan-based hydrogels, especially when it is extracted from mushrooms, preferably under ambient temperature conditions below 25 ° C.
The hydrogel obtained at the end of these different stages 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, in particular the degree of acetylation, the molar mass and the concentration. It is in the form of a visco-elastic block, the mechanical properties of which depend on the intrinsic characteristics of the starting chitosan, in particular once again the degree of acetylation, the molar mass and the concentration.
The hydrogel obtained is easy to handle, it detaches without difficulty and without tearing from the flat surface on which it was made.
The observation in conventional Scanning Electron Microscopy of the dehydrated hydrogel shows a 3D, porous fibrillar structure, similar to that of a living tissue as shown in Figure 2. The observation in Scanning Electronic Cryo-Microscopy of the hydrated hydrogel, as shown in Figure 1, shows a pore size of 1-3 µm not allowing cell penetration into the hydrogel but allowing free diffusion of nutrients and cell waste.
Example 2: Synthesis of particles / fragments of chitosan hydrogel in order to develop the 3D structure (structure M1 and structure M2).
Structure M1:
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 ground using an ultra-turrax, at 6,000 to 17,000 rpm, for 10 seconds 2-4 times. In order to obtain particles of uniform size and expected diameter, the grinding is preferably carried out at 6000 rpm for 10 seconds at 3 times, making it possible to reach particle sizes of: 400-700 μm (50% ), or even 250-900 pm (> 80%), with an average of around 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 quantity of chitosan particles which will be brought into contact with the chondrogenic cells. Preliminary tests made it possible to validate the reproducibility of the measurement.
M2 structure:
In order to reinforce the viscoelastic properties of the 3D structure in which the chondrocytes are seeded, the inventors also produced a second structure (M2), by adding an anionic constituent, interacting with the cationic functions of chitosan. The polymer chosen is hyaluronic acid, preferably of bacterial origin because such a constituent is known for its better biocompatibility properties, in order to avoid allergies or possible rejections. The molecular mass by weight of the hyaluronic acid used in the production of the structure M2 is approximately 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 document FR2965278 (Univ. De Caen Basse-Normandie, et al).
The hydrogel particles obtained at the end of Example 2, with hyaluronic acid (3D structure M2) or else without (3D structure M1), are sterilized, for example at 121 ° C. for 15 min, before the put in contact with the cells. A few mini-spoons of hydrogel particles are taken, preferably 2 mini-spoons corresponding to 80 to 84 particles, which are introduced into the well of a 24-well culture plate previously covered with an insert (pore size 8 pm). The cells are added to it, 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 spontaneously adhere to the particles of chitosan hydrogel. The amount of cells falling to the bottom of the well is considered negligible.
As a control, 6.10 ⁵ cells, obtained as described above, are cultured in monolayers on plastic in 24-well plates with culture conditions identical to those described above for 3D structures (controlled atmosphere in an oven at 37 ° C, with a CO2 level of 5%, normoxia). Cells also adhere to it spontaneously.
Example 4: Cell proliferation step.
In this step, the culture medium chosen favors the multiplication of cells.
The medium chosen is a solution of 50/50 DMEM-HAM F12 + 1% AB (streptomycin / penicillin) + 10 SVF added with a solution called "Fl" comprising FGF-2 at 5ng / ml + Insulin at 5pg / ml. (Claus et al; 2012).
The mixture described in the previous step is covered, after a short time, with this culture medium known to be favorable for the proliferation of cells.
During this amplification phase, the culture medium is renewed 3 times a week, for cultures in 3D structures as well as for cultures in monolayers. The proliferation period lasts between one and two weeks for obtaining a sufficient number of cells. The inventors observed that the amplification phase lasted for approximately two weeks, when the cells were seeded in a structure made up 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).
In addition, the initial quantity of cells of 6.10 ⁵ cells / insert can quite be increased, if the condition of the number of cells / mass of hydrogel or number of cells / volume of hydrogel, or number of cells / number of hydrogel particles is observed. For example, it is entirely possible to seed 1 to 1.5 x 10 ⁶ cells / insert, provided that the necessary quantity is added in 3D structure, in order to obtain more than 4 x 10 ⁶ cells / insert at the end of the process, or even 10.5 x 10 ⁶ cells / insert, or even more.
Monitoring by light microscopy:
Reverse phase optical microscopy monitoring was performed. It made it possible to validate that the cells adhere well to the particles of chitosan hydrogel and that this environment is favorable for their culture. The culture conditions (three-dimensional structure M1 or M2, and culture medium F1) promote the proliferation and division of the chondrocytes.
The cells observed can proliferate either in isolation or in a cluster. Cultures in 3D structures of hydrogel particles show predominantly round cells. The elongated shape, characteristic of fibroblasts, is not observed in 3D structures M1 and M2, except sometimes at the periphery, that is to say at the interface between the structure and the external environment.
As a control, the inventors carried out in parallel cultures in monolayers. After 24 hours of culture, in the same F1 medium as the cells seeded in the M1 and M2 structures, the chondrocytes adopt an elongated morphology characteristic of fibroblastic cells.
Viability of cells:
The viability of the chondrocytes seeded in the three-dimensional structures was measured with the Live and Dead kit on culture fractions at 7 days (in the course of amplification, in F1 medium). The enumeration of dead cells (red) and living cells (green) is carried out with the ImageJ software from images taken under fluorescence microscopy at x20. The percentage of dead cells is calculated for each condition by making the ratio of dead cells / total cells. The viability is greater than 93%, or even greater than 97%, which shows good biocompatibility of the 3D structure.
Figure 4 illustrates the results obtained.
Proliferation tests:
Proliferation tests were carried out by total cell counting with the Cellometer T4 after detachment of the cells with trypsin and staining of the dead cells with trypan blue.
The measurements were carried out after 1 day (D1), 14 days (D14) and 21 days (D21) of culture after seeding of primary chondrocytes on D0.
Figure Fig.5 illustrates the evolution of the cell population.
After 7 days, 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).
In three-dimensional structure, M1 or M2, the cells remain round during the multiplication stage while they adopt an elongated shape, of fibroblastic type, in monolayers.
In conclusion, at the end of this proliferation step, it is observed that the amplification of the cells in three-dimensional structure composed of particles of chitosan hydrogel (structure M1) is almost as productive as in monolayers (MC), which is the reference protocol for the multiplication of cells such as chondrocytes.
In addition, Figure 6 illustrates that in monolayers, the cell population is identical from 7 days, regardless of the initial density of seeded cells.
The addition of hyaluronic acid to the three-dimensional structure of chitosan hydrogel particles (structure M2) results in a very strong acceleration of cell proliferation, much greater than the structure M1 or the culture in monolayers, in particular of a factor 2.
Example 5 Differentiation and Production of an Extracellular Matrix
In the context of the present invention, the stages of multiplication and differentiation are preferably distinct: firstly the cells multiply and secondly, they differentiate and produce the extracellular matrix. The culture medium used for the previous stage of multiplication is modified after 15 days. The "F1" culture medium is replaced by a "BIT" medium in order to promote the step of cell differentiation and production of extracellular matrix.
The culture medium is then preferably composed of: 50/50 DMEM-HAM F12 + 1% AB (streptomycin / penicillin) + 10 SVF, to which is added a BIT solution composed of: BMP-2 at 200ng / ml + Insulin at 5pg / ml + triiodothyronine T3 at 100mM (Claus et al, 2012).
This new culture medium is renewed every three days. The re-differentiation and chondrogenesis period preferably lasts 3 weeks. As a control, the same medium is changed for the cells cultivated in monolayers.
Monitoring by light microscopy:
It is known that cells with a fibrillary appearance in monolayers in F1 medium round off after 1 week of culture in BIT medium. The cells cultured in monolayers (corresponding to the control) change their appearance after passing through BIT medium, which indicates that the change in culture medium does induce a change in cell behavior.
In hydrogels (structures M1 and M2), the cells are predominantly round at the end of the amplification phase and continue to be so throughout the production phase of extracellular matrix. This point is illustrated in particular in Figure Fig.7.
At the end of culture, at D35, the cells are perfectly round in hydrogels (structures M1 and M2), less in monolayers.
In three-dimensional structures, cells can clump together the hydrogel particles and form a more or less compact kind of "ball". This observation is made in most conditions containing the hydrogels, not in the monolayers, however, which serve as controls. This observation is evidence of the high production of extracellular matrix that has accumulated around the cells. Cells make more extracellular matrix in a three-dimensional environment than in monolayers.
It should be noted that the composition consisting of said beads, however, remains, under certain conditions, injectable, despite the synthesis of a large amount of extracellular matrix. Anyway, the composition is implantable.
PCR tests:
By PCR, the transcription rate of the following proteins was quantified: COLI, COLII and GAPDH. The transcription rate is used as a benchmark to compare the COLI and COLII transcription levels. Indeed, it is well known that the chondrocyte phenotype and the production of extracellular matrix is accompanied by a strong synthesis of COLII transcripts, while COLI transcripts generally accompany the process of de-differentiation, in particular towards a fibroblastic phenotype. The results are shown in Figure 8.
The COLII / GAPDH results produced at the end of the MEC synthesis stage, show that there are more COLII transcripts for hydrogel cultures than for 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 illustrated in Figure Fig. 9. It shows that there is a much better ratio, and therefore a much better re-differentiation after de-differentiation, within 3D structures made up of chitosan hydrogel particles compared to monolayers. The three-dimensional environment tested therefore promotes well 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 chondrocytic phenotype.
The culture conditions (three-dimensional structure based on chitosan hydrogel, and BIT culture medium) therefore favor the re-differentiation of the chondrocytes and the production of cartilage matrix, compared to a culture in monolayers.
Western Blot tests:
After checking the transcription rates of the Coll and ColII genes, as a function of the culture conditions (3D or monolayers), the rate of synthesis of the corresponding proteins was checked, by the Western Blot technique. The results of the various Western Blots are illustrated in Fig. 10.
The results of the anti-COLII Western Blot show the presence of COLII under all conditions. The results of the anti-COLI WB show more intense spots in monolayers. This observation corroborates what has been revealed in Q-PCR: the COLII / COLI ratio is higher in 3D hydrogel structures than in monolayers.
The Western Blot analysis shows the expression of proteins characteristic of articular cartilage in the 3D structure.
Immunohistochemistry results:
The compositions obtained were then observed in immunohistochemistry, in order to compare the implementation of the new process, in three-dimensional structure, and the traditional culture in monolayers, at the level of the synthesis of ECM, of proteoglycans, of collagen types I and II . The results are illustrated by the photos in FIG. 11.
We can clearly observe the greater presence of proteoglycans in three-dimensional structures (M1 and M2 in Figure 11) than in monolayers (MC in Figure 11) thanks to the staining with Safranin O (SO), which highlights the presence of GAG. Furthermore, the significant production of extracellular matrix and type II collagen is observed on the immunohistochemistry images of the cells in three-dimensional environment, marked respectively by the HES staining, which highlights the nuclei and the ECM, and by Immunolabelling of Collagen II, which highlights the presence of COLII. The coloration of the cells within the matrix by Collagen I immunostaining further confirms the virtual absence of collagen I, when the chondrocytes are cultured within three-dimensional structures.
EXAMPLE 6 Chondrocyte Transplant
The set of cells and 3D structure (either the M1 structure made up of chitosan hydrogel particles, or the M2 structure made up of chitosan hydrogel particles supplemented with an anionic molecule of hyaluronic acid type) at the end of culture , i.e. between 3 and 6 weeks, a cartilage neo-tissue which can be injected or implanted by arthroscopy.
It is obviously possible to increase the number of cells in an insert by increasing the quantity of hydrogel, while respecting the same conditions of number of cells relative to the mass of hydrogel, or to the number of particles of hydrogel, constituting the 3D structure.
For example, for a sample of 0.3g-0.5g of human cartilage, 11.5 x 10 ⁶ cells (chondrocytes) can be extracted. Insofar as the inventors have shown, in the previous 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 hydrogel structure, corresponding at 80-84 hydrogel particles, the following concentration data are obtained:
Initial concentration of 6.7 x 10 ⁶ cells / g of biomaterial,
Final concentration greater than 40 x10 ⁶ cells / g of biomaterial (3D structure).
For a sample of 1 to 1.5 .10 ⁶ cells, we can therefore reach more than 3.10 ⁶ cells, or even more than 10.5. 10 ⁶ cells, at the end of the process, in 2-5 weeks, which is amply sufficient for a transplant where the recommended amounts are 3.2-6.5 x 10 ⁶ cells.
In conclusion of the previous examples, the following points are observed:
During the amplification / multiplication phase:
- an equivalent or even higher yield in three-dimensional structure comprising particles of pure chitosan hydrogel than in monolayers,
- a much higher yield in three-dimensional structure comprising hydrogel particles supplemented with hyaluronic acid than in monolayers.
During the differentiation and production phase of MEC:
- re-differentiation of cells into 3D structures and monolayers as proven by PCR, WB and immunohistochemistry analyzes;
- a significantly higher ratio of COLII / COLI messenger RNAs in three-dimensional structure than in monolayers,
- a significantly higher COLII / COLI protein ratio in three-dimensional structure than in monolayers
- a stable chondrocyte phenotype in 3D structure
- the production of abundant cartilage matrix in 3D structure
The succession of stages, in the same three-dimensional medium, comprising particles of chitosan hydrogel with / without structuring molecule, of amplification then of differentiation / chondrogenesis is very favorable for obtaining an injectable or implantable cartilage tissue. with excellent mechanical and biological properties.
Adding hyaluronic acid improves the system by speeding up the cell amplification process and increasing the number of cells to implant or saving a week on the overall protocol.
The configuration of the structure optimizes the contact surface with the cells.
Example 7: Comparison of the different 3D structures.
The inventors reproduced the 3D structures described in the previous 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 illustrated in Table 1. The value 1 was assigned to the structure M1 corresponding to particles of several hundred microns, obtained from chitosan from fungi.
The proliferation rate obtained with the M2 structure (mushroom extract and supplemented with 2M hyaluronic acid) is 2 times higher compared to the M1 structure (mushroom extract not supplemented with Hyaluronic acid), which itself makes it possible to obtain a proliferation rate 1.5 times greater compared to chitosan extracted from squid, or else with particles of the order of tens of μm.
Hundreds of ym fragments of Fragments tens of ym of Chitosan mushroom extract 2 ** supplemented with HA 2M Chitosan mushroom extract 1.2 supplemented with HA 1M Chitosan mushroom extract Γ 0.67 Chitosan extract from squid 0.67
* structure M1; ** M2 structure
Table 1: Proliferation ratios.
REFERENCES :
Claus S, Mayer N, Aubert-Foucher E, Chajra H, Perrier-Groult E, Lafont J, Piperno M, Damour O, Mallein-Gerin F. Cartilage-characteristic matrix reconstruction by sequential addition of soluble factors during expansion of human articular chondrocytes and their cultivation in collagen sponges. Tissue Eng Part C Methods. 2012; 18 (2): 104-12.
Correia CR, et al. Chitosan scaffolds containing hyaluronic acid for cartilage tissue engineering. Tissue Eng Part C Methods. 2011 Jul; 17 (7): 717-30.
Denuziere A, Ferrier D, Damour O, Domard A. Chitosan-chondroitin sulfate and chitosanhyaluronate polyelectrolyte complexes: biological properties. Biomaterials. 1998; 19 (14): 1275-85. Griffon DJ, Sedighi MR, Schaeffer DV, Eurell JA, Johnson AL. Chitosan scaffolds: interconnective pore size and cartilage engineering. Acta Biomater. 2006 May; 2 (3): 313-20.
Hao T, Wen N, Cao JK, Wang HB, Lü SH, Liu T, Lin QX, Duan CM, Wang CY. The support of matrix accumulation and the promotion of sheep articular cartilage defects repair in vivo by chitosan hydrogels. Osteoarthritis Cartilage. 2010 Feb; 18 (2): 257-65.
Hautier A, et al. Bone morphogenetic protein-2 stimulates chondrogenic expression in human nasal chondrocytes expanded in vitro. Growth Factors. 2008; 26 (4): 201-11.
Hoemann CD, Sun J, Légaré A, McKee MD, Buschmann MD. Tissue engineering of cartilage using an injectable and adhesive chitosan-based cell-delivery vehicle. Osteoarthritis Cartilage. 2005; 13 (4): 318-29.
Lahiji A, Sohrabi A, Hungerford DS, Frondoza CG. Chitosan supports the expression of extracellular matrix proteins in human osteoblasts and chondrocytes. J Biomed Mater Res. 2000; 51 (4): 586-95.
Liu G, et al. Optimal combination of soluble factors for tissue engineering of permanent cartilage from cultured human chondrocytes. J Biol Chem. 2007 Jul 13; 282 (28): 20407-15.
Montembault A, Tahiri K, Korwin-Zmijowska C, Chevalier X, Corvol MT, Domard A. A material decoy of biological media based on chitosan physical hydrogels: application to cartilage tissue engineering. Biochemistry. 2006 May; 88 (5): 551-64.
Park H, Choi B, Hu J, Lee M. Injectable chitosan hyaluronic acid hydrogels for cartilage tissue engineering. Acta Biomater. 2013 Jan; 9 (1): 4779-86.
Suh JK, Matthew HW. Application of chitosan-based polysaccharide biomaterials in cartilage tissue engineering: a review. Biomaterials. 2000; 21 (24): 2589-98.
Tallheden T, et al. Proliferation and differentiation potential of chondrocytes from osteoarthritic patients. Arthritis Res Ther. 2005; 7 (3): R560-8.

权利要求:
Claims (15)
[1" id="c-fr-0001]
Claims:
1. Three-dimensional structure formed by particles of physical hydrogel of chitosan or of derivative of chitosan, associated with an anionic polymer, where said particles of hydrogel are of average size ranging between 10 μm and 1.5 mm.
[2" id="c-fr-0002]
2. Three-dimensional structure according to claim 1, wherein said anionic polymer is a polymer of hyaluronic acid, or a derivative of hyaluronic acid or a complex of hyaluronic acid.
[3" id="c-fr-0003]
3. Three-dimensional structure according to claim 1 or 2, wherein said hydrogel particles are of average size between 400 µm and 700 µm.
[4" id="c-fr-0004]
4. Three-dimensional structure according to any one of claims 1 to 3 wherein the chitosan has a weight-average molar mass greater than 150 kDa, preferably between 150 and 220 kDa.
[5" id="c-fr-0005]
5. Three-dimensional structure according to any one of claims 1 to 4 wherein the chitosan is extracted from fungi, preferably from Agaricus bisporus.
[6" id="c-fr-0006]
6. Three-dimensional structure according to any one of claims 1 to 5 wherein said physical chitosan hydrogel is synthesized without crosslinking agent.
[7" id="c-fr-0007]
7. Three-dimensional structure according to any one of claims 1 to 6 wherein said physical chitosan hydrogel has a water content of at least 70%, preferably at least 80%.
[8" id="c-fr-0008]
8. Three-dimensional structure according to any one of claims 1 to 7 wherein the hyaluronic acid is extracted from bacterial fermentation, with a weight-average molar mass greater than 1MDa, or greater than 2 MDa.
[9" id="c-fr-0009]
9. Three-dimensional structure according to any one of claims 1 to 8, characterized in that the relative proportion of the anionic polymer vis-à-vis the hydrogel of chitosan or derivative of chitosan, is between 1% and 10% , preferably between 1% and 3%.
[10" id="c-fr-0010]
10. Structure according to any one of claims 1 to 9, in which the viability rate of cells seeded in the structure is greater than 93%, or even greater than 97% at 7 days.
[11" id="c-fr-0011]
11. Structure according to any one of claims 1 to 10, ensuring an optimal spatial distribution of the seeded cells.
[12" id="c-fr-0012]
12. Use of a three-dimensional structure according to any one of claims 1 to 11, for seeding chondrocytes, or stem cells differentiated into chondrocytes, or chondrocyte precursors, or induced pluripotent cells (IPS), or mesenchymal stem cells, or co-cultures of these cells, where said cells are not human embryonic stem cells.
[13" id="c-fr-0013]
13. Use of a three-dimensional structure according to any one of claims 1 to 11, for seeding cells, preferably bone cells, fibroblasts, keratinocytes or combinations of some of these cells.
[14" id="c-fr-0014]
14. Use of a three-dimensional structure according to claim 12 or 13, for the in vitro seeding of cells for the purpose of proliferation and synthesis of extracellular matrix.
[15" id="c-fr-0015]
15. Three-dimensional structure according to any one of claims 1 to 11, for use in cartilage repair or for use as an implant, or as a graft or as a vector of active principle in humans or animals.
1/5

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优先权:

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申请号 | 申请日 | 专利标题

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FR1461746A|FR3029116B1|2014-12-01|2014-12-01|PROCESS FOR OBTAINING A CARTILAGE GEL FOR CARTILAGE REPAIR COMPRISING CHITOSAN AND CHONDROCYTES|

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FR1461746|2014-12-01|

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PCT/FR2015/053271|WO2016087762A1|2014-12-01|2015-12-01|Cartilage gel for cartilage repair, comprising chitosan and chondrocytes|

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FR1850498A|FR3062064B1|2014-12-01|2018-01-23|THREE-DIMENSIONAL STRUCTURE INCLUDING PARTICLES OF CHITOSAN HYDROGEL|FR1850498A| FR3062064B1|2014-12-01|2018-01-23|THREE-DIMENSIONAL STRUCTURE INCLUDING PARTICLES OF CHITOSAN HYDROGEL|