Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/18—Growth factors; Growth regulators
  • A61K38/1825—Fibroblast growth factor [FGF]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/18—Growth factors; Growth regulators
  • A61K38/1858—Platelet-derived growth factor [PDGF]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/18—Growth factors; Growth regulators
  • A61K38/1858—Platelet-derived growth factor [PDGF]
  • A61K38/1866—Vascular endothelial growth factor [VEGF]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/18—Growth factors; Growth regulators
  • A61K38/1875—Bone morphogenic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
  • A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
  • A61L24/10—Polypeptides; Proteins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/14—Macromolecular materials
  • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
  • A61L27/24—Collagen
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
  • A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
  • A61L27/46—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix with phosphorus-containing inorganic fillers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
  • A61L27/52—Hydrogels or hydrocolloids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
  • A61L27/54—Biologically active materials, e.g. therapeutic substances
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
  • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
  • A61L2300/412—Tissue-regenerating or healing or proliferative agents
  • A61L2300/414—Growth factors

Definitions

• 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.
• BMPs Bone Morphogenetic Proteins
• OPs Osteogenic Proteins
• 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).
• BMPs 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.
• 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.
• 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 lumbar vertebrae and the bone regeneration of the tibia for so-called non-union fractures.
• 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.
• OP-1 Implant 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 impermeable to limit entry of surrounding soft tissue cells and also prevent the entry of inflammatory cells ([0030], 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).
• a calcium salt or a 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 the 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.
• 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,
• a soluble salt of at least divalent cation A soluble salt of at least divalent cation
• Said organic support does not comprise a demineralized bone matrix.
• 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.
• a demineralized matrix may also be prepared in an inactive form after extraction with chaotropic agents.
• organic support is meant a support consisting of an organic matrix and / or a hydrogel.
• 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 which can be used for the constitution of an organic matrix are described in Hoffman's review Hydrogels for Biomedical Applications (Adv Drug Deliv Rev, 2002, 43, 3-12).
• the matrix is chosen from the matrices based on purified natural collagen, sterilized, preferably crosslinked.
• 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 may be used as a mixture to obtain materials which may be in the form of a material with sufficient mechanical properties to be shaped or molded or in the form of a "putty" where collagen or a hydrogel acts as a 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.
• 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.
• 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.
• 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.
• 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.
• the natural polymer is chosen from the group of hydrogel-forming polysaccharides, among them hyaluronic acid, alginic acid and their biologically acceptable salts.
• said composition is in the form of lyophilisate.
• the at least one divalent cation soluble salt is a divalent cation soluble salt selected from calcium, magnesium or zinc cations.
• the at least one divalent cation soluble salt is a calcium salt.
• 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.
• 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.
• the divalent cation soluble salt is a magnesium salt whose counterion is selected from the group consisting of
• D-gluconate formate, D-saccharate, acetate, L-lactate, glutamate, aspartate, propionate, fumarate, sorbate, bicarbonate, bromide or ascorbate.
• 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.
• the divalent cation soluble salt is calcium chloride.
• the soluble cation salt is a soluble multivalent cation salt.
• multivalent cations are meant species carrying more than two positive charges such as iron, aluminum, cationic polymers such as polylysine, spermine, protamine, fibrin.
• osteogenic growth factor or BMP alone or in combination is meant a BMP selected from the group of therapeutically active BMPs (Bone Morphogenetic Proteins).
• 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.
• the osteogenic protein is BMP-2 (Dibotermin-alpha).
• 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).
• the hydrogel can be prepared just prior to implantation.
• the hydrogel may be prepared and stored in a pre-filled syringe for subsequent implantation.
• 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.
• a lyophilizate is very rapid and allows easy obtaining of a ready-to-use formulation, said formulation being rehydrated before implantation by the addition of blood or implanted in its dehydrated form, the rehydration intervening then, after implantation, by contact with biological fluids.
• osteogenic growth factors In addition to these osteogenic growth factors, it is possible to add other proteins and in particular angiogenic growth factors such as PDGF, VEGF or FGF.
• angiogenic growth factors such as PDGF, 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).
• the size of the matrix and the amount of osteogenic growth factor are a function of the volume of the site to be filled.
• 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.
• the doses currently accepted in the literature are between 8 and
• 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.
• doses administered will be less than 1 mg.
• the divalent cation solutions have concentrations of between 0.01 and 1 M, preferably between 0.05 and 0.2 M.
• 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 method for preparing an implant according to the invention which comprises at least the following steps: a) a solution comprising an osteogenic growth factor is available, b) an organic matrix is available and or a hydrogel, c) the solution containing the growth factor is added to the organic matrix and / or the hydrogel, and the mixture is optionally homogenized, d) the implant obtained in c) is added to solution of a soluble salt of at least divalent cation,
• the invention also relates to the method for preparing an implant according to the invention which comprises at least the following steps: a) a solution comprising an osteogenic growth factor is available, b) an organic matrix is available and / or a hydrogel, c) adding to the organic matrix and / or hydrogel b) a solution of a soluble salt of cation at least divalent, d) adding the solution containing the growth factor to the organic matrix and / or the hydrogel obtained in c) and the mixture is optionally homogenized, e) the lyophilization of the implant obtained in step d) is optionally carried out.
• the organic matrix is a matrix consisting of crosslinked hydrogels and / or collagen.
• the matrix is chosen from the matrices based on purified natural collagen, sterilized, preferably crosslinked.
• 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.
• 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.
• the natural polymer is chosen from the group of hydrogel-forming polysaccharides, among which hyaluronic acid, alginic acid, dextran, pectin, cellulose and its derivatives, pullulan, xanthan, carrageenan, chitosan, chondroitin and their biologically acceptable salts.
• the natural polymer is chosen from the group of hydrogel-forming polysaccharides, among which hyaluronic acid, alginic acid and their biologically acceptable salts.
• the at least divalent cation soluble salt solution is a divalent cation solution.
• 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.
• the divalent cation soluble salt is calcium chloride.
• 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
• 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
• the at least divalent cation soluble salt solution is a multivalent cation solution.
• the multivalent cations are selected from the group consisting of multivalent cations of iron, aluminum, cationic polymers such as polylysine, spermine, protamine, fibrin.
• an organic matrix of the formulation obtained in step c) is impregnated and then the at least divalent cation solution is added.
• 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.
• said composition may be used in combination with a prosthetic device of the type vertebral prosthesis or vertebral fusion cage.
• Implant 1 40 ⁇ l of a solution of rhBMP-2 at 0.05 mg / ml are introduced sterilely, in a sterile 200 mm 3 cross-linked collagen sponge of the Helistat type (Integra Life Sciences, Plainsboro, New Jersey). The solution is allowed to incubate for 30 minutes in the collagen sponge before use.
• the dose of rhBMP-2 is 2 ⁇ g.
• Implant 2 II is prepared as implant 1 with 40 ⁇ l of a solution of rhBMP-2 at 0.5 mg / ml.
• the dose of rhBMP-2 is 20 ⁇ g.
• Implant 3 40 ⁇ l of a solution of rhBMP-2 at 1.5 mg / ml are introduced sterilely into a sterile 200 mm3 cross-linked collagen sponge of the type Helistat (Integra LifeSciences, Plainsboro, New Jersey). The solution is incubated for 30 minutes in the collagen sponge before adding 100 ⁇ l of a solution of calcium chloride at a concentration of 18.3 mg / ml. The sponge is ready for use after 15 minutes. The dose of rhBMP-2 is 20 ⁇ g.
• Implant 4 40 ⁇ l of a solution of rhBMP-2 at 0.15 mg / ml are introduced sterilely into a Helistat type sterile 200 mm 3 cross-linked collagen sponge (Integra Life Sciences, Plainsboro, New Jersey). The solution is incubated for 30 minutes in the collagen sponge before adding 100 ⁇ l 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 rhBMP-2 is 2 ⁇ g.
• Implant 5 II is prepared as implant 4 with 40 ⁇ l of a solution of rhBMP-2 at 1.5 mg / ml.
• the dose of rhBMP-2 is 20 ⁇ g.
• 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.
• Gel 2 615 ⁇ l of a solution of rhBMP-2 at 0.57 mg / ml are prepared by diluting a solution of rhBMP-2 at 1.35 mg / ml in an Infuse-type buffer with water sterile. This rhBMP-2 solution is transferred to a sterile 10 ml syringe. 2.9 ml of 4% sodium hyaluronate gel 1 containing calcium chloride at a concentration of 13.1 mg / ml are transferred to a sterile 10 ml syringe.
• the rhBMP-2 solution 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 final gel is transferred to a 10 ml Falcon.
• the concentration of rhBMP-2 in gel 2 is 0.10 mg / ml.
• rhBMP-2 implanted 200 ⁇ l of the gel 2 are injected per implantation site.
• the dose of rhBMP-2 implanted is 20 ⁇ g.
• 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 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.
• the animals are anesthetized with an injection of tiletamine-zolazepam (ZOLETI L ® 25-50 mg / kg, IM, Virbac, France).
• the animals are then euthanized by injecting a dose of pentobarbital (DOLETHAL ®, VÉTOQUINOL, France).
• DOLETHAL ® pentobarbital
• VÉTOQUINOL pentobarbital
• a macroscopic observation of each site is then made, any sign of local intolerance (inflammation, necrosis, haemorrhage) and the presence of bone tissue and / or cartilaginous is recorded and scored according to the following scale: O: absence, 1: weak, 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.
• This in vivo experiment makes it possible to measure the osteoinductive effect of rhBMP-2 by placing the implant in a muscle of the back of a rat.
• This non-osseous site is said to be ectopic.
• a dose of 2 ⁇ g of rhBMP-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 ⁇ g of rhBMP-2 in a collagen sponge (Implant 2) does not have sufficient osteoinductive power to obtain, after 21 days, ossified implants with an average weight of 38 mg.
• the addition of calcium salts in the collagen sponge containing rhBMP-2 makes it possible to increase the osteogenic activity of rhBMP-2.
• the average mass of ossified implants 3 is twice that of implants 2.
• lyophilization makes it possible to increase the effect of the calcium salt on the osteogenic activity of rhBMP-2 (Implant 5).
• the average mass of freeze-dried implants containing rhBMP-2 and CaCl 2 is approximately four times that of implants containing only rhBMP-2 (implant 2).
• the bone score is equivalent between these implants.
• rhBMP-2 for a dose of rhBMP-2 of 2 ⁇ g, rhBMP-2 in the presence of freeze-dried CaCl 2 in the collagen sponge (Implant 4) makes it possible to generate ossified implants contrary to rhBMP-2 alone at the same dose.
• the sodium hyaluronate gel containing rhBMP-2 (gel 2) 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 3 times greater than that of the explants obtained with the collagen implants containing 20 ⁇ g of rhBMP-2 alone (Implant 2).

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