EP2288370A1

EP2288370A1 - Osteogenic composition including a complex growth factor/amphiphilic polymer, a soluble cation salt, and an organic substrate

Info

Publication number: EP2288370A1
Application number: EP09732932A
Authority: EP
Inventors: Rémi SOULA; Olivier Soula; Gérard Soula
Original assignee: Adocia SAS
Current assignee: Adocia SAS
Priority date: 2008-04-14
Filing date: 2009-04-14
Publication date: 2011-03-02
Also published as: WO2009127940A1; US20110159068A1; IL208139A0; ZA201006627B; MX2010011267A; AU2009237414A1; JP2011519292A; CN102065882A; KR20110014588A; CA2720203A1; US20090291114A1; BRPI0906553A2

Abstract

The invention relates to an open implant comprising an osteogenic composition including at least: • a complex osteogenic/polysaccharide amphiphilic anionic growth factor, • a soluble cation salt that is at least divalent, and • an organic substrate • said organic substrate does not including any demineralized bone matrix. In one embodiment, said implant is in freeze-dried form. The invention also relates to the preparation method thereof.

Description

OSTEOGENIC COMPOSITION COMPRISING AMPHIPHILIC GROWTH FACTOR / POLYMER COMPLEX, SOLUBLE CATION SALT AND

ORGANIC SUPPORT

The present invention relates to the field of osteogenic formulations and more particularly to formulations of osteogenic proteins belonging to the family of Bone Morphogenetic Proteins, BMPs.

Bone Morphogenetic Proteins (BMPs) are growth factors involved in osteoinduction mechanisms. BMPs also referred to as Osteogenic Proteins (OPs) were initially characterized by Urist in 1965 (Urist MR Science 1965; 150,893). These proteins isolated from cortical bone have the ability to induce bone formation in a large number of animals (Urist MR Science 1965, 150, 893).

BMPs are expressed as propeptides which, after post-translational processing, have a length of between 104 and 139 residues. They have a great homology of sequences between them and have similar three-dimensional structures. In particular, they have 6 cysteine residues involved in intramolecular disulfide bonds forming a "cysteine knot" (Scheufler C. 2004 J. Mol Biol 1999, 287, 103, Schlunegger MP, J. Mol Biol 1993, 231, 445). Some of them have a 7 ^th cysteine also involved in an intermolecular disulfide bridge responsible for dimer formation (Scheufler C. Mol Biol 2004 J. 1999; 287:... 103).

In their active form, BMPs assemble into homodimers or even heterodimers as described by Israel et al. (Israel Dl, Growth Factors, 1996, 13 (3-4), 291). Dimeric BMPs interact with BMPR transmembrane receptors (Mundy et al., Growth Factors, 2004, 22 (4), 233). This recognition is at the origin of a cascade of intracellular signaling involving Smad proteins in particular resulting in the activation or repression of target genes.

BMPs, with the exception of BMPs 1 and 3, play a direct and indirect role in the differentiation of mesenchymal cells causing their differentiation into osteoblasts (Cheng H., J. Bone and Joint Surgery, 2003, 85A 1544-1552). They also possess chemotaxis properties and induce proliferation and differentiation.

Certain human recombinant BMPs, and in particular rhBMP-2 and rhBMP-7, have clearly demonstrated an ability to induce bone formation in vivo in humans and have been approved for certain medical applications. Thus, recombinant human BMP-2, dibotermin alfa according to the international nonproprietary name, is formulated in products sold under the name Infuse ^® in the US and InductOs ^® in Europe. This product is prescribed in the fusion of the lumbar vertebrae and bone regeneration of the tibia for so-called non-union fractures. In the case of InFUSE ^® for fusion of the lumbar vertebrae, the surgical procedure consists first of all, to soak a collagen sponge with a solution of rhBMP-2, then to place the sponge in a hollow cage, LT Cage, previously implanted between the vertebrae.

Human recombinant BMP-7, eptotermin alpha according to the international nonproprietary name, has the same therapeutic indications as BMP-2 and is the basis of two products: OP-1 Implant for open fractures of the tibia and OP-1 Putty for the fusion of the lumbar vertebrae. OP-1 Implant consists of a powder containing rhBMP-7 and collagen to be taken up in 0.9% saline solution. The paste obtained is then applied to the fracture during a surgical procedure. OP-1 Putty comes in the form of two powders: one containing rhBMP-7 and collagen, the other carboxymethylcellulose (CMC). During surgery, the CMC solution is reconstituted with 0.9% saline and mixed with rhBMP-7 and collagen. The paste thus obtained is applied to the site to be treated.

Patent application US2008 / 014197 discloses an osteoinductive implant consisting of a support (scaffold) containing a mineral ceramic, a solid membrane integrally bonded to the support and an osteogenic agent. The support is preferably a collagen sponge. The mineral ceramic comprises a calcium derivative, preferably a water-insoluble mineral matrix such as biphasic calcium phosphate ([0024], p2). The solid membrane integrally bound to the implant must be impervious so as to limit the entry of surrounding soft tissue cells and also prevent the entry of inflammatory cells ([003O] ₁ p 3). The entry of these cells into the implant is described as possibly leading to a reduction in bone growth and treatment failure ([0007], p 1). This invention is focused on adding a membrane to the implant to improve osteogenesis.

US2007 / 0254041 discloses a sheet-shaped device containing a demineralized bone matrix (DBM), collagen particles and a physically cross-linked polysaccharide matrix. This implant may also contain an osteogenic substance such as a growth factor. The physically cross-linked polysaccharide serves as a stabilizer for the demineralized bone particles ([0026], p 3). The alginate-based polysaccharide is crosslinked by addition of calcium chloride.

The patent application WO96 / 39203 describes an osteogenic and biocompatible composite material with a physical resistance. This osteoinductive material is composed of demineralized bone, osteoinduction can only take place in the presence of demineralized bone, or in the presence of protein extracts of demineralized bone, or in the presence of these two elements according to the authors (lines 2-5, p 2). To this material is added a calcium salt or a mineral salt. The mineral salt is described as possibly being sodium hydroxide, sodium chloride, magnesium chloride or magnesium hydroxide (lines 4-9, p 17). The calcium salt may be a soluble salt or not (lines 20-21, p 17) and is preferably calcium hydroxide. The selection of the hydroxides of different cations, in particular of calcium, to be added is justified by the effect of increasing the pH of the matrix favorable to the increase of collagen synthesis in this environment (lines 7-11, p 15 ).

This invention covers the formation of new demineralized bone implants whose physical and osteogenic properties would be improved by increasing the pH of the implant.

It has also been shown that it is particularly advantageous to form complexes between a growth factor and a polymer in order to stabilize it, increase its solubility and / or increase its activity. Thus, in the patent application FR0705536 in the name of the applicant, it has been possible to demonstrate that the complex formation between BMP-2 and an amphiphilic polymer notably makes it possible to increase the solubility of this highly hydrophobic and poorly soluble pH-sensitive protein. physiological.

In the patent application FR0705536, the Applicant has also demonstrated the increase in the biological activity of BMP-2 in the presence of a derivative of dextran functionalized with a hydrophobic substituent. In vitro, this complex of BMP-2 appears in every point superior to BMP-2 alone.

However, it remains essential to find a formulation to improve the performance of these BMP growth factors in order to, for example, reduce the quantities to be administered.

This problem is common to many growth factor formulations, since these proteins are generally used in doses that are several orders of magnitude higher than the physiological doses.

It is the merit of the Applicant to have found a formulation of growth factors to improve their activity by adding a solution of a soluble salt of an at least divalent cation to a hydrogel containing said growth factors, said soluble cation salt, at least divalent, potentiating the effect of the growth factor.

Surprisingly, this new formulation makes it possible to produce the same osteogenic effect with lesser amounts of growth factors.

The invention relates to an open implant consisting of an osteogenic composition comprising at least:

An osteogenic growth factor / amphiphilic anionic polysaccharide complex,

A soluble salt of at least divalent cation, and

An organic support, said organic support not comprising a demineralized bone matrix. The term "open implant" means an implant having no membrane or envelope capable of limiting or regulating exchanges with the tissues surrounding the implant and substantially homogeneous in its constitution.

Demineralized bone matrix is defined as

DBM) a matrix obtained by acid extraction of autologous bone, leading to the loss of the majority of the mineralized components but to the preservation of collagenic or non-collagenic proteins, including growth factors. Such a demineralized matrix may also be prepared in an inactive form after extraction with chaotropic agents.

By organic support is meant a support consisting of an organic matrix and / or a hydrogel.

By organic matrix is meant a matrix consisting of cross-linked hydrogels and / or collagen.

The organic matrix is a hydrogel obtained by chemical crosslinking of polymer chains. Interchain covalent bonds defining an organic matrix. Polymers that can be employed for the constitution of an organic matrix are described in Hoffman's review Hydrogels for Biomedical Applications (Ad v. Drug Deliv Rev, 2002, 43, 3-12).

In one embodiment, the matrix is chosen from the matrices based on purified, sterilized natural collagen. Natural polymers such as collagen are components of the extracellular matrix that promote attachment, migration and cell differentiation. They have the advantage of being extremely biocompatible and are degraded by enzymatic digestion mechanisms. Collagen-based matrices are obtained from fibrillar type I or IV collagen extracted from tendon or beef or pork bone. These collagens are first purified before being crosslinked and then sterilized.

The organic supports according to the invention can be used as a mixture to obtain materials which can be in the form of a material with sufficient mechanical properties to be formed or molded or in the form of a "putty" or the collagen or hydrogel plays a role of binder.

Mixed materials can also be used, for example a matrix which combines collagen and inorganic particles and which can be in the form of a composite material with reinforced mechanical properties or in the form of a "putty" or the collagen plays a role of binder.

Usable inorganic materials include essentially calcium phosphate ceramics such as hydroxyapatite (HA), tricalcium calcium phosphate (TCP), biphasic calcium phosphate (BCP) or amorphous calcium phosphate (ACP) which have as their main interest a chemical composition very close to that of the bone. These materials have good mechanical properties and are immunologically inert. These materials can be in various forms such as powders, aggregates or blocks. These materials have very different degradation rates depending on their compositions and hydroxyapatite is degraded very slowly (several months) while tricalcium calcium phosphate degrades more quickly (several weeks). It is for this purpose that biphasic calcium phosphates have been developed because they have intermediate resorption rates. These inorganic materials are known to be primarily osteoconductive.

The term hydrogel means a hydrophilic three-dimensional network of polymer capable of adsorbing a large quantity of water or biological fluids (Peppas et al., Eur J Pharm Biopharm 2000, 50, 27-46). Such a hydrogel consists of physical interactions and is therefore not obtained by chemical crosslinking of the polymer chains.

Among these polymers, synthetic polymers can be found as well as natural polymers. Hydrogen-forming polysaccharides are described, for example, in the article entitled Polysaccharide hydrogels for modified release formulations (Coviello et al., J. Control Release, 2007, 119, 5-24). In one embodiment, the crosslinked or non-crosslinked hydrogel-forming polymer is selected from the group of synthetic polymers, among which are copolymers of ethylene glycol and lactic acid, copolymers of ethylene glycol and glycolic acid, poly (N-vinyl pyrrolidone), polyvinyl acids, polyacrylamides, polyacrylic acids.

In one embodiment, the hydrogel-forming polymer is chosen from the group of natural polymers among which hyaluronic acid, keratane, pullulan, pectin, dextran, cellulose and cellulose derivatives, alginic acid , xanthan, carrageenan, chitosan, chondroitin, collagen, gelatin, polylysine, fibrin and their biologically acceptable salts.

In one embodiment, the natural polymer is chosen from the group of hydrogel-forming polysaccharides, among which hyaluronic acid, aiginic acid, dextran, pectin, cellulose and its derivatives, pullulan, xanthan, carrageenan, chitosan, chondroitin and their biologically acceptable salts.

In one embodiment, the natural polymer is chosen from the group of hydrogel-forming polysaccharides, among them hyaluronic acid, alginic acid and their biologically acceptable salts.

Amphiphilic polysaccharide is understood to mean a polysaccharide chosen from the group of polysaccharides functionalized with hydrophobic derivatives.

These polysaccharides consist mainly of (1, 4) and / or (1, 3) and / or (1, 2) glycosidic linkages. They can be neutral, that is to say they can not carry acidic or anionic functions and carry acid functions.

They are functionalized by at least one derivative of tryptophan, noted Trp: said tryptophan derivative being grafted or bound to the polysaccharides by coupling with an acid function, said acid function possibly being an acid function of an anionic polysaccharide and / or an acid function carried by a linker R linked to the polysaccharide by a function F said function F resulting from the coupling between the linker R and a function -OH of the neutral or anionic polysaccharide,

F being either an ester, thioester, amide, carbonate, carbamate, ether, thioether or amine function,

- R being a chain comprising between 1 and 18 carbons, optionally branched and / or unsaturated comprising one or more heteroatoms, such as O, N and / or S, and having at least one acid function,

Trp being a residue of a tryptophan derivative, L or D, produces coupling between the tryptophan amine and the at least one acid carried by the R group and / or an acid carried by the anionic polysaccharide.

According to the invention, the polysaccharide predominantly comprising glycoside bonds of the type (1, 4), (1, 3) and / or (1, 2) functionalized with at least one tryptophan derivative may correspond to the following general formula I :

polysaccharide

F [Trp 1

[Trp] n

Formula

the polysaccharide predominantly consisting of (1,4) and / or (1,3) and / or (1,2) glycosidic bonds, F resulting from the coupling between the linker R and an OH function of the neutral or anionic polysaccharide, being either an ester, thioester, amide, carbonate, carbamate, ether, thioether or amine function,

R being a chain comprising between 1 and 18 carbons, optionally branched and / or unsaturated comprising one or more heteroatoms, such as O, N or / and S, and having at least one acidic function

Trp being a residue of a tryptophan derivative, L or D, produces coupling between the amine of the tryptophan derivative and the at least one acid carried by the R group and / or an acid carried by the anionic polysaccharide. n represents the molar fraction of the Rs substituted by Trp and is between 0.05 and 0.7.

0 represents the mole fraction of the acid functions of the polysaccharides substituted with Trp and is between 0.05 and 0.7.

1 represents the mole fraction of acid functions carried by the group R per saccharide unit and is between 0 and 2, j represents the mole fraction of acid functional groups carried by the anionic polysaccharide per saccharide unit and is between 0 and 1,

(i - + j) represents the mole fraction of acid functions per saccharide unit and is between 0.1 and 2,

- When R is not substituted by Trp, then the acid or acids of the R group are cation carboxylates, alkali preferably as Na or K.

when the polysaccharide is an anionic polysaccharide, when one or more acid functions of the polysaccharide are not substituted by

Trp, then they are salified by a cation, alkaline preferably as Na ⁺ or K ⁺ , said polysaccharides being amphiphilic at neutral pH.

In one embodiment, F is either an ester, a carbonate, a carbamate or an ether.

In one embodiment, the polysaccharide consists predominantly of glycoside bonds of the type (1, 4). In one embodiment, the polysaccharide predominantly composed of glycoside bonds of type (1, 4) is selected from the group consisting of pullulan, alginate, hyaluronan, xylan, galacturonan or a cellulose soluble in the water. In one embodiment, the polysaccharide is a pullulan.

In one embodiment, the polysaccharide is an alginate.

In one embodiment, the polysaccharide is a hyaluronan.

In one embodiment, the polysaccharide is a xylan.

In one embodiment, the polysaccharide is a galacturonan. In one embodiment, the polysaccharide is a water-soluble cellulose.

In one embodiment, the polysaccharide consists predominantly of glycoside bonds of type (1, 3). In one embodiment, the polysaccharide consisting predominantly of glycoside bonds of type (1, 3) is a curdlane.

In one embodiment, the polysaccharide consists predominantly of glycoside bonds of type (1, 2). In one embodiment, the polysaccharide consisting predominantly of glycoside bonds of type (1, 2) is an inulin.

In one embodiment, the polysaccharide consists predominantly of glycoside bonds of type (1, 4) and (1, 3). In one embodiment, the polysaccharide predominantly composed of glycoside bonds of (1,4) and (1,3) type is a glucan.

In one embodiment, the polysaccharide consists predominantly of (1,4) and (1,3) and (1,2) glycosidic linkages. In one embodiment, the polysaccharide predominantly composed of glycoside bonds of (1,4) and (1,3) and (1,2) type is mannan.

In one embodiment, the polysaccharide according to the invention is characterized in that the group R is chosen from the following groups:

or their alkali metal salts.

In one embodiment, the polysaccharide according to the invention is characterized in that the tryptophan derivative is selected from the group consisting of tryptophan, tryptophanol, tryptophanamide, 2-indole ethylamine and their alkaline cation salts.

In one embodiment, the polysaccharide according to the invention is characterized in that the tryptophan derivative is chosen from the esters of tryptophan of formula II.

Formula II

E being a group that can be:

A linear or branched C1 to C8 alkyl.

A linear or branched C 6 -C 20 alkylaryl or arylalkyl.

The polysaccharide may have a degree of polymerization m of between 10 and 10,000.

In one embodiment, it has a degree of polymerization m of between 10 and 1000. In another embodiment, it has a degree of polymerization m of between 10 and 500. In one embodiment, the polysaccharides are chosen from the group of dextrans functionalized with hydrophobic amino acids such as tryptophan and tryptophan derivatives as described in application FR 07/02316.

According to the invention, the functionalized dextran may correspond to the following general formula III:

Formula

R being a chain comprising between 1 and 18 carbons, optionally branched and / or unsaturated comprising one or more heteroatoms, such as O, N or / and S, and having at least one acid function

F resulting from the coupling between the linker R and a function -OH of the neutral or anionic polysaccharide, being either an ester, thioester, amide, carbonate, carbamate, ether, thioether or amine function,

AA being a hydrophobic amino acid residue, L or D, produces coupling between the amino acid amine and an acid carried by the R group.

t represents the mole fraction of substituent F-R- [AA] n per glycoside unit and is between 0.1 and 2, p represents the mole fraction of Rs substituted with AA and is between 0.05 and 1.

When R is not substituted by AA, then the acid or acids of the R group are cationic carboxylates, preferably alkaline, such as Na ⁺ , K ⁺ , said dextran being amphiphilic at neutral pH.

In one embodiment, the alkaline cation is Na ⁺ .

In one embodiment, F is either an ester, a carbonate, a carbamate or an ether.

In one embodiment, the polysaccharide according to the invention is a carboxymethyl dextran of formula IV.

Formula IV or the corresponding acid.

In one embodiment, the polysaccharide according to the invention is a monosuccinic ester of dextran of formula V:

Formula V or the corresponding acid.

or their alkali metal salts. In one embodiment, the dextran according to the invention is characterized in that the hydrophobic amino acid is chosen from tryptophan derivatives, such as tryptophan, tryptophanol, tryptophanamide and 2-indole ethylamine and their salts. alkaline cation.

In one embodiment, the dextran according to the invention is characterized in that the tryptophan derivatives are chosen from the tryptophan esters of formula II as defined above.

In one embodiment, the dextran according to the invention is a tryptophan modified carboxymethyldextran of formula VI:

Formula Vl

In one embodiment, the dextran according to the invention is a monosuccinic ester of dextran modified with tryptophan of formula VII:

Form VII

In one embodiment, the dextran according to the invention is characterized in that the hydrophobic amino acid is selected from phenylalanine, leucine, isoleucine and valine and their alcohol, amide or decarboxylated derivatives. In one embodiment, the dextran according to the invention is characterized in that the derivatives of phenylalanine, leucine, isoleucine and valine are chosen from the esters of these amino acids of formula VIII.

Form VIII

E being defined as before.

In one embodiment, the dextran according to the invention is characterized in that the hydrophobic amino acid is phenylalanine, and its alcohol, amide or decarboxylated derivatives.

Dextran may have a degree of polymerization m of between 10 and 10,000.

In one embodiment, it has a degree of polymerization m of between 10 and 1000.

In another embodiment, it has a degree of polymerization m of between 10 and 500.

In one embodiment, the polysaccharides are chosen from the group of polysaccharides comprising carboxyl functional groups such as those described in application FR 08/05506, at least one of which is substituted by a hydrophobic alcohol derivative, denoted by Ah: hydrophobic alcohol (Ah) being grafted or bound to the anionic polysaccharide by a coupling arm R, said coupling arm being bonded to the anionic polysaccharide by a function F 'said function F' resulting from the coupling between the amine function of the linker R and a carboxyl function of the anionic polysaccharide, and said coupling arm being bonded to the hydrophobic alcohol by a function G resulting from the coupling between a carboxyl, isocyanate, thioacid or alcohol function of the coupling arm and a function of the hydrophobic alcohol, the carboxyl functions of the unsubstituted anionic polysaccharide being in the form of a cation carboxylate, preferably alkaline, such as Na ⁺ or K ⁺ .

F 'being an amide function,

G being either an ester, thioester, carbonate or carbamate function,

R being a chain comprising between 1 and 18 carbons, optionally branched and / or unsaturated, optionally comprising one or more heteroatoms, such as O, N or / and S, and having at least one acid function,

Ah being a residue of a hydrophobic alcohol, produces coupling between the hydroxyl function of the hydrophobic alcohol and at least one electrophilic function carried by the group R.

said polysaccharide having carboxyl functional groups being amphiphilic at neutral pH.

The polysaccharide having carboxyl functional groups partially substituted by hydrophobic alcohols is chosen from polysaccharides comprising carboxyl functional groups of general formula IX:

Form IX

in which q represents the mole fraction of the carboxyl functions of the polysaccharide substituted with FRG-Ah and is between 0.01 and 0.7, - F ', R ₁ G and Ah as defined above, and when the carboxyl function of the polysaccharide is not substituted by F'-RG-Ah, then the carboxyl functional group (s) of the polysaccharide are carboxylates of cation, alkali preferably as Na ⁺ or K ⁺ .

In one embodiment, the polysaccharides comprising carboxyl functional groups are polysaccharides naturally carrying carboxyl functional groups and are chosen from the group consisting of alginate, hyaluronan and galacturonan.

In one embodiment, the polysaccharides comprising carboxyl functional groups are synthetic polysaccharides obtained from polysaccharides naturally having carboxyl functional groups or from neutral polysaccharides on which at least 15 carboxyl functional groups per 100 saccharide units have been grafted with general formula X.

polysaccharide

Q

X

the natural polysaccharides being chosen from the group of polysaccharides consisting mainly of (1, 6) and / or (1, 4) and / or (1, 3) and / or (1, 2) glycoside bonds,

L being a bond resulting from the coupling between the linker Q and an OH function of the polysaccharide and being either an ester, thioester, carbonate, carbamate or ether function,

r represents the mole fraction of the L-Q substituents per saccharide unit of the polysaccharide

Q being a chain comprising between 1 and 18 carbons, optionally branched and / or unsaturated comprising one or more heteroatoms, such as O, N and / or S, and comprising at least one carboxyl functional group, - CO ₂ H In one embodiment, the polysaccharide consists predominantly of glycoside bonds of (1, 6) type.

In one embodiment, the polysaccharide consisting predominantly of glycoside bonds of type (1, 6) is dextran.

In one embodiment, the polysaccharide consists predominantly of glycoside bonds of (1, 4) type.

In one embodiment, the polysaccharide predominantly composed of glycoside bonds of type (1, 4) is selected from the group consisting of pullulan, alginate, hyaluronan, xylan, galacturonan or a cellulose soluble in the water.

In one embodiment, the polysaccharide is a pullulan. In one embodiment, the polysaccharide is an alginate.

In one embodiment, the polysaccharide is a hyaluronan. In one embodiment, the polysaccharide is a xylan. In one embodiment, the polysaccharide is a galacturonan. In one embodiment, the polysaccharide is a water-soluble cellulose.

In one embodiment, the polysaccharide consists predominantly of glycoside bonds of type (1, 3).

In one embodiment, the polysaccharide consisting predominantly of glycoside bonds of type (1, 3) is a curdlane.

In one embodiment, the polysaccharide consists predominantly of glycoside bonds of type (1, 2).

In one embodiment, the polysaccharide consisting predominantly of glycoside bonds of type (1, 2) is an inulin.

In one embodiment, the polysaccharide consists predominantly of (1,4) and (1,3) glycosidic linkages.

In one embodiment, the polysaccharide predominantly composed of glycoside bonds of (1,4) and (1,3) type is a glucan. In one embodiment, the polysaccharide consists predominantly of (1,4) and (1,3) and (1,2) glycosidic linkages.

In one embodiment, the polysaccharide predominantly composed of glycoside bonds of (1,4) and (1,3) and (1,2) type is mannan.

In one embodiment, the polysaccharide according to the invention is characterized in that the group Q is chosen from the following groups:

In one embodiment, r is between 0.1 and 2.

In one embodiment, r is 0.2 to 1.5.

In one embodiment, the group R according to the invention is characterized in that it is chosen from amino acids.

In one embodiment, the amino acids are selected from alpha amino acids.

In one embodiment, the alpha amino acids are selected from alpha natural amino acids.

In one embodiment, the natural alpha amino acids are chosen from leucine, alanine, iso-leucine, glycine, phenylalanine, tryptophan and valine.

In one embodiment, the hydrophobic alcohol is selected from fatty alcohols.

In one embodiment, the hydrophobic alcohol is chosen from alcohols consisting of an unsaturated or saturated alkyl chain comprising from 4 to 18 carbons. In one embodiment, the fatty alcohol is selected from meristyl, cetyl, stearyl, cetearyl, butyl, oleyl and lanolin.

In one embodiment, the hydrophobic alcohol is selected from cholesterol derivatives.

In one embodiment, the cholesterol derivative is cholesterol.

In one embodiment, the hydrophobic alcohol Ah is selected from tocopherols.

In one embodiment, the tocopherol is alpha tocopherol.

In one embodiment, alpha tocopherol is the racemic alpha tocopherol.

In one embodiment, the hydrophobic alcohol is selected from alcohols bearing aryl groups.

In one embodiment, the aryl group bearing alcohol is selected from benzyl alcohol, phenethyl alcohol.

In one embodiment, it has a degree of polymerization m of between 10 and 1000.

In one embodiment, said composition is in the form of lyophilisate. In one embodiment, the at least one divalent cation soluble salt is a divalent cation soluble salt selected from calcium, magnesium or zinc cations.

In one embodiment, the at least one divalent cation soluble salt is a soluble calcium salt.

The term "soluble salt of at least divalent cation" means a salt whose solubility is equal to or greater than 5 mg / ml, preferably 10 mg / ml, preferably 20 mg / ml.

In one embodiment, the divalent cation soluble salt is a calcium salt whose counterion is selected from chloride, D-gluconate, formate, D-saccharate, acetate, L-lactate, glutamate, aspartate, propionate, fumarate, sorbate, bicarbonate, bromide or ascorbate.

In one embodiment, the divalent cation soluble salt is a magnesium salt whose counterion is selected from chloride, D-gluconate, formate, D-saccharate, acetate, L-lactate, glutamate, aspartate, propionate, fumarate, sorbate, bicarbonate, bromide or ascorbate.

In one embodiment, the divalent cation soluble salt is a zinc salt whose counterion is selected from chloride, D-gluconate, formate, D-saccharate, acetate, L-lactate, glutamate, aspartate, propionate, fumarate, sorbate, bicarbonate, bromide or ascorbate.

In one embodiment, the divalent cation soluble salt is calcium chloride.

In one embodiment, the soluble cation salt is a soluble multivalent cation salt.

By multivalent cations are meant species carrying more than two positive charges such as iron, aluminum, cationic polymers such as polylysine, spermine, protamine, fibrin. By osteogenic growth factor or BMP alone or in combination is meant a BMP selected from the group of therapeutically active BMPs (Bone Morphogenetic Proteins).

More particularly, the osteogenic proteins are selected from the group consisting of BMP-2 (Dibotermin-alpha), BMP-4, BMP-7 (Eptotermin-alpha), BMP-14 and GDF-5.

In one embodiment, the osteogenic protein is BMP-2 (Dibotermin-alpha). In one embodiment, the osteogenic protein is GDF-5.

The BMPs used are recombinant human BMPs, obtained according to the techniques known to those skilled in the art or purchased from suppliers such as, for example, Research Diagnostic Inc. (USA).

In one embodiment, the hydrogel can be prepared just prior to implantation.

In one embodiment, the hydrogel may be prepared and stored in a pre-filled syringe for subsequent implantation.

In one embodiment, the hydrogel may be prepared by rehydrating a lyophilisate just prior to implantation or implanted in dehydrated form.

Lyophilization is a water sublimation technique allowing dehydration of the composition. This technique is commonly used for protein storage and stabilization.

The rehydration of a lyophilizate is very rapid and makes it possible to easily obtain a ready-to-use formulation, said formulation being able to be rehydrated before implantation or implanted in its dehydrated form, the rehydration then taking place, after implantation, by contact with biological fluids. In addition to these osteogenic growth factors, it is possible to add other proteins and in particular angiogenic growth factors such as PDGF _1, VEGF or FGF.

The invention therefore relates to a composition according to the invention characterized in that it further comprises angiogenic growth factors selected from the group consisting of PDGF, VEGF or FGF.

The osteogenic compositions according to the invention are used by implantation for example to fill bone defects, to perform vertebral fusions or maxillofacial repairs or for the treatment of the absence of fracture consolidation (non-union).

In these different therapeutic uses the size of the matrix and the amount of osteogenic growth factor are a function of the volume of the site to be filled.

In one embodiment, the anionic polysaccharide solutions have concentrations of between 0.1 mg / ml and 100 mg / ml, preferably 1 mg / ml at 75 mg / ml, more preferably between 5 and 50 mg / ml.

In one embodiment, for a vertebral implant the osteogenic growth factor doses will be between 0.05 mg to 8 mg, preferably between 0.1 mg and 4 mg, more preferably between 0.1 mg and 2 mg. , while the doses currently accepted in the literature are between 8 and 12 mg.

In one embodiment, for a vertebral implant, the doses of angiogenic growth factor will be between 0.05 mg and 8 mg, preferably between 0.1 mg and 4 mg, more preferably between 0.1 mg and 2 mg. mg.

For example, in the cases of maxillofacial repair or in the treatment of non-union, doses administered will be less than 1 mg.

In one embodiment, the divalent cation solutions have concentrations of between 0.01 and 1 M, preferably between 0.05 and 0.2 M. In one embodiment, the anionic polysaccharide solutions have concentrations of between 0.1 mg / ml and 100 mg / ml, preferably 1 mg / ml at 75 mg / ml, more preferably between 5 and 50 mg / ml.

The invention also relates to the process for preparing an implant according to the invention which comprises at least the following steps: a) a solution comprising an osteogenic growth factor / anionic polysaccharide complex and an organic matrix and or a hydrogel, b) adding the solution containing the complex to the organic matrix and / or the hydrogel, and optionally mixing the mixture, c) adding to the implant obtained in b) a solution of a soluble salt of at least divalent cation, d) optionally performs the lyophilization of the implant obtained in step c).

The invention also relates to the process for preparing an implant according to the invention which comprises at least the following steps:

a) a solution comprising an osteogenic growth factor / amphiphilic anionic polysaccharide complex and an organic matrix and / or a hydrogel, b) is added to the organic matrix and / or the hydrogel a) a solution of a soluble salt of at least divalent cation, c) adding the solution containing the growth factor to the organic matrix and / or the hydrogel obtained in b) and optionally homogenizing the mixture, d) proceeding optionally lyophilization of the implant obtained in step c).

In one embodiment, the organic matrix is a matrix consisting of crosslinked hydrogels and / or collagen. In one embodiment, the matrix is chosen from the matrices based on purified natural collagen, sterilized, preferably crosslinked.

In one embodiment, in step a), the crosslinked or non-crosslinked hydrogel-forming polymer is selected from the group of synthetic polymers, among which are copolymers of ethylene glycol and lactic acid, copolymers of ethylene glycol and glycolic acid, poly (N-vinyl pyrrolidone), polyvinyl acids, polyacrylamides, polyacrylic acids.

In one embodiment, in step a), the crosslinked or non-crosslinked hydrogel-forming polymer is chosen from the group of natural polymers, among which hyaluronic acid, keratane, pectin, dextran, cellulose and cellulose derivatives, alginic acid, xanthan, carrageenan, chitosan, chondroitin, collagen, gelatin, polylysine, fibrin and their biologically acceptable salts.

In one embodiment, the natural polymer is chosen from the group of polysaccharides forming hydrogels, among which hyaluronic acid, alginic acid, dextran, pectin, cellulose and its derivatives, pullulan, xanthan, carrageenan, chitosan, chondroitin and their biologically acceptable salts.

In one embodiment, in step a), the natural polymer is chosen from the group of hydrogel-forming polysaccharides, among them hyaluronic acid, alginic acid and their biologically acceptable salts.

In one embodiment in step b) or c), the at least divalent cation soluble salt solution is a divalent cation solution.

In one embodiment, the soluble salts of divalent cation are calcium salts whose counterion is chosen from among chloride, D-gluconate, formate, D-saccharate, acetate, L-lactate, glutamate, aspartate, propionate, fumarate, sorbate, bicarbonate, bromide or ascorbate. In one embodiment, the divalent cation soluble salt is calcium chloride.

In one embodiment, the soluble salts of divalent cation are magnesium salts whose counterion is chosen from chloride,

D-gluconate, formate, D-saccharate, acetate, L-lactate, glutamate, aspartate, propionate, fumarate, sorbate, bicarbonate, bromide or ascorbate.

In one embodiment, the soluble salts of divalent cation are zinc salts whose counterion is chosen from chloride, D-gluconate, formate, D-saccharate, acetate, L-lactate, glutamate, aspartate, propionate, fumarate, sorbate, bicarbonate, bromide or ascorbate.

In one embodiment in step b) or c), the at least divalent cation soluble salt solution is a multivalent cation solution.

In one embodiment the multivalent cations are selected from the group consisting of multivalent cations of iron, aluminum, cationic polymers such as polylysine, spermine, protamine, fibrin.

In one embodiment in step a), a solution of a non-osteogenic growth factor is also available.

The invention also relates to the use of the composition according to the invention as a bone implant.

In one embodiment, said composition may be used in combination with a prosthetic device of the type of vertebral prosthesis or vertebral fusion cage.

It also relates to therapeutic and surgical methods using said composition in bone reconstruction.

The invention is illustrated by the following examples. Example 1 Preparation of sodium dextranmethylcarboxylate modified with the sodium salt of L-tryptophan

Polymer 1 is a sodium dextranmethylcarboxylate modified with the sodium salt of L-tryptophan obtained from a dextran with a weight average molar mass of 40 kg / mol, ie a degree of polymerization of 154 (Pharmacosmos) according to the process. described in the patent application FR07.02316. The mole fraction of sodium methylcarboxylates, whether or not modified with tryptophan, is t in formula I, is 1.03. The molar fraction of tryptophan-modified sodium methylcarboxylates, p in formula III, is 0.36.

Example 2 Preparation of a sodium dextranethylcarboxylate modified with the ethyl ester of L-tryptophan

Polymer 2 is a sodium dextranethyl carboxylate modified with the ethyl ester of L-tryptophan obtained from a dextran with a weight average molar mass of 40 kg / mol, ie a degree of polymerization of 154 (Pharmacosmos) according to the process. described in the patent application FR07.02316. The mole fraction of sodium methylcarboxylates, whether or not modified with the ethyl ester of tryptophan, is t in formula III, is 1.07. The molar fraction of tryptophan ethyl ester-modified sodium methylcarboxylates, p in formula III, is 0.49.

Example 3 Preparation of a sodium dextranethylcarboxylate modified with the decyl ester of L-glycine

Polymer 3 is a sodium dextranethyl carboxylate modified with the decyl ester of L-glycine obtained from a dextran with a weight average molar mass of 40 kg / mol, ie a degree of polymerization of 154.

(Pharmacosmos) according to the process described in the patent application FR08.05506.

The molar fraction of sodium methylcarboxylates, modified or unmodified by the L-glycine decyl ester, in the formula X, is 1.04. The mole fraction of L-glycine decyl ester-modified sodium methylcarboxylates, q in formula IX, is 0.09. EXAMPLE 4 Preparation of an octanoic ester-modified sodium dextranmethylcarboxylate of L-phenylalanine

Polymer 4 is a sodium dextranethyl carboxylate modified with the octanoic ester of L-phenylalanine obtained from a dextran with a weight average molar mass of 40 kg / mol, ie a degree of polymerization of 154 (Pharmacosmos) according to US Pat. process described in the patent application FR08.05506. The mole fraction of sodium methylcarboxylates, modified or not with the octanoic ester of L-phenylalanine, ie r in formula X, is 1.07. The molar fraction of octanoic ester-modified sodium methylcarboxylates of L-phenylalanine, q in formula IX, is 0.08.

Example 5 Preparation of rhGDF-5 / Polymer 3 Complex

Formulation 1: 50 μl of a solution of rhGDF-5 at 2.0 mg / ml in 5 mM PHCI are mixed with 50 μl of a solution of Polymer 3 at 61.1 mg / ml. The polymer solution is buffered with 20 mM phosphate (pH 7.2). The GDF-5 / Polymer 3 complex solution is at pH 6.4 and contains 10 mM phosphate. The molar ratio GDF-5 / Polymer 3 is 1/20. This solution is finally filtered on 0.22 μm. The final solution is clear and is characterized by Dynamic Diffusion of Light. Most of the objects present measure less than 10 nm.

Example 6 Preparation of rhGDF-5 / Polymer 4 Complex

Formulation 2: 679 μl of a solution of rhGDF-5 at 3.7 mg / ml in 10 mM HCl are mixed with 1821 μl of a solution of Polymer 4 at 42.3 mg / ml (pH 7 , 3). The GDF-5 / Polymer 4 complex solution is at pH 6.5 and contains 1 mg / ml of GDF-5 and 30.8 mg / ml of Polymer 4. The molar ratio of GDF-5 / Polymer 4 is 1/20. This solution is finally filtered on 0.22 μm. The final solution is clear and is characterized by Dynamic Diffusion of Light. Most of the objects present measure less than 10 nm. Example 7: Preparation of collagen sponge implants / rhBMP-2

Implant 1: 40 μl of a solution of rhBMP-2 at 0.05 mg / ml are introduced sterilely in a sterile 200 mm3 crosslinked collagen sponge of the Helistat type (Integra LifeSciences, Plainsboro, New Jersey). The solution is allowed to incubate for 30 minutes in the collagen sponge before use.

The dose of BMP-2 is 2 μg.

Implant 2: It is prepared as implant 1 with 40 μl of a solution of rhBMP-2 at 0.5 mg / ml. The dose of BMP-2 is 20 μg.

Example 8 Preparation of the rhBMP-2 / Polymer 1 Complex

Formulation 3: 50 μl of a RhBMP-2 solution at 0.15 mg / ml is mixed with 100 μl of a 37.5 mg / ml solution of Polymer 1. The solutions of rhBMP-2 and Polymer 1 are buffered at pH 7.4. This solution is incubated for two hours at ^{40 °} C. and sterile filtered through 0.22 μm.

Formulation 4: It is prepared as Formulation 3 by mixing 50 μl of a solution of rhBMP-2 at 1.5 mg / ml with 100 μl of a solution of Polymer 1 at 37.5 mg / ml.

Example 9 Preparation of Implants Collagen Sponge / BMP-2 Complex / Polymer 1 in the Presence of Calcium Chloride, Freeze Dried

Implant 3: 40 μl of Formulation 4 are introduced into a sterile 200 mm ³ crosslinked collagen sponge of Helistat type (Integra LifeSciences, Plainsboro, New Jersey). The solution is incubated for 30 minutes in the collagen sponge before adding 100 μl of a solution of calcium chloride at a concentration of 18.3 mg / ml. After 15 minutes, the sponge is ready for use. The dose of BMP-2 is 20 μg. EXAMPLE 10 Preparation of Implants Collagen Sponge / BMP-2 Complex / Polymer 1 in the Presence of Calcium Chloride, Freeze Dried

Implant 4: 40 μl of Formulation 3 are introduced into a sterile 200 mm ³ crosslinked collagen sponge of the Helistat type (Integra LifeSciences, Plainsboro, New Jersey). The solution is incubated for 30 minutes in the collagen sponge before adding 100 μl of a solution of calcium chloride at a concentration of 18.3 mg / ml. The sponge is then frozen and sterilized lyophilized. The dose of BMP-2 is 2 μg.

Implant 5: II is prepared as implant 4 with 40 μl of Formulation 4. The dose of BMP-2 is 20 μg.

Example 11 Evaluation of the Osteoinductive Power of the Various Formulations

The objective of this study is to demonstrate the osteoinductive power of different formulations in a model of ectopic bone formation in rats. Male rats of 150 to 250 g (Sprague Dawley OFA - SD, Charles River Laboratories France, B.P. 109, 69592 ArbresIe) are used for this study.

Analgesic treatment (buprenorphine, Temgesic ^® , Pfizer, France) is given before surgery. The rats are anesthetized by inhalation of a mixture of O2 isoflurane (1-4%). The fur is removed by shaving over a wide dorsal area. The skin of this dorsal zone is disinfected with a solution of povidone iodine (Vetedine ^® solution, Vetoquinol, France).

Paravertebral incisions of about 1 cm are made to clear the left and right paravertebral dorsal muscles. Access to the muscles is performed by transfacial incision. Each of the implants is placed in a pocket in such a way that no compression on them can be exerted. Four implants are implanted per rat (two implants per site). The opening of the implants is then sutured using a polypropylene wire (Prolene 4/0, Ethicon, France). The skin is closed with a non-absorbable suture. The rats are then returned to their respective cages and kept under observation during their recovery. At 21 days, the animals are anesthetized with an injection of tiletamine-zolazepam (ZOLETIL ^® 25-50 mg / kg, IM, Virbac, France).

The animals are then euthanized by injecting a dose of pentobarbital (DOLETHAL ^®, VÉTOQUINOL, France). A macroscopic observation of each site is then carried out, any sign of local intolerance (inflammation, necrosis, haemorrhage) and the presence of bone and / or cartilage tissue is recorded and scored according to the following scale: 0: absence, 1: low, 2: moderate, 3: marked, 4: important.

Each of the implants is removed from its implantation site and macroscopic photographs are taken. The size and weight of the implants are then determined. Each implant is then stored in a 10% buffered formalin solution.

Results:

This in vivo experiment measures the osteoinductive effect of BMP-2 by placing the implant in a muscle of the back of a rat. This non-osseous site is said to be ectopic.

Macroscopic observations of explants allow us to evaluate the presence of bone tissue and the mass of implants.

A dose of 2 μg of BMP-2 in a collagen sponge (Implant 1) does not have sufficient osteoinductive power to be able to find the collagen implants after 21 days.

A dose of 20 μg of BMP-2 in a collagen sponge (Implant 2) leads to the production of ossified implants of 38 mg average mass after 21 days. For the same dose of BMP-2 of approximately 20 μg, the BMP-2 / Polymer 1 (Implant 3) complex in the presence of CaCl ₂ in solution in the collagen sponge makes it possible to increase the osteogenic activity of the BMP. -2. The average mass of the implants 3 is about 3 times greater than that of the implants.

Lyophilization makes it possible to amplify this increase in osteogenic activity since the average mass of the implants containing 20 μg of BMP-2 in the form of a complex with Polymer 1 in the presence of freeze-dried CaCb in the collagen sponge (implant 5) is two greater than that of implants in which the BMP-2 / Polymer 1 complex in the presence of CaCl 2 is in solution (implant 3).

For a 10-fold lower dose of BMP-2, the BMP-2 complex in the presence of freeze-dried CaCl ₂ in the collagen sponge (Implant 4) makes it possible to generate ossified implants with a mass 2 times greater with a bone score equivalent to those with the BMP-2 alone. This new formulation makes it possible to strongly reduce the doses of BMP-2 to be administered while maintaining the osteogenic activity of this protein.

Example 12: Preparation of formulations containing the rhBMP-2 / Polymer 1 complex

Formulation 5: 552 μl of a solution of rhBMP-2 at 1.35 mg / ml are mixed with 619 μl of a polymer solution 1 at 60.0 mg / ml. The volume of formulation 5 is completed at 1300 μl by addition of sterile water. This solution is incubated for two hours at 4 ° C. and sterile filtered through 0.22 μm. The concentration of rhBMP-2 in formulation 5 is 0.571 mg / ml and that in polymer 1 is 28.6 mg / ml.

Formulation 6: It is prepared as Formulation 5 by mixing 175 μl of a solution of rhBMP-2 at 1.47 mg / ml with 1224 μl of a solution of polymer 1 at 60.0 mg / ml. The volume of formulation 6 is completed at 1800 μl by addition of sterile water. The concentration of rhBMP-2 in formulation 6 is 0.14 mg / ml and that in polymer 1 is 40.8 mg / ml. Formulation 7: It is prepared as Formulation 5 by mixing 26.5 μl of a solution of rhBMP-2 at 1.46 mg / ml at 321.7 μl of a solution of polymer 1 at 60.0 mg / ml . The formulation volume is completed at

772 μl per day of sterile water. The concentration of rhBMP-2 in formulation 7 is 0.05 mg / ml and that in polymer 1 is 25 mg / ml.

Example 13 Preparation of a Sodium Hyaluronate Gel Containing Calcium Chloride

Gel 1: 10.62 ml of sterile water are introduced into a 50 ml Falcon.

0.44 g of sodium hyaluronate (Pharma grade 80, Kibun Food Chemifa, LTD) are added with vigorous vortexing. 0.14 g of calcium chloride are then added to the sodium hyaluronate gel, also with stirring. The concentration of calcium chloride in the gel is 13.1 mg / ml.

Example 14 Preparation of a sodium hyaluronate gel containing rhBMP-2 / Polymer 1 complex and calcium chloride

Gel 2: 1230 μl of Formulation 5 are transferred to a sterile 10 ml syringe. 5.8 ml of 4% sodium hyaluronate gel 1 containing calcium chloride at a concentration of 13.1 mg / ml are transferred into a sterile 10 ml syringe. The formulation solution 5 is added to the gel 1 by coupling the two syringes and the gel obtained is homogenized by several passages from one syringe to the other. The opaque gel obtained is transferred into a 50 ml Falcon. The concentration of rhBMP-2 in gel 2 is 0.10 mg / ml and that in polymer 1 is 5.0 mg / ml.

200 μl of the gel 2 are injected per implantation site. The dose of rhBMP-2 implanted is 20 μg. Example 15 Preparation of a sodium hyaluronate gel containing the rhBMP-2 / Polymer 1 complex and calcium chloride

Gel 3: This gel is prepared as described in Example 13 using

1697 μl of formulation 6 and 8 ml of 4% sodium hyaluronate gel containing calcium chloride at a concentration of 15.8 mg / ml. The concentration of rhBMP-2 in gel 3 is 0.025 mg / ml and that in polymer 1 is 7.14 mg / ml. 200 μl of the gel 3 are injected per implantation site. The dose of rhBMP-2 implanted is 5 μg.

EXAMPLE 16 Preparation of a Sodium Alginate Gel Containing the RhBMP-2 / Polymer 1 Complex and Calcium Chloride

Gel 4: This gel is prepared using 772 μl of formulation 7 and

386 μl of sodium alginate gel at 40 mg / ml. 40 μl of a 45.5 mg / ml calcium chloride solution are added to 60 μl of the sodium alginate gel containing the rhBMP-2 / Polymer 1 complex. The concentration of rhBMP-2 in the gel 4 is of 0.02 mg / ml and that of polymer 1 of 10.0 mg / ml.

100 μl of the gel 4 are injected per implantation site. The dose of rhBMP-2 implanted is 2 μg.

Example 17: Preparation of a Collagen Implant Containing a Sodium Alginate Gel Containing the RhBMP-2 / Polymer 1 Complex and Calcium Chloride

Implant 6: The gel 5 is prepared using 645 .mu.l of formulation 7 and 323 .mu.l of 40 mg / ml sodium alginate gel. 60 μl of the sodium alginate gel containing the rhBMP-2 / Polymer 1 complex are added to a Helistat type sterile 200 mm ³ cross-linked collagen sponge (Integra LifeSciences, Plainsboro, New Jersey). 40 μl of a solution of calcium chloride 45.5 mg / ml are also added to this sponge. After 30 minutes of contact time, the sponge is then frozen and lyophilized. This sponge can be directly implanted in the rat. The dose of rhBMP-2 in implant 1 is 2 μg, that of Polymer 1 is 1 mg.

Example 18 Evaluation of the Ostoinductive Power of the Various Formulations

The osteoinductive power is evaluated according to the protocol described in Example 11.

Results: This in vivo experiment makes it possible to measure the osteoinductive effect of rhBMP-2 placed in a muscle of the back of a rat. This non-osseous site is said to be ectopic. The results of the various examples are summarized in the following table.

A dose of 2 μg of rhBMP-2 in a collagen sponge (Implant 1) does not have sufficient osteoinductive power to allow explants to be found after 21 days.

A dose of 20 μg of rhBMP-2 in a collagen sponge

(Implant 2) leads to obtaining ossified explants of 38 mg average mass after 21 days.

For the same dose of rhBMP-2 of 20 μg, the sodium hyaluronate gel containing the rhBMP-2 / Polymer 1 (Gel 2) complex in the presence of calcium chloride makes it possible to increase the osteogenic activity of rhBMP-2. . The average mass of the explants obtained with the gel 2 is approximately 6 times greater than that of the explants obtained with the collagen implants containing 20 μg of rhBMP-2 alone (Implant 8). For a 4-fold lower dose of rhBMP-2, ie 5 μg of rhBMP-2, the rhBMP-2 / Polymer 1 complex in the presence of CaCl ₂ in the sodium hyaluronate gel (Gel 3) makes it possible to generate ossified explants of mass 9 times higher with a bone score equivalent to explants obtained with collagen implants containing 20 μg of rhBMP-2 alone (Implant 8). This new formulation makes it possible to strongly reduce the doses of BMP-2 while maintaining the osteogenic activity of this protein.

For a 10-fold lower dose of rhBMP-2, rhBMP-.2 / Polymer 1 in a sodium alginate gel containing calcium chloride (Gel 4) makes it possible to generate ossified explants with a mass slightly greater than those obtained. with collagen implants containing 20 μg of rhBMP-2 alone (Implant 8). This new formulation makes it possible to strongly reduce rhBMP-2 doses while maintaining the osteogenic activity of this protein.

The alginate gel containing the rhBMP-2 / Polymer 1 complex can also be deposited in a collagen sponge which serves as a support for the growth of bone cells. In this case also, 2 μg of rhBMP-2 (Implant 6) makes it possible to obtain ossified explants with a mass greater than those obtained with collagen implants containing 20 μg of rhBMP-2 alone (Implant 8).

Claims

An open implant consisting of an osteogenic composition comprising at least:

• an osteogenic growth factor,

A soluble salt of at least divalent cation, and

• an organic support

Said organic support does not comprise a demineralized bone matrix.

2. Implant according to claim 1, characterized in that the support consists of an organic matrix and / or a polymer forming a hydrogel.

3. Implant according to any one of the preceding claims, characterized in that the organic matrix is a matrix consisting of crosslinked hydrogels and / or collagen.

4. Implant according to any one of the preceding claims, characterized in that the matrix is selected from the matrices based on purified natural collagen, sterilized preferably crosslinked.

5. Implant according to any one of the preceding claims, characterized in that the crosslinked or non-crosslinked hydrogel-forming polymer is chosen from the group of synthetic polymers among which the copolymers of ethylene glycol and lactic acid, the copolymers of ethylene glycol and glycolic acid, poly (N-vinyl pyrrolidone), polyvinyl acids, polyacrylamides, polyacrylic acids.

6. Implant according to any one of claims 1 to 5, characterized in that the polymer forming a crosslinked or non-crosslinked hydrogel is selected from the group of natural polymers among which hyaluronic acid, keratane, pullulan, pectin, dextran, cellulose and cellulose derivatives, alginic acid, xanthan, carrageenan, chitosan, chondroitin, collagen, gelatin, polylysine, fibrin and their biologically acceptable salts.

7. Implant according to claim 6, characterized in that the natural polymer is selected from the group of polysaccharides forming hydrogels, among which hyaluronic acid, alginic acid, dextran, pullulan, pectin, cellulose and its derivatives, xanthan, carrageenan, chitosan, chondroitin and their biologically acceptable salts.

8. Implant according to claim 6, characterized in that the natural polymer is selected from the group of polysaccharides forming hydrogels, among which hyaluronic acid, alginic acid and their biologically acceptable salts

9. Implant according to any one of the preceding claims, characterized in that said composition is in the form of lyophilisate.

10. Implant according to any one of the preceding claims, characterized in that the osteogenic growth factor is selected from the group of therapeutically active BMPs (Bone Morphogenetic Proteins).

11. Implant according to any one of the preceding claims, characterized in that the osteogenic growth factor is selected from the group consisting of BMP-2 (Dibotermine-alpha), BMP-4, BMP-7 (Eptotermin-1). alpha), BMP-14 and GDF-5.

12. Implant according to any one of the preceding claims, characterized in that the osteogenic protein is BMP-2 (Dibotermine-alpha).

13. Implant according to any one of the preceding claims, characterized in that the osteogenic protein is GDF-5.

14. Implant according to any one of the preceding claims, characterized in that it further comprises angiogenic growth factors selected from the group consisting of PDGF, VEGF or FGF.

15. Implant according to any one of the preceding claims, characterized in that the at least divalent cation is a divalent cation selected from the group consisting of calcium, magnesium or zinc cations.

16. Implant according to any one of the preceding claims, characterized in that the soluble salt of divalent cation is a calcium salt whose counterion is selected from chloride, D-gluconate, formate, D-saccharate , acetate, L-lactate, glutamate, aspartate, propionate, fumarate, sorbate, bicarbonate, bromide or ascorbate.

17. Implant according to any one of the preceding claims, characterized in that the soluble salt of divalent cation is calcium chloride.

18. Implant according to any one of claims 1 to 15, characterized in that the at least divalent cation is a multivalent cation selected from the group consisting of iron cations, aluminum or cationic polymers selected from polylysine , spermine, protamine and fibrin, alone or in combination.

19. Implant according to any one of the preceding claims, characterized in that the amphiphilic polysaccharide is selected from the group consisting of polysaccharides functionalized with hydrophobic derivatives.

20. Implant according to any one of the preceding claims, characterized in that the amphiphilic polysaccharide is selected from the group consisting of anionic polysaccharides predominantly comprising glycoside bonds of (1, 4), (1, 3) and / or 1, 2), functionalized with at least one tryptophan derivative corresponding to the following general formula I:

polysaccharide

F [Trp I R [Trp] n

Formula I The polysaccharide predominantly consisting of (1,4) and / or (1,3) and / or (1,2) glycosidic linkages resulting from the coupling between the linking arm R and a functional group; OH of the neutral or anionic polysaccharide, being either an ester, thioester, amide, carbonate, carbamate, ether, thioether or amine function,

R being a chain comprising between 1 and 18 carbons, optionally branched and / or unsaturated comprising one or more heteroatoms, such as O, N and / or S, and having at least one acidic function

(i + j) represents the mole fraction of acid functions per saccharide unit and is between 0.1 and 2,

when the polysaccharide is an anionic polysaccharide, when one or more acid functions of the polysaccharide are not substituted with Trp, then they are salified by a cation, alkaline preferably as Na ⁺ or K ⁺ . said polysaccharides being amphiphilic at neutral pH.

21. Implant according to any one of the preceding claims, characterized in that the amphiphilic polysaccharide is selected from the group consisting of functionalized anionic polysaccharides of general formula III below:

Formula III

AA being a hydrophobic amino acid residue, L or D, produces coupling between the amino acid amine and an acid carried by the R group. Said hydrophobic amino acid is chosen from tryptophan derivatives, such as tryptophan, tryptophanol, tryptophanamide, 2-indole ethylamine and their alkali cation salts or selected from phenylalanine, leucine, isoleucine and valine and their alcohol, amide or decarboxylated derivatives.

t represents the mole fraction of FR- [AA] n substituent per glycosidic unit and is between 0.1 and 2, p represents the mole fraction of Rs substituted by AA and is between 0.05 and 1. When R is not substituted by AA, then the acid or acids of the R group are cationic carboxylates, alkali preferably as Na ^"1" , K ⁺ , said dextran being amphiphilic at neutral pH.

22. Implant according to any one of the preceding claims, characterized in that the amphiphilic polysaccharide is selected from the group consisting of polysaccharides having carboxyl functional groups partially substituted with hydrophobic alcohols of general formula IX:

Polysaccharide + carboxyl

F ¹

I

R

G Ah

Form IX

in which, q represents the mole fraction of the carboxyl functions of the polysaccharide substituted with F-R-G-Ah and is between 0.01 and 0.7,

F 'being an amide function,

G being either an ester, thioester, carbonate or carbamate function,

Ah being a residue of a hydrophobic alcohol, produces coupling between the hydroxyl function of the hydrophobic alcohol and at least one electrophilic function carried by the group R. - When the carboxyl function of the polysaccharide is not substituted by F'-RG-Ah, then the carboxyl functional group (s) of the polysaccharide are cationic carboxylates, alkali preferably as Na ⁺ or K ⁺ . said polysaccharide having carboxyl functional groups being amphiphilic at neutral pH.

23. A method for preparing an implant according to the invention comprising at least the following steps: a) a solution comprising an osteogenic growth factor is available; b) an organic matrix and / or an (c) adding the solution containing the growth factor to the organic matrix and / or the hydrogel, and optionally mixing the mixture, d) adding to the implant obtained in c) a solution of a soluble salt of at least divalent cation, e) the lyophilization of the implant obtained in step d) is optionally carried out.

24. The method of claim 23, characterized in that the organic matrix is a matrix consisting of a crosslinked hydrogel and / or collagen.

25. The method of claim 23, characterized in that the matrix is selected from the matrices based on purified natural collagen sterilized preferably crosslinked.

26. The method of claim 23, characterized in that the crosslinked or non-crosslinked hydrogel-forming polymer is chosen from the group of synthetic polymers among which the copolymers of ethylene glycol and lactic acid, the copolymers of the ethylene glycol and glycolic acid, poly (N-vinyl pyrrolidone), polyvinyl acids, polyacrylamides, polyacrylic acids.

27. Method according to any one of claims 23 to 26 characterized in that the polymer forming a crosslinked or non-crosslinked hydrogel is selected from the group of natural polymers among which hyaluronic acid, keratane, pectin, dextran, cellulose and cellulose derivatives, alginic acid, xanthan, carrageenan, chitosan, chondroitin, collagen, gelatin, polylysine, fibrin and their biologically acceptable salts.

28. The method of claim 23 characterized in that the natural polymer is selected from the group of hydrogel-forming polysaccharides, consisting of hyaluronic acid, alginic acid, dextran, pectin, cellulose and its derivatives, the pullulan, xanthan, carrageenan, chitosan, chondroitin and their biologically acceptable salts.

29. The method of claim 23 characterized in that the natural polymer is selected from the group of polysaccharides forming hydrogels, consisting of hyaluronic acid, alginic acid and their biologically acceptable salts.

30. Process according to any one of claims 23 to 29, characterized in that the soluble solution of at least divalent cation salt is a divalent cation solution.

31. The method of claim 30 characterized in that the soluble salt of divalent cation is selected from magnesium salts whose counterion is chloride, D-gluconate, formiat, D-saccharate, acetate, L-lactate, glutamate, aspartate, propionate, fumarate, sorbate, bicarbonate, bromide or ascorbate.

32. The method of claim 31 characterized in that the soluble salt of divalent cation is selected from chloride, D-gluconate, formate, D-saccharate, acetate, L-lactate, glutamate, aspartate, propionate, fumarate, sorbate, bicarbonate, bromide or ascorbate.

33. The method of claim 31 characterized in that in step d) the soluble salt of divalent cation is calcium chloride.

34. Method according to any one of claims 23 to 35, characterized in that in step a), a solution of a non-osteogenic growth factor is also available.