POROUS BONE SUBSTITUTION MATERIAL

Abstract

"POROUS BONE SUBSTITUTION MATERIAL" The present invention relates to a porous bone substitute material intended for bone repair, in particular the repair of a cavity bone defect and / or the repair of a segmental bone defect.

Description

Title of the invention: POROUS BONE SUBSTITUTION MATERIAL technical field

[0001] The present invention belongs to the field of bone substitute materials for bone repair, in particular the repair of a cavity bone defect and / or the repair of a segmental bone defect. Previous art

[0002] Bone is constantly undergoing a process of renewal and repair. Indeed, our bone mass adapts to the biomechanical stresses of our lives by replacing old tissue with new tissue. Bones are composed of cells, called osteocytes, surrounded by a mineralized extracellular matrix. This matrix is ​​renewed through the balance between the action of two types of cells: osteoblasts and osteoclasts. Osteoblasts synthesize the bone matrix, while osteoclasts remove aging bone tissue under the influence of various hormones and mechanical stresses. This process gives bone remarkable self-repair properties, enabling it to regenerate in the event of injury.Thus, after a fracture, realignment and maintenance of the limb are generally sufficient for healing: by generating new tissue, the osteogenesis process fills the gap caused by the fracture, restoring the functional efficiency of the bone.

[0003] However, in some cases, this natural self-repair process is insufficient: approximately one in ten times, mechanical or biological problems prevent the self-repair of a fracture. Furthermore, certain bone injuries encountered in victims of domestic or road accidents, terrorist attacks, certain pathologies (such as pseudo-osteoarthritis), or surgical interventions (removal of tumors, cysts, or infectious foci) can result in significant bone loss that natural osteogenesis will not be sufficient to fill. Bone reconstruction must then be assisted.

[0004] One proposed solution for repairing bone is to graft an autologous bone fragment. This is called an autograft. An autograft does not produce an immune response, since the tissue comes from the patient. However, it results in significant cell death in the transplanted tissue. The graft's ability to produce new bone cells can compensate for this loss, but it depends in particular on the graft's vascularization. This vascularization is essential for bone reconstruction: the vessels supply the energy and nutrients necessary for cell proliferation. Furthermore, an autograft requires two surgical procedures. (grafting followed by transplantation) can cause complications (pain, abscesses, neuralgia). The size of the graft required for filling represents another significant limitation.

[0005] Another solution considered for repairing a bone is to graft a bone fraction from a donor.

[0006] These two solutions are not satisfactory. Therefore, it appears necessary to develop a bone substitute material with properties similar to natural bone, but also capable of promoting osteosynthesis associated with growth factors and progenitor cells.

[0007] To enable the repair of a bone defect, the bone substitute material must possess two important properties: - osteoconduction, which refers to the ability of the material to serve as a passive support for bone regrowth, and - osteo-induction, a property of a material containing proteins, the release of which induces the biological cascade necessary for bone formation.

[0008] It must also be porous and absorbable.

[0009] Several types of materials are used as passive supports for cellular and tissue colonization: these may include natural or synthetic ceramics, or various materials of natural origin whose chemical composition is similar to that of the mineral phase of bone. The materials of natural origin used come from diverse sources: for example, one can cite ceramicized bovine bone or the exoskeleton of coral (porites), a calcium carbonate that exhibits interesting osteoconductive and biomechanical properties. The synthetic materials most often used for filling bone defects are hydroxyapatite and tricalcium phosphates, two mineral species from the phosphate family, either pure or in mixtures. They can be prepared in the form of blocks or granules. Controlling the density, grain size, and porosity will determine the material's behavior in vivo.

[0010] However, when used, these materials cannot be used alone and require combination with growth factors and progenitor cells. Furthermore, these materials do not allow for the restoration of bone continuity and structure in significant bone defects and rarely combine osteoconduction and osteoinduction properties with controlled and complete biodegradability, in harmony with bone regeneration kinetics.

[0011] The inventors have therefore developed a new porous bone substitute material exhibiting good osteoconduction and osteoinduction, while also displaying good biocompatibility and degradation suitable for bone regeneration. Furthermore, the porous bone substitute material is easily handled. for the surgeon, and easily malleable to adapt to all types of bone defects, including significant bone defects. Summary of the invention

[0012] Thus, the present invention relates to a porous bone substitute material comprising: - at least one porous elastomeric matrix, and - particles of decellularized bone.

[0013] The present invention also relates to the use of said porous bone substitute material in bone repair, preferably the repair of a cavity bone defect and / or the repair of a segmental bone defect.

[0014] The present invention also relates to a bone repair kit comprising the porous bone substitute material.

[0015] The present invention also relates to a method for preparing a bone substitute material. Detailed description of the invention

[0016] The present invention therefore relates to a porous bone substitute material comprising: - at least one porous elastomeric matrix, and - particles of decellularized bone.

[0017] For the purposes of the present invention, "bone substitute material" means a physical support on which osteoprogenitor cells can adhere, migrate, proliferate and differentiate into osteoblasts, the cells responsible for bone formation, on the surface and inside the bone substitute material.

[0018] Advantageously, the bone substitute material according to the invention is a composite material comprising at least one porous elastomeric matrix and decellularized bone particles, the individual properties of which combine to form a heterogeneous material (the bone substitute material) having greatly improved overall performance, properties which cannot be observed with at least one elastomeric matrix or decellularized bone particles when used individually.

[0019] The inventors have shown, surprisingly, that the porous bone substitute material comprising at least an elastomeric matrix and decellularized bone particles according to the invention, exhibits: - sufficient mechanical properties to withstand stress forces, but also to support the regeneration process in the area to be repaired and to provide support for bone tissue in that area, - porosity and interconnectivity allowing the circulation of progesterone cells nitrogens, nutrients and other molecules involved in regulating these processes, while allowing internal vascularization of the porous bone substitute material of the invention.

[0020] In particular, the inventors have shown that the porous bone substitute material is mechanically stable (Young's modulus E of 100-300 kPa), non-toxic (metabolic activity of mesenchymal stromal cells greater than 90% after 24 hours of incubation with the porous bone substitute material according to the invention), biodegradable (lifespan at 37°C between 12 and 65 months), and osteoconductive. Indeed, the inventors have shown that the porous bone substitute material allows the adhesion of progenitor cells as well as their proliferation, including in depth, followed by the differentiation of progenitor cells into osteoblasts.

[0021] For the purposes of the present invention, the term "elastomeric matrix" means a structure consisting of a porous elastomeric system, said structure being capable of incorporating decellularized bone particles. Advantageously, at least one elastomeric matrix according to the present invention exhibits good biodegradability, good biocompatibility, and good mechanical properties.

[0022] For the purposes of the present invention, "elastomer" means one or more cross-linked polymers exhibiting "rubber-like elasticity" properties. In a particular embodiment of the invention, the elastomer must be biocompatible and biodegradable.

[0023] For the purposes of the present invention, a “biocompatible” elastomeric matrix is ​​understood to be an elastomeric matrix that is advantageously both compatible for implantation in a patient, i.e. that this implantation presents a favorable benefit / risk ratio from a therapeutic point of view, for example within the meaning of Directive 2001 / 83 / EC, i.e. a reduced or even non-existent risk for the patient, versus the therapeutic benefit concerned; and compatible for the inclusion of decellularized bone particles, i.e. that it allows the inclusion of decellularized bone particles, that it does not degrade or only slightly degrades the activity of the decellularized bone particles included in the matrix, and that is suitable for bone reconstruction once the biomaterial is implanted in a patient, human or animal.

[0024] For the purposes of the present invention, by "biodegradable" elastomeric matrix means an elastomeric matrix which is bioresorbable and / or biodegradable and / or bioabsorbable, with a common purpose of progressive disappearance, with one or more different or complementary mechanisms of degradation, solubilization or absorption of the elastomeric matrix in the human or animal patient in whom the material has been implanted.

[0025] In a particular embodiment of the invention, at least one matrix elastomer according to the invention comprises a poly(urea-urethane ester) based elastomer.

[0026] In a particularly advantageous embodiment of the invention, at least one elastomeric matrix of the porous bone substitute material according to the invention comprises a poly(ester-urea-urethane) based elastomer, the ester being selected from caprolactone (PCL) oligomers, lactic acid (PLA) oligomers, glycolic acid (PGA) oligomers, hydroxybutyrate (PHB) oligomers, hydroxyvalerate (PVB) oligomers, dioxanone (PDO) oligomers, poly(ethylene adipate) (PEA) oligomers, poly(butylene adipate) (PBA) oligomers or combinations thereof.

[0027] In one particular embodiment, at least one elastomeric matrix of the porous bone substitute material is a matrix comprising a poly(caprolactone-urea-urethane) elastomer. In another particular embodiment, at least one elastomeric matrix of the porous bone substitute material is a matrix comprising a poly(lactic acid-urea-urethane) elastomer. In yet another particular embodiment, at least one elastomeric matrix of the porous bone substitute material is a matrix comprising a poly(glycolic acid-urea-urethane) elastomer. In yet another particular embodiment, at least one elastomeric matrix of the porous bone substitute material is a matrix comprising a poly(hydroxyvalerate-urea-urethane) elastomer.In another particular embodiment, at least one elastomeric matrix of the porous bone substitute material is a matrix comprising a poly(hydroxybutyrate-urea-urethane) elastomer. In another particular embodiment, at least one elastomeric matrix of the porous bone substitute material is a matrix comprising a poly(dioxanone-urea-urethane) elastomer. In another particular embodiment, at least one elastomeric matrix of the porous bone substitute material is a matrix comprising a poly(ethylene adipate-urea-urethane) elastomer. In yet another particular embodiment, at least one elastomeric matrix of the porous bone substitute material is a matrix comprising a poly(butylene adipate-urea-urethane) elastomer.

[0028] In a particular embodiment, at least one elastomeric matrix of the porous bone substitute material is a matrix comprising a poly(caprolactone-urea-urethane) and poly(lactic acid-urea-urethane) elastomer. In a particular embodiment, at least one elastomeric matrix of the porous bone substitute material is a matrix comprising a poly(caprolactone-urea-urethane) and poly(glycolic acid-urea-urethane) elastomer. In a particular embodiment, at least one elastomeric matrix of the The porous bone substitute material is a matrix comprising an elastomer based on poly(caprolactone-urea-urethane) and poly(hydroxyvalerate-urea-urethane). In a particular embodiment, at least one elastomeric matrix of the porous bone substitute material is a matrix comprising an elastomer based on poly(caprolactone-urea-urethane) and poly(hydroxybutyrate-urea-urethane). In a particular embodiment, at least one elastomeric matrix of the porous bone substitute material is a matrix comprising an elastomer based on poly(caprolactone-urea-urethane) and poly(dioxanone-urea-urethane). In a particular embodiment, at least one elastomeric matrix of the porous bone substitute material is a matrix comprising an elastomer based on poly(caprolactone-urea-urethane) and poly(ethylene adipate-urea-urethane).In a particular embodiment, at least one elastomeric matrix of the porous bone substitute material is a matrix comprising a poly(caprolactone-urea-urethane) and poly(butylene adipate-urea-urethane) based elastomer.

[0029] In a particular embodiment, at least one elastomeric matrix of the porous bone substitute material is a matrix comprising an elastomer based on poly(lactic acid-urea-urethane) and poly(glycolic acid-urea-urethane). In a particular embodiment, at least one elastomeric matrix of the porous bone substitute material is a matrix comprising an elastomer based on poly(lactic acid-urea-urethane) and poly(hydroxybutyrate-urea-urethane). In a particular embodiment, at least one elastomeric matrix of the porous bone substitute material is a matrix comprising an elastomer based on poly(lactic acid-urea-urethane) and poly(hydroxybutyrate-urea-urethane). In a particular embodiment, at least one elastomeric matrix of the porous bone substitute material is a matrix comprising a poly(lactic acid-urea-urethane) and poly(dioxanone-urea-urethane) based elastomer.In a particular embodiment, at least one elastomeric matrix of the porous bone substitute material is a matrix comprising an elastomer based on poly(lactic acid-urea-urethane) and poly(ethylene adipate-urea-urethane). In a particular embodiment, at least one elastomeric matrix of the porous bone substitute material is a matrix comprising an elastomer based on poly(lactic acid-urea-urethane) and poly(butylene adipate-urea-urethane).

[0030] In a particular embodiment, at least one elastomeric matrix of the porous bone substitute material is a matrix comprising a poly(glycolic acid-urea-urethane) and poly(hydroxyvalerate-urea-urethane) elastomer. In a particular embodiment, at least one elastomeric matrix of the porous bone substitute material is a A matrix comprising an elastomer based on poly(glycolic acid-urea-urethane) and poly(hydroxybutyrate-urea-urethane). In a particular embodiment, at least one elastomeric matrix of the porous bone substitute material is a matrix comprising an elastomer based on poly(glycolic acid-urea-urethane) and poly(dioxanone-urea-urethane). In a particular embodiment, at least one elastomeric matrix of the porous bone substitute material is a matrix comprising an elastomer based on poly(glycolic acid-urea-urethane) and poly(ethylene adipate-urea-urethane). In a particular embodiment, at least one elastomeric matrix of the porous bone substitute material is a matrix comprising an elastomer based on poly(glycolic acid-urea-urethane) and poly(butylene adipate-urea-urethane).

[0031] In a particular embodiment, at least one elastomeric matrix of the porous bone substitute material is a matrix comprising an elastomer based on poly(hydroxyvalerate-urea-urethane) and poly(hydroxybutyrate-urea-urethane). In a particular embodiment, at least one elastomeric matrix of the porous bone substitute material is a matrix comprising an elastomer based on poly(hydroxyvalerate-urea-urethane) and poly(dioxanone-urea-urethane). In a particular embodiment, at least one elastomeric matrix of the porous bone substitute material is a matrix comprising an elastomer based on poly(hydroxyvalerate-urea-urethane) and poly(ethylene adipate-urea-urethane). In a particular embodiment, at least one elastomeric matrix of the porous bone substitute material is a matrix comprising a poly(hydroxyvalerate-urea-urethane) and poly(butylene adipate-urea-urethane) based elastomer.

[0032] In a particular embodiment, at least one elastomeric matrix of the porous bone substitute material is a matrix comprising a poly(dioxanone-urea-urethane) and poly(ethylene adipate-urea-urethane) elastomer. In a particular embodiment, at least one elastomeric matrix of the porous bone substitute material is a matrix comprising a poly(dioxanone-urea-urethane) and poly(butylene adipate-urea-urethane) elastomer.

[0033] In a particular embodiment, at least one elastomeric matrix of the porous bone substitute material is a matrix comprising an elastomer based on poly(ethylene adipate-urea-urethane) and poly(butylene adipate-urea-urethane).

[0034] In a particular embodiment, at least one elastomeric matrix of the porous bone substitute material is a matrix comprising an elastomer based on poly(caprolactone-urea-urethane), poly(lactic acid-urea-urethane) and poly(glycolic acid-urea-urethane).

[0035] In a particular embodiment, at least one elastomeric matrix of the porous bone substitute material is a matrix comprising an elastomer based on poly(caprolactone-urea-urethane), poly(lactic acid-urea-urethane), poly(glycolic acid-urea-urethane) and poly(hydroxyvalerate-urea-urethane).

[0036] In a particular embodiment, at least one elastomeric matrix of the porous bone substitute material is a matrix comprising an elastomer based on poly(caprolactone-urea-urethane), poly(lactic acid-urea-urethane), poly(glycolic acid-urea-urethane) and poly(hydroxybutyrate-urea-urethane).

[0037] In a particular embodiment, at least one elastomeric matrix of the porous bone substitute material is a matrix comprising an elastomer based on poly(caprolactone-urea-urethane), poly(lactic acid-urea-urethane), poly(glycolic acid-urea-urethane), poly(hydroxyvalerate-urea-urethane), and poly(hydroxybutyrate-urea-urethane). In a particular embodiment, at least one elastomeric matrix of the porous bone substitute material is a matrix comprising an elastomer based on poly(caprolactone-urea-urethane), poly(lactic acid-urea-urethane), poly(glycolic acid-urea-urethane), poly(hydroxyvalerate-urea-urethane), poly(hydroxybutyrate-urea-urethane), and poly(dioxanone-urea-urethane).

[0038] In a particular embodiment, at least one elastomeric matrix of the porous bone substitute material is a matrix comprising an elastomer based on poly(caprolactone-urea-urethane), poly(lactic acid-urea-urethane), poly(glycolic acid-urea-urethane), poly(hydroxyvalerate-urea-urethane), poly(hydroxybutyrate-urea-urethane), poly(dioxanone-urea-urethane) and poly(butylene adipate-urea-urethane).

[0039] In a particular embodiment, at least one elastomeric matrix of the porous bone substitute material is a matrix comprising an elastomer based on poly(caprolactone-urea-urethane), poly(lactic acid-urea-urethane), poly(glycolic acid-urea-urethane), poly(hydroxyvalerate-urea-urethane), poly(hydroxybutyrate-urea-urethane), poly(dioxanone-urea-urethane), poly(butylene adipate-urea-urethane) and poly(ethylene adipate-urea-urethane).

[0040] These elastomers enable the implementation of the present invention and also have the advantages of being cytocompatible, allowing the restoration of the physiological stresses of the deficient bone, avoiding the need for reoperation after restoration, and allowing for proper reconstruction of the deficient bone. Particularly advantageously, at least one elastomeric matrix of the porous bone substitute material is a matrix comprising a poly(caprolactone-urea-urethane) based elastomer. This poly(caprolactone-urea-urethane) based elastomer also has the advantage of having a elastomer, giving it flexibility, possessing an interconnected porous structure and osteo-induction properties adapted to bone reconstruction.

[0041] In one embodiment according to the invention, the bone particles present in the porous bone substitute material are decellularized bone particles. For the purposes of the present invention, "decellularized bone" means the bone collagen matrix consisting exclusively of collagen, in particular type I collagen, the mineral phase consisting of hydroxyapatite (crystallized calcium phosphate) and calcium carbonate crystals, and osteoinductive proteins. Advantageously, the decellularized bone is obtained from natural cancellous bone. Advantageously, the natural cancellous bone may be a human femoral head.

[0042] In other words, the decellularized bone according to the invention is devoid of all bone cells (osteoblasts, osteoclasts, osteocytes and bone lining cells) and of all potentially pathogenic constituents.

[0043] In a particular embodiment, the proportion of collagen present in a decellularized bone particle is between 10 and 40% by weight, advantageously between 15% and 35%. Advantageously, the decellularized bone particle consists of type I collagen and type III collagen.

[0044] Advantageously, a decellularized bone particle comprises, relative to the total weight of the particle: - a proportion of lipids less than 2% by weight, - a protein content of between 25 and 45% by weight, - a proportion of calcium of 10 to 30% by weight, - a proportion of phosphorus of 5 to 20% by weight, - a water content of less than 15% by weight, the calcium / phosphorus ratio being advantageously from 1 to 2.2.

[0045] In a particular embodiment, the decellularized bone particles of the porous bone substitute material can be obtained from natural bone. Advantageously, the natural bone particles can be obtained from allogeneic or xenogeneic bone, of human or animal origin.

[0046] Advantageously, the decellularized bone particles of the porous bone substitute material can be obtained from natural bone according to one of the processes described in patents FR2798294 or EP0502059.

[0047] In a particular embodiment, the decellularized bone particles of the porous bone substitute material according to the invention are obtained from natural bone of human or animal origin. Advantageously, the decellularized bone particles of the porous bone substitute material according to the invention are obtained from natural spongy bone. Advantageously, the natural spongy bone may be a human femoral head.

[0048] In a particular embodiment, the decellularized bone particles according to the invention have a diameter between 1 µm and 1 mm. Advantageously, the diameter of the decellularized bone particles is between 1 µm and 1 mm, advantageously between 10 nm and 900 µm, advantageously between 100 nm and 800 µm, advantageously between 100 nm and 700 µm, advantageously between 100 nm and 600 µm, advantageously between 100 nm and 500 µm, advantageously between 1 µm and 800 µm. advantageously between 1 p.m. and 7:00 p.m., advantageously between 10 p.m. and 6:00 p.m., advantageously between 10:00 p.m. and 5:50 p.m., advantageously between 2:00 p.m. and 5:00 p.m., advantageously between 3:00 p.m. and 4:50 p.m., advantageously between 3:00 p.m. and 4:00 p.m. Typically, the diameter of decellularized bone particles is between 300 pm and 400 pm.

[0049] As an example of decellularized bone, we can cite in particular Allodyn® and Osteopure® (OST Développement, Clermont Ferrand) for bone of human origin, or the product Laddec®, (OST Développement, Clermont-Ferrand) for bone of animal origin.

[0050] In an advantageous embodiment of the invention, the porous bone substitute material according to the invention comprises: - at least one elastomeric matrix comprising a poly(urea-urethane ester) based elastomer, the ester being selected from caprolactone oligomers (PCL), lactic acid oligomers (PLA), glycolic acid oligomers (PGA), hydroxybutyrate oligomers (PHB), hydroxyvalerate oligomers (PVB), dioxanone oligomers (PDO), poly(ethylene adipate) oligomers (PEA), poly(butylene adipate) oligomers (PBA) or combinations thereof, and - particles of decellularized bone.

[0051] In a first particular embodiment of the invention, the porous bone substitute material according to the invention comprises: - at least one elastomeric matrix comprising a poly(caprolactone-urea-urethane) based elastomer, and - particles of decellularized bone.

[0052] In a second particular embodiment of the invention, the porous bone substitute material according to the invention comprises: - at least one elastomeric matrix comprising a poly(lactic acid-urea-urethane) based elastomer, and - particles of decellularized bone.

[0053] In a third particular embodiment of the invention, the porous bone substitute material according to the invention comprises: - at least one elastomeric matrix comprising a poly(acid)-based elastomer glycolic-urea-urethane), and - particles of decellularized bone.

[0054] In a fourth particular embodiment of the invention, the porous bone substitute material according to the invention comprises: - at least one elastomeric matrix comprising a poly(caprolactone-urea-urethane) and poly(lactic acid-urea-urethane) based elastomer, and - particles of decellularized bone.

[0055] In a fifth particular embodiment of the invention, the porous bone substitute material according to the invention comprises: - at least one elastomeric matrix comprising a poly(caprolactone-urea-urethane) and poly(glycolic acid-urea-urethane) based elastomer, and - particles of decellularized bone.

[0056] In a sixth particular embodiment of the invention, the porous bone substitute material according to the invention comprises: - at least one elastomeric matrix comprising a poly(lactic acid-urea-urethane) and poly(glycolic acid-urea-urethane) based elastomer, and - particles of decellularized bone.

[0057] In a seventh particular embodiment of the invention, the porous bone substitute material according to the invention comprises: - at least one elastomeric matrix comprising an elastomer based on poly(caprolactone-urea-urethane), poly(lactic acid-urea-urethane) and poly(glycolic acid-urea-urethane), and - particles of decellularized bone.

[0058] In an eighth particular embodiment of the invention, the porous bone substitute material according to the invention comprises: - at least one elastomeric matrix comprising a poly(hydroxyvalerate-urea-urethane) based elastomer, and - particles of decellularized bone.

[0059] In a ninth particular embodiment of the invention, the porous bone substitute material according to the invention comprises: - at least one elastomeric matrix comprising a poly(hydroxybutyrate-urea-urethane) based elastomer, and - particles of decellularized bone.

[0060] In a tenth particular embodiment of the invention, the porous bone substitute material according to the invention comprises: - at least one elastomeric matrix comprising a poly(dioxanone-urea-urethane) based elastomer, and - particles of decellularized bone.

[0061] In an eleventh particular embodiment of the invention, the porous bone substitute material according to the invention comprises: - at least one elastomeric matrix comprising a poly(ethylene adipate-urea-urethane) based elastomer, and - particles of decellularized bone.

[0062] In a particular twelfth embodiment of the invention, the porous bone substitute material according to the invention comprises: - at least one elastomeric matrix comprising a poly(butylene adipate-urea-urethane) based elastomer, and

[0063] - decellularized bone particles.

[0064] In an advantageous embodiment of the invention, the porous bone substitute material according to the invention consists solely of: - at least one elastomeric matrix comprising a poly(urea-urethane ester) based elastomer, the ester being selected from caprolactone oligomers (PCL), lactic acid oligomers (PLA), glycolic acid oligomers (PGA), hydroxybutyrate oligomers (PHB), hydroxyvalerate oligomers (PVB), dioxanone oligomers (PDO), poly(ethylene adipate) oligomers (PEA), poly(butylene adipate) oligomers (PBA) or combinations thereof, and - particles of decellularized bone.

[0065] In an advantageous embodiment of the invention, the porous bone substitute material according to the invention consists of: - at least one elastomeric matrix comprising a poly(urea-urethane ester) based elastomer, the ester being selected from caprolactone oligomers (PCL), lactic acid oligomers (PLA), glycolic acid oligomers (PGA), hydroxybutyrate oligomers (PHB), hydroxyvalerate oligomers (PVB), dioxanone oligomers (PDO), poly(ethylene adipate) oligomers (PEA), poly(butylene adipate) oligomers (PBA) or combinations thereof, and - particles of decellularized bone.

[0066] Regardless of the aforementioned embodiment, the decellularized bone particles can be obtained from natural bone of human or animal origin or from synthetic bone. Advantageously, the decellularized bone particles have a diameter between 1 mm and 1 mm, advantageously between 300 µm and 400 µm.

[0067] In a particularly advantageous embodiment of the invention, the porous bone substitute material according to the invention comprises: - at least one elastomeric matrix comprising a poly(caprolactone-urea-urethane) based elastomer, and - particles of decellularized bone.

[0068] Advantageously, the inventors have demonstrated that the specific combination of decellularized bone particles and at least one elastomeric matrix comprising a poly(caprolactone-urea-urethane)-based elastomer confers improved biocompatibility to the porous bone substitute material due to the presence of hydroxyapatite. Indeed, the degradation of the at least one elastomeric matrix comprising a poly(caprolactone-urea-urethane)-based elastomer produces a slightly acidic environment, leading to a decrease in cell proliferation. The addition of decellularized bone particles neutralizes this acidity, thanks to the presence of hydroxyapatite.

[0069] The inventors have also demonstrated that the specific combination of decellularized bone particles and at least one elastomeric matrix comprising a poly(caprolactone-urea-urethane) elastomer allows for increased osteo-induction compared to the use of the elastomeric matrix comprising a poly(caprolactone-urea-urethane) elastomer alone. Indeed, the addition of decellularized bone particles leads to increased cell attachment and mineralization without the addition of exogenous factors, due to modifications in the surface topography of the porous bone substitute material and / or the release of calcium ions.

[0070] Advantageously, the porous bone substitute material according to the invention consists solely of: - at least one elastomeric matrix comprising a poly(caprolactone-urea-urethane) based elastomer, and - particles of decellularized bone.

[0071] Advantageously, the porous bone substitute material according to the invention consists of: - at least one elastomeric matrix comprising a poly(caprolactone-urea-urethane) based elastomer, and - particles of decellularized bone.

[0072] Advantageously, the porous bone substitute material according to the invention comprises: - at least one elastomeric matrix comprising a poly(caprolactone-urea-urethane) based elastomer, and - decellularized bone particles, said decellularized bone particles having been obtained from natural bone of human or animal origin or from synthetic bone.

[0073] Advantageously, the porous bone substitute material according to the invention consists of: - at least one elastomeric matrix comprising a poly(caprolactone-urea-urethane) based elastomer, and - decellularized bone particles, said decellularized bone particles having been obtained from natural bone of human or animal origin or from synthetic bone.

[0074] Advantageously, the porous bone substitute material according to the invention comprises: - at least one porous elastomeric matrix comprising a poly(caprolactone-urea-urethane) based elastomer, and - decellularized bone particles, said decellularized bone particles having a diameter between 1mm and 1mm, advantageously between 300pm and 400pm.

[0075] Advantageously, the porous bone substitute material according to the invention comprises: - at least one porous elastomeric matrix comprising a poly(caprolactone-urea-urethane) based elastomer, and - decellularized bone particles, said decellularized bone particles having a diameter between 1mm and 1mm, advantageously between 300pm and 400pm, said decellularized bone particles having been obtained from natural bone of human or animal origin or from synthetic bone.

[0076] In a particular embodiment of the invention, the decellularized bone particles represent at least 10% by weight of the porous bone substitute material. Advantageously, decellularized bone particles represent at least 11% by weight of the porous bone substitute material, advantageously at least 12%, advantageously at least 13%, advantageously at least 14%, advantageously at least 15%, advantageously at least 16%, advantageously at least 17%, advantageously at least 18%, advantageously at least 19%, advantageously at least 20%, advantageously at least 21%, advantageously at least 22%, advantageously at least 23%, advantageously at least 24%, advantageously at least 25%, advantageously at least 26%, advantageously at least 27%, advantageously at least 28%, advantageously at least 29%, advantageously at least 30%, advantageously at least 31%, advantageously at least 32%, advantageously at least 33%, advantageously at least 34%,advantageously at least 35%, advantageously at least 36%, advantageously at least 37%, advantageously at least 38%, advantageously at least 39%, advantageously at least 40%, advantageously at least 41%, advantageously at least 42%, advantageously at least 43%, advantageously at least 44%, advantageously at least 45%, advantageously at least 46%, advantageously at least 47%, advantageously at least 48%, advantageously at least 49%, advantageously at least , 50% by weight of the porous bone substitute material. Advantageously, decellularized bone particles represent between 10% and 50% by weight of the porous bone substitute material. Advantageously, decellularized bone particles represent between 11% and 50%, advantageously between 12% and 50%, advantageously between 13% and 50%, advantageously between 14% and 50%, advantageously between 15% and 50%, advantageously between 16% and 50%, advantageously between 17% and 50%, advantageously between 18% and 50%, advantageously between 19% and 50%, advantageously between 20% and 50%, advantageously between 21% and 50%, advantageously between 22% and 50%, advantageously between 23% and 50%, advantageously between 24% and 50%, advantageously between 25% and 50%, advantageously between 26% and 50%, advantageously between 27% and 50%, advantageously between 28% and 50%, advantageously between 29% and 50%, advantageously between 30% and 50%, advantageously between 31% and 50%, advantageously between 32% and 50%,advantageously between 33% and 50%, advantageously between 34% and 50%, advantageously between 35% and 50%, advantageously between 36% and 50%, advantageously between 37% and 50%, advantageously between 38% and 50%, advantageously between 39% and 50%, advantageously between 40% and 50% by weight of the porous bone substitute material.

[0077] In a particularly advantageous embodiment of the invention, the decellularized bone particles represent 33% by weight of the porous bone substitute material. In another particularly advantageous embodiment of the invention, the decellularized bone particles represent 50% by weight of the porous bone substitute material.

[0078] In a particular embodiment of the invention, the porous bone substitute material has a multi-scale pore size ranging from 50 pm to 2000 pm. For the purposes of the present invention, the terms "pore size" and "pore diameter" may be used interchangeably.

[0079] Advantageously, the multi-scale pore size of the porous bone substitute material is between 50 pm and 2000 pm, advantageously between 50 pm and 1500 pm, advantageously between 50 pm and 1000 pm, advantageously between 50 pm and 800 pm, advantageously between 100 pm and 1500 pm, advantageously between 100 pm and 1000 pm, advantageously between 100 pm and 800 pm.

[0080] For the purposes of the present invention, macroporosity refers to a pore size greater than 50 nm, microporosity to a pore size less than 2 nm, and mesoporosity to a pore size between 2 nm and 50 nm. Advantageously, the porous bone substitute material exhibits macroporosity.

[0081] In an advantageous embodiment of the invention, the porous bone substitute material has a total porosity greater than or equal to 60%. For the purposes of the In this invention, "total porosity" means the ratio of the volume of voids to the total volume of the porous bone substitute material. Advantageously, the total porosity of the porous bone substitute material is greater than or equal to 60%, advantageously greater than or equal to 61%, advantageously greater than or equal to 62%, advantageously greater than or equal to 63%, advantageously greater than or equal to 64%, advantageously greater than or equal to 65%, advantageously greater than or equal to 66%, advantageously greater than or equal to 67%, advantageously greater than or equal to 68%, advantageously greater than or equal to 69%, advantageously greater than or equal to 70%, advantageously greater than or equal to 71%, advantageously greater than or equal to 72%, advantageously greater than or equal to 73%, advantageously greater than or equal to 74%, advantageously greater than or equal to 75%, advantageously greater than or equal to 76%,advantageously greater than or equal to 77%, advantageously greater than or equal to 78%, advantageously greater than or equal to 79%, advantageously greater than or equal to 80%, advantageously greater than or equal to 81%, advantageously greater than or equal to 82%, advantageously greater than or equal to 83%, advantageously greater than or equal to 84%, advantageously greater than or equal to 85%, advantageously greater than or equal to 86%, advantageously greater than or equal to 87%, advantageously greater than or equal to 88%, advantageously greater than or equal to 89%, advantageously greater than or equal to 90%, advantageously greater than or equal to 91%, advantageously greater than or equal to 92%, advantageously greater than or equal to 93%, advantageously greater than or equal to 94%, advantageously greater than or equal to 95%, advantageously greater than or equal to 96%, advantageously greater than or equal to 97%, advantageously greater than or equal to 98%,advantageously greater than or equal to 99%. In a particularly advantageous embodiment, the porous bone substitute material has a total porosity greater than or equal to 80%.

[0082] Advantageously, the total porosity of the porous bone substitute material is between 60% and 95%, advantageously between 61% and 89%, advantageously between 62% and 88%, advantageously between 63% and 87%, advantageously between 64% and 86%, advantageously between 65% and 85%, advantageously between 66% and 84%, advantageously between 67% and 83%, advantageously between 68% and 82%, advantageously between 69% and 81%, advantageously between 70% and 80%. In a particularly advantageous embodiment, the porous bone substitute material has a total porosity of between 70% and 90%.

[0083] In a particular embodiment of the invention, the porous bone substitute material has an interconnectivity between the pores of between 60% and 100%. Advantageously, the interconnectivity between the pores is between 65% and 100%, advantageously between 70% and 100%, advantageously between 75% and 100%, advantageously between 80% and 100%, advantageously between 85% and 100%, advantageously between 90% and 100%, advantageously between 91% and 100%, advantageously between 92% and 100%, advantageously between 93% and 100%, advantageously between 94% and 100%, advantageously between 95% and 100%, advantageously between 96% and 100%, advantageously between 97% and 100%, advantageously between 98% and 100%, advantageously between 99% and 100%.

[0084] In a particularly advantageous embodiment of the invention, the interconnectivity between the pores is greater than 65%, advantageously greater than 70%, advantageously greater than 75%, advantageously greater than 80%, advantageously greater than 85%, advantageously greater than 90%, advantageously greater than 91%, advantageously greater than 92%, advantageously greater than 93%, advantageously greater than 94%, advantageously greater than 95%, advantageously greater than 96%, advantageously greater than 97%, advantageously greater than 98%, advantageously greater than 99%. In a particularly advantageous embodiment of the invention, the porous bone substitute material exhibits 100% interconnectivity between the pores.

[0085] In a particularly advantageous embodiment, the porous bone substitute material according to the invention has a multiscale pore size ranging from 50 pm to 2000 pm, a total porosity ranging from 60% to 95%, and an interconnectivity between pores ranging from 60% to 100%. Advantageously, the porous bone substitute material according to the invention has a multiscale pore size ranging from 50 pm to 2000 pm, a total porosity ranging from 70% to 85%, and an interconnectivity between pores of 100%.

[0086] In a particularly advantageous embodiment, the porous bone substitute material comprising at least one elastomeric matrix including a poly(caprolactone-urea-urethane) based elastomer and decellularized bone particles has a multiscale pore size of between 50 µm and 2000 µm, a total porosity of between 60% and 95%, and an inter-pore interconnectivity of between 60% and 100%. Advantageously, the porous bone substitute material comprising at least one elastomeric matrix including a poly(caprolactone-urea-urethane) based elastomer and decellularized bone particles has a multiscale pore size of between 50 µm and 2000 µm, a total porosity of between 70% and 85%, and an inter-pore interconnectivity of 100%.The porosity of the material, the size of the pores and their interconnection have a major influence on the ability of the porous bone substitute material to become vascularized and gradually resorb.

[0087] Thus, due to its total porosity of between 70% and 85% and its pore size With a multi-scale pore size ranging from 50 µm to 2000 µm, the porous bone substitute material, comprising at least one elastomeric matrix including a poly(caprolactone-urea-urethane) elastomer and decellularized bone particles, is particularly well-suited to cell migration and bone formation within said porous bone substitute material. Furthermore, the multi-scale pore size, ranging from 50 µm to 2000 µm, and the 100% interconnectivity between the pores, allow for the regulation of angiogenesis and osteogenesis within the porous bone substitute material according to the invention. Indeed, the interconnected porous network guides cell attachment and growth, and thus the growth of newly formed bone.Thus, the porous bone substitute material comprising at least one elastomeric matrix including a poly(caprolactone-urea-urethane) based elastomer and decellularized bone particles allows the migration of progenitor cells and their differentiation into osteoblasts, making it an osteoconductive material.

[0088] The size of the porous bone substitute material depends on the size and thickness of the bone to be reconstructed. In a particular embodiment of the invention, the porous bone substitute material has a size between 10 mm and 20 cm and a thickness between 100 µm and 4 cm.Advantageously, the size of the porous bone substitute material is between 10 mm and 20 cm, advantageously between 50 mm and 20 cm, advantageously between 100 mm and 20 cm, advantageously between 500 mm and 20 cm, advantageously between 1 cm and 20 cm, advantageously between 2 cm and 20 cm, advantageously between 3 cm and 20 cm, advantageously between 4 cm and 20 cm, advantageously between 5 cm and 20 cm, advantageously between 6 cm and 20 cm, advantageously between 7 cm and 20 cm, advantageously between 8 cm and 20 cm, advantageously between 9 cm and 20 cm, advantageously between 10 cm and 20 cm, advantageously between 11 cm and 20 cm, advantageously between 12 cm and 20 cm, advantageously between 13 cm and 20 cm, advantageously between 14 cm and 20 cm, advantageously between 15 cm and 20 cm. In a particular embodiment of the invention, the size of the porous bone substitute material is 10 cm, particularly when the bone to be reconstructed is a long bone.

[0089] Advantageously, the thickness of the porous bone substitute material is between 100 µm and 4 cm, advantageously between 200 µm and 4 cm, advantageously between 500 µm and 4 cm, advantageously between 1 mm and 4 cm, advantageously between 1 cm and 4 cm, advantageously between 1 cm and 3 cm. In one advantageous particular embodiment, the thickness of the porous bone substitute material is between 1 cm and 3 cm, particularly when the bone to be reconstructed is a long bone. In another advantageous particular embodiment, the thickness of the porous bone substitute material is between 100 µm and 1 cm, particularly when the bone to be reconstructed is a flat bone.

[0090] In a particular embodiment of the invention, the porous bone substitute material has a volume of at least 0.1 cm3. Advantageously, the porous bone substitute material has a volume of at least 0.2 cm3, advantageously at least 0.3 cm3, advantageously at least 0.4 cm3, advantageously at least 0.5 cm3, advantageously at least 0.6 cm3, advantageously at least 0.7 cm3, advantageously at least 0.8 cm3, advantageously at least 0.9 cm3, advantageously at least 1 cm3, advantageously at least 2 cm3, advantageously at least 3 cm3, advantageously at least 4 cm3, advantageously at least 5 cm3, advantageously at least 6 cm3, advantageously at least 7 cm3, advantageously at least 8 cm3, advantageously at least 9 cm3, advantageously at least 10 cm3, advantageously at least 20 cm3, advantageously at least 30 cm3, advantageously at least 40 cm3, advantageously at least 50 cm3, advantageously at least 60 cm3, advantageously at least 70 cm3,advantageously at least 80 cm³, advantageously at least 90 cm³, advantageously at least 100 cm³, advantageously at least 150 cm³, advantageously at least 200 cm³, advantageously at least 250 cm³, advantageously at least 300 cm³, advantageously at least 350 cm³, advantageously at least 400 cm³. In an advantageous embodiment, the porous bone substitute material has a volume between 0.1 and 400 cm³.

[0091] In one embodiment of the invention, the bone substitute material can be in various forms, advantageously cylindrical, flat, or prismatic. Advantageously, the bone substitute material can be in the form of a flexible porous sponge, a flexible porous membrane, or a flexible porous film.

[0092] Another aspect of the invention relates to the porous bone substitute material according to the invention for its use in bone repair. For the purposes of the present invention, "bone repair" means the reconstruction of damaged bone by inducing osteogenesis. The porous bone substitute material according to the invention can be useful for repairing a variety of orthopedic lesions. Advantageously, the porous bone substitute material according to the invention can be used for the repair of a cavity bone defect and / or the repair of a segmental bone defect.Advantageously, the porous bone substitute material according to the invention can be used for the repair of a maxillofacial bone defect.

[0093] For the purposes of the present invention, a "cavity bone defect" is understood to mean a bone loss with a volume of at least 0.1 cm³ without loss of continuity with respect to the total surface area of ​​the bone without the defect. Advantageously, the bone loss without loss of continuity has a volume of at least 0.1 cm³. Advantageously, the porous bone substitute material has a volume of at least 0.2 cm3, advantageously at least 0.3 cm3, advantageously at least 0.4 cm3, advantageously at least 0.5 cm3, advantageously at least 0.6 cm3, advantageously at least 0.7 cm3, advantageously at least 0.8 cm3, advantageously at least 0.9 cm3, advantageously at least 1 cm3, advantageously a volume of at least 2 cm3, advantageously a volume of at least 3 cm3, advantageously at least 4 cm3, advantageously at least 5 cm3, advantageously at least 6 cm3, advantageously at least 7 cm3, advantageously at least 8 cm3, advantageously at least 9 cm3,advantageously at least 10 cm³, advantageously at least 20 cm³, advantageously at least 30 cm³, advantageously at least 40 cm³, advantageously at least 50 cm³, advantageously at least 60 cm³, advantageously at least 70 cm³, advantageously at least 80 cm³, advantageously at least 90 cm³, advantageously at least 100 cm³, advantageously at least 150 cm³, advantageously at least 200 cm³, advantageously at least 250 cm³, advantageously at least 300 cm³, advantageously at least 350 cm³, advantageously at least 400 cm³. In an advantageous embodiment, the bone loss without loss of continuity has a volume between 1 and 400 cm³ relative to the total surface area of ​​the bone without defects.

[0094] For the purposes of the present invention, a "segmental bone defect" is understood to mean a loss of bone along the length of the bone of at least 10 mm with loss of continuity with respect to the bone without the defect. Advantageously, the bone loss with loss of continuity is at least 10 mm, advantageously at least 50 mm, advantageously at least 100 mm, advantageously at least 500 mm, advantageously at least 1 cm, advantageously at least 2 cm, advantageously at least 3 cm, advantageously at least 4 cm, advantageously at least 5 cm, advantageously at least 6 cm, advantageously at least 7 cm, advantageously at least 8 cm, advantageously at least 9 cm, advantageously at least 10 cm, advantageously at least 11 cm, advantageously at least 12 cm, advantageously at least 13 cm, advantageously at least 14 cm, advantageously at least 15 cm.

[0095] Examples of situations where such defects may exist include post-traumatic conditions with segmental bone loss, bone tumor surgery where bone has been excised, and after total joint arthroplasty (e.g., impaction grafting, etc.), bone loss due to infection, and congenital defects. The porous bone substitute material according to the invention can be used as Prosthetic bone remodeling or replacement implants, for example in orthopedic surgery, including hip revisions; replacement of bone loss, for example in traumatology; remodeling in maxillofacial surgery; or filling of periodontal defects and dental extraction sockets, including ridge augmentation and sinus lifts. The porous bone substitute material according to the invention can thus be used to correct any number of bone defects at a bone repair site.

[0096] In a particular embodiment of the invention, the porous bone substitute material according to the invention can be used to repair any type of bone, whether of human or animal origin. In a particular embodiment, the porous bone substitute material according to the invention can be used to repair a long bone. Examples of long bones include the humerus, femur, tibia, fibula, radius, and ulna. In a particular embodiment, the porous bone substitute material according to the invention can be used to repair a short bone. Examples of short bones include the vertebrae, patella, carpal bone, and tarsal bone. In a particular embodiment, the porous bone substitute material according to the invention can be used to repair a flat bone. Examples of flat bones include the ribs, the skull bone, the iliac bone, the shoulder blade, and the sternum.Advantageously, the porous bone substitute material according to the invention can be used to repair maxillofacial bone.

[0097] In a particular embodiment of the invention, the bone repair is greater than or equal to 5% by volume of the volume of the bone to be repaired. Advantageously, bone repair is greater than or equal to 6% by volume of the volume of bone to be repaired, advantageously greater than or equal to 7%, advantageously greater than or equal to 8%, advantageously greater than or equal to 9%, advantageously greater than or equal to 10%, advantageously greater than or equal to 11%, advantageously greater than or equal to 12%, advantageously greater than or equal to 13%, advantageously greater than or equal to 14%, advantageously greater than or equal to 15%, advantageously greater than or equal to 16%, advantageously greater than or equal to 17%, advantageously greater than or equal to 18%, advantageously greater than or equal to 19%, advantageously greater than or equal to 20%, advantageously greater than or equal to 21%, advantageously greater than or equal to 22%, advantageously greater than or equal to 23%,advantageously greater than or equal to 24%, advantageously greater than or equal to 25%, advantageously greater than or equal to 26%, advantageously greater than or equal to 27%, advantageously greater than or equal to 28%, advantageously greater than or equal to 29%, advantageously greater than or equal to 30%, advantageously greater than or equal to 31%, advantageously greater than or equal to 32%, advantageously greater than or equal to 33%, advantageously greater than or , equal to 34%, advantageously greater than or equal to 35%, advantageously greater than or equal to 36%, advantageously greater than or equal to 37%, advantageously greater than or equal to 38%, advantageously greater than or equal to 39%, advantageously greater than or equal to 40%, advantageously greater than or equal to 41%, advantageously greater than or equal to 42%, advantageously greater than or equal to 43%, advantageously greater than or equal to 44%, advantageously greater than or equal to 45%, advantageously greater than or equal to 46%, advantageously greater than or equal to 47%, advantageously greater than or equal to 48%, advantageously greater than or equal to 49%. Advantageously, the bone repair is greater than or equal to 50% by volume of the volume of bone to be repaired.

[0098] In a particularly advantageous embodiment of the invention, the porous bone substitute material according to the invention can be used for bone repair in humans or animals. By way of example, the animal may be a horse, a pony, a dog, a cat, a rat, a mouse, a pig, a sow, a cow, an ox, a bull, a calf, a goat, a ewe, a ram, a lamb, a donkey, a camel, a dromedary, the list not being exhaustive.

[0099] Another aspect of the invention relates to a bone repair kit comprising the porous bone substitute material according to the invention and a fixation element. Advantageously, the bone repair kit comprises the porous bone substitute material comprising at least one elastomeric matrix comprising a poly(caprolactone-urea-urethane) based elastomer and decellularized bone particles according to the invention, and a fixation element. For the purposes of the present invention, "fixation element" means a metal plate or a tubular, circular, internal, or external fixator intended to hold the porous bone substitute material according to the invention in place on the bone to be repaired while the bone heals. Advantageously, the fixator may be a locking plate with threaded screw holes, allowing the insertion of counter-screws, preventing any screw retraction, such as the Surfix® fixator.In another embodiment, the fixative can be a Polyetheretherketone (PEEK) plate from RiSystem or a steel plate.

[0100] Another aspect of the invention relates to a method for preparing a porous bone substitute material according to the invention. In a particular embodiment of the invention, the porous bone substitute material according to the invention is obtained by the poly-HIPE method (formation and polymerization / crosslinking of high internal phase emulsions). High internal phase or HIPE emulsions consist of immiscible liquid / liquid dispersed systems, in which the volume of the internal phase, also called the dispersed phase, occupies a volume greater than approximately 74-75% of the total volume of the emulsion, i.e., a volume greater than what is geometrically possible for the compact packaging of monodisperse spheres.

[0101] In a particular embodiment, the process for preparing a porous bone substitute material comprises the following steps: a) prepare an organic phase comprising the compounds necessary for the synthesis of poly(urea-urethane ester), b) Add water and decellularized bone particles to the organic phase from step a) to form an emulsion, c) polymerize / crosslink the emulsion containing the decellularized bone particles from step c) to obtain said porous bone substitute material, d) wash said porous bone substitute material obtained in step c), and e) dry said porous bone substitute material obtained in step d).

[0102] In one embodiment of the invention, step a) consists of preparing an organic phase comprising the compounds necessary for the synthesis of poly(urea-urethane ester). Advantageously, the organic phase further comprises an oligoester, an organic solvent for the oligoester, a crosslinking agent, a catalyst, and a surfactant. Advantageously, the organic phase comprises toluene, polycaprolactone triol oligomer, the surfactant Span80, the crosslinking agent hexamethylene dii-socyanate (HMDI), and the catalyst dibutyltin dilaurate (DBTDL).

[0103] In one particular embodiment, step a) comprises a first step a1) of solubilizing polycaprolactone triol oligomer and Span80 surfactant in toluene, followed by a second step a2) of adding HMDI crosslinking agent and DBTDL catalyst to the solution from step a1) to form the organic phase. In an advantageous embodiment of the invention, 7 mL of toluene, 1.3 g of polycaprolactone triol oligomer, 1.3 g of Span80 surfactant, 1.04 mL of HMDI crosslinking agent, and 12 drops of DBTDL catalyst are used. Advantageously, a person skilled in the art will be able to adjust the quantities of toluene, polycaprolactone triol oligomer, Span80 surfactant, HMDI crosslinking agent and DBTDL catalyst according to the desired pore size for the porous bone substitute material.

[0104] In a particular embodiment, step b) of the process consists of adding water to the organic phase to form the emulsion, and then adding decellularized bone particles to the emulsion. Advantageously, the water and the decellularized bone particles are introduced gradually and simultaneously under stirring until an emulsion is obtained. Advantageously, the water is sterile distilled water. Advantageously, a person skilled in the art will be able to adjust the quantity of water according to the desired pore size for the porous bone substitute material. Advantageously, the quantity of water added is 34 mL.

[0105] In a particular embodiment, step c) of the process consists of polymerizing / The emulsion obtained in step b) is crosslinked to obtain the porous bone substitute material. Advantageously, the polymerization / crosslinking is carried out in a mold to give the porous bone substitute material the desired shape. Advantageously, the emulsion obtained in step b) is placed at a temperature between 30°C and 80°C for 10 to 30 hours. Advantageously, the emulsion obtained in step b) is placed at a temperature between 35°C and 65°C, advantageously at a temperature between 40°C and 60°C, advantageously at a temperature between 45°C and 65°C, advantageously at a temperature between 50°C and 60°C, advantageously at a temperature of 55°C.Advantageously, the emulsion obtained in step b) is placed at a temperature between 30°C and 80°C for 10 to 30 hours, advantageously for 11 to 29 hours, advantageously for 12 to 29 hours, advantageously for 13 to 28 hours, advantageously for 14 to 27 hours, advantageously for 15 to 27 hours, advantageously for 16 to 27 hours, advantageously for 17 to 27 hours, advantageously for 18 to 26 hours, advantageously for 19 to 25 hours, advantageously for 20 to 24 hours, advantageously for 22 hours. Advantageously, a person skilled in the art will be able to adjust the temperature according to the desired pore size for the porous bone substitute material.

[0106] In a particular embodiment of the invention, the porous bone substitute material obtained in step c) is annealed prior to step d). Advantageously, the porous bone substitute material obtained in step c) is annealed for at least 1 hour at a temperature of at least 50°C. Advantageously, the porous bone substitute material obtained in step c) is annealed for 2 hours at a temperature of 100°C.

[0107] In a particular embodiment, the washing step of step d) removes the reagents necessary for the synthesis of poly(urea-urethane ester) that did not react during polymerization, as well as any remaining surfactant and catalyst. Advantageously, the washing in step d) is carried out using one of the following products: dichloromethane, dichloromethane / hexane, hexane, water, a mixture of these products, or the successive application of these products. Advantageously, the washing in step d) is carried out by contacting the dried porous bone substitute material with dichloromethane for at least 24 hours, followed by a washing step with dichloromethane / hexane (50%vol / 50%vol) for at least 24 hours, followed by a washing step with hexane for at least 24 hours, and then a final washing with distilled water for at least 24 hours.

[0108] In a particular embodiment, the process according to the invention may further comprise a drying step between step c) and step d). Advantage Generally, this drying stage can be carried out by air drying or in an oven. A person skilled in the art will know how to adjust the oven temperature according to the material being dried. Advantageously, drying is carried out by air drying for at least 7 days.

[0109] In a particular embodiment, the drying in step e) can be carried out by air drying or in an oven. A person skilled in the art will know how to adjust the oven temperature according to the material to be dried. Advantageously, the drying is carried out by air drying for at least 15 days.

[0110] In a particular embodiment, the process according to the invention may further comprise a sterilization step (f) following the washing step (d) of said porous bone substitute material. Advantageously, the sterilization is carried out after vacuum washing in an aqueous medium.

[0111] In one embodiment, sterilization step e) is carried out as follows: f1) contacting the porous bone substitute material in sterile water for one hour under vacuum, f2) replacing the sterile water and contacting the porous bone substitute material in the replaced sterile water for 4 hours under vacuum, f3) contacting the porous bone substitute material from step f2) in 70% ethanol for 1 hour under vacuum, f4) replacement of 70% ethanol with sterile water and contacting the porous bone substitute material from step f3) with the sterile water overnight at ambient pressure, f5) sterilization by autoclave of the porous bone substitute material from step f4) in water.

[0112] In another embodiment, the sterilization step f) can be carried out by gamma radiation.

[0113] In another embodiment, the sterilization step f) can be carried out by contacting the biomaterial with ethylene oxide.

[0114] In another embodiment, the sterilization step f) can be carried out by bringing the biomaterial into contact with a plasma phase from a gas.

[0115] In a particular embodiment, the process according to the invention may further comprise a step (g) of preserving said porous bone substitute material after step (e) of sterilization. Advantageously, step (g) of preserving said porous bone substitute material is carried out by placing the porous bone substitute material in 70% ethanol until its use.

[0116] In a particular embodiment of the invention, the process for preparing a porous bone substitute material comprises the following steps: a) prepare an organic phase comprising the compounds necessary for the synthesis of poly(urea-urethane ester), b) Add water and decellularized bone particles to the organic phase from step a) to form an emulsion, c) polymerize / crosslink the emulsion obtained in step b) to obtain said porous bone substitute material, and d) wash said porous bone substitute material obtained in step c) e) dry said porous bone substitute material obtained in step d) e) sterilization of the porous bone substitute material obtained from step e), and f) optionally, preservation of the porous bone substitute material.

[0117] In a particular embodiment of the invention, the process for preparing a porous bone substitute material comprises the following steps: a) prepare an organic phase comprising the compounds necessary for the synthesis of poly(urea-urethane ester), b) simultaneously add water and decellularized bone particles to the organic phase of step a) to form an emulsion, c) polymerize / crosslink the emulsion obtained in step b) to obtain said porous bone substitute material, and d) wash said porous bone substitute material obtained in step c) e) dry said porous bone substitute material obtained in step d) e) sterilization of the porous bone substitute material obtained from step e), and f) optionally, preservation of the porous bone substitute material.

[0118] In a particularly advantageous embodiment of the invention, the process for preparing a porous bone substitute material comprises the following steps: a) prepare an organic phase comprising the compounds necessary for the synthesis of poly(urea-urethane ester), said step a) comprising a first step a1) consisting of solubilizing in toluene, the polycaprolactone triol oligomer and the surfactant Span80, then a second step a2) consisting of adding the crosslinking agent HMDI and the catalyst DBTDL to the solution of step a1) to form the organic phase, b) Add water and decellularized bone particles to the organic phase from step a) to form an emulsion, c) polymerize / crosslink the emulsion obtained in step b) to obtain said porous bone substitute material, and d) wash said porous bone substitute material obtained in step c) e) dry said porous bone substitute material obtained in step d) for at least 15 days, f) sterilization of the porous bone substitute material obtained in step e) and, g) optionally, storage of the porous bone substitute material. Figures

[0119] [Fig. 1] Figure 1 shows the allogeneic bone granules homogeneously distributed within and on the surface of the porous bone substitute material according to the invention. The images were obtained by 3D microscopy (VHX Keyence) of the porous bone substitute material according to the invention after washing without staining the bone granules, the arrows indicating the presence of bone granules (A, B).

[0120] [fig.2] Figure 2 represents the analysis by Infrared Transform Spectroscopy of Fourier (FTIR) of bone granule alone, of poly(caprolactone-urea-urethane) elastomer matrix alone and of porous bone substitute material according to the invention (composite).

[0121] [fig.3] Figure 3 shows the stress-strain curves of the matrix poly(caprolactone-urea-urethane) elastomer alone and porous bone substitute material according to the invention (composite) during compression tests (left: entire curves; right: enlargement at the beginning of the curves).

[0122] [fig.4] Figure 4 shows the mass loss of the elastomer matrix poly(caprolactone-urea-urethane) alone and the porous bone substitute material according to the invention (composite) during in vitro degradation at 37°C and accelerated to 90°C.

[0123] [fig.5] Figure 5 represents the cellular activity determined by MTT test after in cubation with the extraction media of the poly(caprolactone-urea-urethane) elastomer matrix alone and of the porous bone substitute material according to the invention (composite). The "Blank" represents the result for control cells under normal conditions, and the "positive control" represents the result for cells in the presence of a cytotoxic molecule (chosen here as HMDI).

[0124] [fig.6] Figure 6 shows cell viability determined by blue staining of trypan during indirect cytotoxicity testing of the poly(caprolactone-urea-urethane) elastomer matrix alone and of the porous bone substitute material according to the invention (composite). "Blank" represents the result for control cells under normal conditions, and "positive control" represents the result for cells in the presence of a cytotoxic molecule (chosen here as HMDI).

[0125] [fig.7] Figure 7 represents the migration of mesenchymal stromal cells obtained from canine adipose tissue from day 10 (D10) to day 40 (D40) within the poly(caprolactone-urea-urethane) elastomer matrix alone and the porous bone substitute material according to the invention (composite).

[0126] [fig.8] Figure 8 represents a critically sized segmental defect on a femur of rat.

[0127] [fig.9A] Figure 9A represents the cylinders of the porous bone substitute material according to the invention before implantation in conditioning medium.

[0128] [fig.9B] Figure 9B represents the cylinders of the porous bone substitute material according to the invention after acquisition by microtomography.

[0129] [fig.lOA] Figure 10A represents the radiological follow-up of a segmental femoral defect maintained by an osteosynthesis plate, with partial reconstruction after 61 days.

[0130] [fig.lOB] Figure 10B represents the radiological follow-up of a segmental femoral defect maintained by an osteosynthesis plate, with the appearance of a failure of the system after 31 days.

[0131] [fig.l 1] Figure 11 represents the concentrations of red blood cells and blood platelets for the control (Non-operated (Non op)), control (Empty Defect), poly(caprolactone-urea-urethane) elastomer matrix alone (Elastomer), bone substitute material according to the invention (Composite), decellularized bone and positive control (C pos) (non-critical) at the different study times.

[0132] [fig.12] Figure 12 shows the concentrations of white blood cells and lymphocytes for the control (Non-operated (Non op)) batches, Control (Empty Defect), poly(caprolactone-urea-urethane) elastomer matrix alone (Elastomer), bone substitute material according to the invention (Composite), decellularized bone and positive control (C pos) (non-critical) at the different study times.

[0133] [fig.13] Figure 13 shows the concentrations of serum markers of the me bone scaling (CTX: bone resorption; P1PN and Oc: bone synthesis) for control lots (Non-operated (Non op)), Control (Empty Defect), poly(caprolactone-urea-urethane) elastomer matrix alone (Elastomer), bone substitute material according to the invention (Composite), decellularized bone and positive control (C pos) (non-critical) at the different study times.

[0134] [fig.l4A] Figure 14A represents the amount of bone formed after 1 or 3 months inside the area of ​​the bone defect for the control (Non-operated (Non Op)), control (Empty Defect), poly(caprolactone-urea-urethane) elastomer matrix alone (Elastomer), bone substitute material according to the invention (Compo) and positive control (C pos) (non-critical) lots at the different study times.

[0135] [fig.l4B] Figure 14B represents the ratio of quantity of bone / initial volume of the bone defect for the control (Non-operated), Control (Empty Defect), poly(caprolactone-urea-urethane) elastomer matrix alone (Elastomer), bone substitute material according to the invention (Compo) and positive control (C pos) (non-critical) lots at the different study times.

[0136] [fig.15A] Figure 15A represents the surface of the bone formed after 1 or 3 months at the inside of the bone defect area for the control (Non-operated), Control (Empty Defect) lots, poly(caprolactone-urea-urethane) elastomer matrix alone (Elastomer), bone substitute material according to the invention (Compo) and positive control (C pos) (non-critical) at the different study times.

[0137] [fig. 15B] Figure 15B represents the bone surface area / initial volume ratio of the bone defect for the control (Non-operated), control (Empty defect), poly(caprolactone-urea-urethane) elastomer matrix alone (Elastomer), bone substitute material according to the invention (Compo) and positive control (C pos) (non-critical) lots at the different study times.

[0138] [fig. 16] Figure 16 represents the bone defect that remained empty after 3 months for the "Control" group, ((A): superior view in 3D microtomography and (B): lateral view in 3D microtomography).

[0139] [fig.l7A] Figure 17A represents the non-critical size bone defect for the "Positive Control" group, in superior view in 3D microtomography after 3 months.

[0140] [fig.l7B] Figure 17B represents the non-critical size bone defect for the "Positive Control" group, in lateral view in 3D microtomography after 3 months.

[0141] [fig.l7C] Figure 17C represents the non-critical size bone defect for the "Positive Control" group, in histological section with Masson's trichrome staining (magnification x5) after 1 month.

[0142] [fig.17D] Figure 17D shows the detail of the central area of ​​the bone defect for the "Positive Control" lot (image inset C, magnification x40).

[0143] [fig.18A] Figure 18A represents the critical size bone defect for the poly(caprolactone-urea-urethane) elastomer matrix lot alone, in superior view in 3D microtomography after 1 month.

[0144] [fig.18B] Figure 18B represents the critical size bone defect for the poly(caprolactone-urea-urethane) elastomer matrix lot alone, in superior view in 3D microtomography after 3 months.

[0145] [fig.18C] Figure 18C represents the critical size bone defect for the poly(caprolactone-urea-urethane) elastomer matrix lot alone, in histological section with Sudan Black staining (magnification x2.5) after 1 month.

[0146] [fig.18D] Figure 18D represents the detail of the central area of ​​the bone defect for the poly(caprolactone-urea-urethane) elastomer matrix lot alone (inset image C, magnification x40), with Masson's trichrome staining after 1 month.

[0147] [fig. 19] Figure 19 represents the bone defect after 3 months for the poly(caprolactone-urea-urethane) elastomer matrix lot alone ((A): lateral view in 2D microtomography, (B): lateral view in 3D microtomography and (C): inferior view in 3D microtomography).

[0148] [fig.20] Figure 20 represents the bone defect after 3 months for the “bone detection” group "Lularized", in lateral view using 3D microtomography.

[0149] [fig.21A] Figure 21A represents the critical size bone defect for the batch porous bone substitute material according to the invention "Composite", in top view in 3D microtomography after 1 month.

[0150] [fig.21B] Figure 21B represents the critical size bone defect for the batch porous bone substitute material according to the invention "Composite", in top view in 3D microtomography after 3 months.

[0151] [fig.21C] Figure 21C represents the critical size bone defect for the batch porous bone substitute material according to the invention "Composite", in histological section with Masson trichrome staining after 1 month (magnification x2.5).

[0152] [fig.21D] Figure 21D shows the detail of the central area of ​​the bone defect for the batch of porous bone substitute material according to the invention "Composite" (image inset C, magnification x40).

[0153] [fig.21E] Figure 21E represents the non-critical size bone defect for the batch porous bone substitute material according to the invention "Composite", in histological section with Masson trichrome staining after 1 month (magnification x2.5).

[0154] [fig.21F] Figure 21F shows the detail of the external area of ​​the bone defect for the batch of porous bone substitute material according to the invention "Composite" (image inset C, magnification x40). Examples

[0155] Example 1: Formulation and synthesis of the porous bone substitute material according to the invention

[0156] Initially, the allogeneic material Allodyn® (decellularized bone) was ground to produce granules with a diameter ranging from 50 to 500 µm. To obtain particles of controlled size, the mixture of granules obtained after grinding was sieved using a sieve shaker (Fisher AS 200 TAP). The granules were thus separated into size ranges of 50 to 100 µm, 100 to 200 µm, 200 to 300 µm, and 300 to 400 µm. Granules larger than 400 µm were not included in this analysis.

[0157] Subsequently, these granules were incorporated into the elastomer matrix comprising a poly(caprolactone-urea-urethane) based elastomer during the synthesis of the latter by the poly-HIPE method (formation and polymerization / crosslinking of high internal phase emulsions). Several elastomer matrix / allogeneic bone ratios were tested.

[0158] These steps have been validated for a human-derived source of decellularized allogeneic bone: Allodyn®. Due to availability issues in sufficient quantities at During the study, a source of decellularized xenogeneic bone of bovine origin, Laddec®, was also tested without observing any variation.

[0159] The selected compositions are: - Bone substitute material A, with a ratio of 100% elastomer matrix / allogeneic bone (Ig / lg), i.e. a mass fraction of bone of 50% and a particle size between 300 and 400 sqm, - Bone substitute material B, with a ratio of 50% elastomer matrix / allogeneic bone (lg / 0.5g), i.e. a mass fraction of bone of 33% and granulometry between 300 and 400 qm.

[0160] A proportion of bone greater than 50% results in a loss of structure of the bone substitute material.

[0161] Example 2: Physico-chemical and mechanical properties of the bone substitute material according to the invention.

[0162] The physicochemical properties of the bone substitute materials according to the invention were tested by: - Fourier transform infrared (FTIR) spectroscopy, for the analysis of chemical functions present in the synthesized substitute materials; - Scanning Electron Microscopy (SEM) for the morphological observation of substitute materials, coupled with elemental analysis (EDX); - Measurement of volumetric absorption rates to determine the interconnectivity of the porous structure; - Alizarin red staining to assess and visualize the incorporation of allogeneic bone particles within the replacement material. This dye is a specific marker of calcium deposits.

[0163] For clarity, the detailed results below are given for the bone substitute material B having a bone mass fraction of 33%, and comprising bone particles of diameter 300 to 400 µm.

[0164] L Interconnectivity / Porosity

[0165] After washing, the density of the bone substitute material was evaluated by pycnometry. The value found, 1.29, compared to that of the matrix alone (1.05) or bone alone (2.59), indicates that bone particles are still present in the elastomeric matrix. A bone mass fraction of 31% was found after washing, compared to the initial mass fraction of 33% introduced into the emulsion. The decellularized bone particles appear to be homogeneously distributed within and on the surface of the bone substitute material (Figure 1). Furthermore, measurements of the volumetric absorption rate showed that the interconnectivity of the porous structure was only very slightly modified by the incorporation of the bone particles and remains above 80%. This value is consistent with good liquid penetration and cell migration (rv poly(caprolactone-urea-urethane) elastomer matrix alone = 100.8 ± 8% vs. rv Composite = 86.4 ± 2%).

[0166] 2, Chemical composition

[0167] EDX (Table 1) and FTIR (Figure 2) analyses confirm the presence of calcium and phosphorus due to decellularized bone particles in the porous bone replacement material.

[0168] [Tables 1] Mass percentage of elements %C %O %P %Ca Theory Poly(caprolactone-urea-urethane) matrix 70.78 29.22 - - Porous bone substitute material according to the invention (composite) 60.2 31.0 3.1 5.7 Experimental Poly(caprolactone-urea-urethane) matrix 75.5 + 3.4 24.5 + 3.42 - - Decellularized bone particles 50.5 + 0.4 32.7 + 0.5 5.9 + 0.1 10.9 + 0.1 Porous bone substitute material according to the invention (composite) 65.8 + 1.2 27.4 + 1.3 1.5 + 0.2 5.3 + 0.3

[0169] Table 1: Elemental composition obtained by EDX analysis of decellularized bone particles alone, poly(caprolactone-urea-urethane) elastomer matrix alone and bone substitute material according to the invention (composite) ('nitrogen not taken into account for comparison with experimental limited by sensitivity of the device).

[0170] Elemental analysis shows a good correlation between the theoretical values ​​expected according to the amount of bone incorporated during the synthesis of the material, and the experimental values ​​of the material after washing. This further proves the presence of bone particles in the elastomer matrix.

[0171] The chemical structure of the elastomer matrix has not been modified since all the peaks associated with this matrix are still present (Figure 2).

[0172] 2.3. Mechanical properties

[0173] The study of mechanical properties required the production of larger diameter samples and the validation of the synthesis steps. Once this was completed, the elastomeric nature of the bone substitute material according to the invention and of the poly(caprolactone-urea-urethane) elastomeric matrix alone was demonstrated by mechanical compression tests: the shape of the Stress vs. Strain curve of the bone substitute material according to the invention is similar to that of the poly(caprolactone-urea-urethane) elastomeric matrix alone, proving the elastomeric nature of the materials (Figure 3).

[0174] The Young's modulus E of the porous material can be defined on the first linear portion of the curve. A value of 228 kPa was found for the bone substitute material according to the invention, which is within the range of elastomeric foams (1 < E < 1000 kPa). In the second linear portion of the curve, the Young's modulus E of the non-porous material, when the pores are all crushed, can be defined. A value of 19 MPa was found for the bone substitute material according to the invention. These values ​​are higher than those found for the poly(caprolactone-urea-urethane) elastomeric matrix alone. Thus, the decellularized bone particles contribute to a slight increase in the moduli of the bone substitute material according to the invention, while retaining the elastomeric character of the polymer matrix.

[0175] 2.4, Degradation kinetics

[0176] An important criterion when developing a bone substitute material for tissue engineering is its resorbability, since it must be replaced over time by newly formed bone. It has been suggested that for bone tissue regeneration, the biomaterial should have reduced hydrophilicity so that the degradation rate can exceed 18 months. In vitro degradation studies were carried out according to ISO 10993-13 standards. The degradation kinetics were evaluated, in particular, by measuring mass loss. Accelerated degradation tests at 90°C showed that the bone substitute material degrades slightly faster than the poly(caprolactone-urea-urethane) elastomer matrix alone (Figure 4). This is due to an increase in the hydrophilicity of the materials, as evidenced by the water contact angle measurements:

[0177] 0= 121+10° for the poly(caprolactone-urea-urethane) elastomer matrix alone vs. 0= 83+22° for the bone substitute material.

[0178] Thus, the poly(caprolactone-urea-urethane) elastomer matrix alone is stable for 14 days at 90°C. Applying the "rule of ten," which gives the relationship between the increase in the degradation rate when the temperature is raised by ten degrees, and taking a Q10 factor of 2-2.5 [ASTM F 1980-02: Standard Guide for Accelerated Aging of Sterile Medical Device Packages], the shelf life of the poly(caprolactone-urea-urethane) elastomer matrix alone at 37°C is estimated to be between 19.4 and 63.4 months.

[0179] For the bone substitute material, the lifespan at 90°C is 10 days, leading to an estimated lifespan at 37°C of between 13.0 and 42.3 months. This stability is suitable for using the bone substitute material as a scaffold for bone regeneration.

[0180] Example 3: Interactions between the porous bone substitute material according to the invention and mesenchymal stromal cells obtained from canine adipose tissue (MSC)

[0181] 3.1. Cytotoxicity tests

[0182] Initially, the cytotoxicity of extraction products possibly released by the bone substitute material according to the invention was studied in accordance with ISO 10993-5 and 10993-12 standards.

[0183] Thus, the bone substitute material as obtained in Example 1 and the poly(caprolactone-urea-urethane) elastomer matrix alone are incubated in standard culture medium for 24 h at 37°C. This extraction medium is then deposited onto a CMS mat at 80% confluence. After 24 h of incubation, the metabolic activity of the cells is measured by an MTT assay.

[0184] The results are presented in (Figure 5).

[0185] The standard sets a cell viability limit of 70% for a product to be considered non-cytotoxic.

[0186] The results obtained show no difference in metabolic activity between the control cells and those exposed to the extraction medium of the poly(caprolactone-urea-urethane) elastomer matrix alone or to the bone substitute material according to the invention (composite). This metabolic activity is greater than 90%, and therefore exceeds the limit set by the standard. These results demonstrate, under these conditions, the absence of cytotoxicity of the material extracts. Subsequently, we evaluated the effect of cytotoxic byproduct release using an indirect cytotoxicity test. To do this, the materials are deposited, without direct contact, on top of the MSCs at 80% confluence. After 24 hours of incubation at 37°C, cell viability is measured by trypan blue staining.

[0187] The results are presented in (Figure 6).

[0188] The cell viability obtained is greater than 80% and comparable between the control and cells exposed to the poly(caprolactone-urea-urethane) elastomer matrix alone or to the bone substitute material according to the invention (composite). The indirect cell-scaffold interaction does not generate cytotoxic compounds in the culture medium after 24 hours.

[0189] 3. 2, Interactions between MSC cells and bone substitute materials.

[0190] Colonization trials with canine MSCs were carried out to test the The "attracting" power of the bone substitute material according to the invention (composite) and of the poly(caprolactone-urea-urethane) elastomer matrix alone was determined. The bone substitute material according to the invention (composite) and the poly(caprolactone-urea-urethane) elastomer matrix alone were respectively deposited onto a MSC mat at 80% confluence. Cell migration was determined at days 10, 20, 30, and 40.

[0191] The results are presented in (Figure 7).

[0192] A cell count of the cells present on and within the bone substitute material according to the invention (composite) and of the poly(caprolactone-urea-urethane) elastomer matrix alone is performed after detachment of the cells by enzymatic treatment. The results obtained show that the cells are capable of migrating within the materials.

[0193] Conclusions: All of these results demonstrate that the bone substitute material according to the invention (composite) is not toxic, that cells are able to adhere to it and proliferate therein and that, regardless of the culture conditions, they colonize it even in depth, demonstrating its osteoconduction properties.

[0194] Example 4: In vivo study of the repair power of bone substitute material (composite) on a rat model of segmental defect.

[0195] The study is based on the reconstruction of a segmental bone defect in rats (7-9 week old male Lewis, January breeding). It allows for the evaluation of the biocompatibility, biodegradability, and efficacy of biomaterials under real-world conditions, this type of lesion being similar to those frequently encountered in military personnel or civilians who are victims of attacks or road accidents...

[0196] The segmental bone defect model consists of removing a section of bone, eliminating all continuity between the two resulting bone segments and preventing spontaneous repair. The critical size of this bone defect is defined as being equal to (at least) 1.5 to 2 times the diameter of the bone concerned.

[0197] In order to evaluate the effectiveness of the bone substitute material according to the invention (composite) compared to the poly(caprolactone-urea-urethane) elastomeric matrix alone, several groups of animals were monitored for up to 3 months post-injury / implantation. The effectiveness of bone repair was assessed by microtomography and histology on femurs harvested after euthanasia. Blood from the animals was used for complete blood counts / differentials and ELISA assays (on serum) of markers of bone formation and resorption.

[0198] Groups of 6 animals were used for each time period, 1 and 3 months, for a total of 60 animals. The right femur is operated on, the left femur serves as a reference.

[0199] • "Control" lot: empty defect, without implant • Batch “poly(caprolactone-urea-urethane) elastomer matrix only” • Batch “bone substitute material according to the invention (composite)” (formula 33% bone, granules 300-400 µm) • Batch “Positive control”: non-critical size defect (empty) • A "control group" (non-op.) corresponds to animals that have not been operated on, but having undergone anesthesia and analgesia.

[0200] The rat model of segmental bone defect involves creating a section in the femoral shaft that prevents spontaneous repair. The critical size of this bone defect is 5 to 6 mm, a length well established in this rodent model. The bone is cut at the level of the shaft, and the two bone ends are stabilized by a fixator adapted to the rat femur (Figure 8). This fixator is held to the superior surface of the femur by four 1.1 mm diameter stainless steel screws (Synthes).

[0201] 5.1. Surgical procedure

[0202] Anesthesia / Analgesia: Anesthesia: Ketamine / Medetomidine: 60 / 0.5 mg / kg intraparenterally.

[0203] Awakening: Atipamezole, 1 mg / kg intramuscularly

[0204] Analgesia: Buprenorphine 0.05mg / kg post-operative (waking up then 3 times a day for 3 days), subcutaneously (SC).

[0205] Surgical procedure:

[0206] • Clipping of the hind leg + back; Disinfection with betadine; Marking of the external aspect of the operated thigh;

[0207] • Incision of the skin and aponeurosis of the external face (20-30 mm) and separation with a surgical chisel with rounded ends of the muscular planes to expose the central part of the femur (diaphysis) without vascular injury; • Placement of the fixator (steel or PEEK plate, L=23 mm) held on the upper face of the femur using a needle holder; • Drilling of the holes intended to receive the screws under continuous saline irrigation (1 mm diameter drill bit); Placement of the 4 transcortical screws; • Transverse section of the bone with an oscillating saw (ConMed) and removal of the bone segment (figure 11), the inside of the hole is cleaned with a delicate injection of saline solution; • Placement of the implant in the bone defect; • Bringing the muscle layers together. If necessary, add 2 to 3 interrupted stitches (absorbable suture). Closure of the skin layers with 5 mm staples; • Local application of betadine; • Upon waking, the animals are placed in lateral recumbency on a dry mat in their cage, on a heating plate; • Post-operative care: subcutaneous analgesia during awakening and then for 3 days (3 times a day).

[0208] The animals used their operated limb immediately upon waking. The animals were monitored daily: the wounds were clean, and no external signs of inflammation, lameness, or behavioral changes were noted while the osteosynthesis system was stable. Weight curves were similar for all groups throughout the study, with a recovery phase of approximately 15 days post-surgery.

[0209] 5.2, Implant preparation

[0210] The biomaterials (bone substitute material according to the invention (composite) and the poly(caprolactone-urea-urethane) elastomer matrix alone) are cylinders 10 to 15 mm long and 4 mm in diameter (Figure 9A). They are prepared the day before implantation under sterile conditions in conditioning medium and incubated in an oven at 37°C and 5% CO2. They are cut during placement in the bone defect to obtain dimensions adapted to each animal. Some batches of composites were scanned by microtomography to verify the size and homogeneity of the granule distribution (Figure 9B) before implantation.

[0211] The conditioning medium used contains DMEM (Dulbecco's Modified Eagle Medium); Penicillin / Streptomycin 0.8%; Fungizone 1%.

[0212] The biomaterials retain their integrity, without disintegrating at the time of implantation.

[0213] Both types of biomaterial - the poly(caprolactone-urea-urethane) elastomer matrix alone and the porous bone substitute material according to the invention (Composite) - appear radio-transparent, which will allow easier radiological monitoring, by enabling visualization of the newly synthesized bone.

[0214] Only the Laddec® granules inside the composite are radio-opaque and detectable by microtomography (figure 9B).

[0215] 5.3. Radiological follow-up

[0216] Radiological monitoring is performed using a surface-mounted radiograph (SARRP) in imaging mode at 1, 3, 6, and 12 days, then every 10 to 15 days, to verify the integrity of the osteosynthesis system and the appearance of mineralized bone within the bone defect (Figure 10A). In the event of failure of the osteosynthesis system, as illustrated in Figure 10B where the most distal screw is loosening after 31 days, the animals are euthanized and the femurs are retrieved for analysis.

[0217] Qualitative analysis of the images shows no bone formation until one month post-surgery. At three months, a more or less significant bone callus is visible inside the defect, restoring continuity between the two fragments in half of the animals in the "bone substitute material according to the invention (composite)" group.

[0218] 5.4. Blood tests

[0219] Blood was collected in EDTA-K3 via intracardiac puncture from anesthetized rats prior to sacrifice, for a complete blood count (using a Procyte DX-IDEXX veterinary hematology analyzer) to detect abnormal inflammation or any effect of the biomaterials on the blood count. Serum was also prepared from the collected blood without anticoagulant and centrifuged at 1500 g for 10 minutes for bone repair marker assay by ELISA.

[0220] Regarding red blood cell count, production remained high at one and three months compared to unoperated animals, but no significant variation was detected for each group and at each time point. Platelet concentration did not vary (Figure 11).

[0221] The overall level of inflammation is similar for all groups (Figure 12) and similar to the baseline level of non-operated rats; no significant difference was recorded: the poly(caprolactone-urea-urethane) elastomer matrix alone or the bone substitute material according to the invention (composite) are therefore well tolerated by the body and do not amplify the inflammatory reaction due to bone trauma at these late times in this rat model.

[0222] Direct markers of bone remodeling (Figure 13) indicate bone synthesis (Oc, P1NP) and bone resorption (CTX1) activity. However, interpreting their levels is challenging in animals, as they can, for example, be dependent on the diurnal / noctinal cycles. The values ​​observed for P1NP (procollagen 1 type N-terminal propeptide) primarily reflect the osteoblast proliferation phases. These values ​​are 4 to 5 times lower than those of non-operated animals (53.20 ± 2.89 ng / mL). However, a value doubled is observed for the group with decellularized bone at 3 months (25.52 ± 11.34 ng / mL).

[0223] Osteocalcin (Oc) concentrations, a serum marker of bone synthesis (reflecting mineralization activity by osteoblasts), remained lower than those of non-operated rats (1031.35 ± 59.06 ng / mL) for all groups at one and three months. However, it should be noted that the values ​​for groups implanted with the different biomaterials at three months were similar to those found for the positive controls (625.65 ± 41.15 ng / mL) where complete bone reconstruction was underway. These values ​​were three times higher than those observed for the control group (empty defect) in which no bone reconstruction was observed (221.40 ± 19.50 ng / mL).

[0224] All of these values ​​would reflect high activity of osteoblasts produced when a biomaterial is placed, even if their number remains lower than that found in non-operated animals. CTX1 (type 1 carboxy-terminal telopeptide of collagen) is a marker of the level of bone resorption via the cathepsin K pathway, the predominant pathway of bone remodeling. When the bone defect remains empty (7.89 ± 0.87 ng / mL), its concentration is lower than that of non-operated animals (9.24 ± 0.52 ng / mL), reflecting low remodeling activity linked to low neosynthesis of bone tissue. This concentration is equivalent to that of non-operated animals when the poly(caprolactone-urea-urethane) elastomer matrix alone, the porous bone substitute material according to the invention (composite), or decellularized bone are implanted after one and three months, indicating that remodeling of the newly formed mineralized tissue is underway.

[0225] 5.5. Microtomographic analyses

[0226] While the femurs are fixed in Burckhardt solution for subsequent histological analyses, they are analyzed by microtomography (Skyscan 1174) with the following parameters: 50 kV; 800 pA to visualize and quantify bone neomineralization at the segmental defect. A rapid acquisition (40 to 70 min) is performed on the complete femurs with their fixative at a resolution of 50 to 60 µm. Then, depending on the level of mineralization and the "solidity" observed at the time of sampling, the fixatives are removed and a finer acquisition is performed (resolution of 14 to 20 µm, 2 to 3 hours) for analysis. The measurement area corresponds to the area of ​​the initial bone defect.

[0227] The acquired data are processed by a quantification software (CTan, Skyscan), and allow visualization and quantification of bone reconstruction at the level of the bone defect by the BV / TV ratio (Bone Volume / Tissue Volume) which translates the quantity of newly formed bone in a given volume.

[0228] Figure 14A quantifies the bone present in the area of ​​the bone defect. This value remains three times lower at both one and three months in control animals compared to non-operated animals (31.11 mm³), when the defect is left empty. This reflects the absence of bone synthesis and therefore of repair in this model. When the poly(caprolactone-urea-urethane) elastomer matrix alone is implanted, this value is equivalent to that of control animals at one month (10.8 ± 1.1 mm³), but is equivalent (24.75 ± 7.29 mm³) to that of non-operated animals after three months.

[0229] The placement of the bone substitute material according to the invention (composite) results in a quantity of bone formed at three months 2.5 times greater (68.60 + 26.02 mm³) than that of non-operated animals. This value is close to that found for the positive controls where the synthesis is 3 times greater (80.03 + 23.99 mm³).

[0230] However, when this quantity of bone formed is compared to the initial volume of the defect area (Figure 14B), the overall values ​​obtained for all groups remain lower than that found for unoperated animals (41.1). This value reflects the proportion of bone in a defined area and can be used to assess the phases of the ongoing repair process. Indeed, bone repair involves an active synthesis phase followed by a more or less lengthy remodeling phase of the bone callus. During this final phase, the bone is remodeled and restructured according to the area of ​​the bone concerned, here in cortical bone, to allow the restoration of mechanical properties.

[0231] The mirror image of the bone surface area (BS) to bone defect volume (BV) ratio (BS / BV) reflects the level of structure of the bone formed (Figures 15A and 15B). For control animals, the high ratio (15.10 + 5.49 mm¹) indicates a rather thin and spread-out mineralized structure, whereas for positive controls, a lower value represents a more compact bone (5.26 + 0.36 mm¹). The intermediate values ​​found with the poly(caprolactone-urea-urethane) elastomer matrix alone (7.82 + 1.65 mm¹) and the bone substitute material according to the invention (composite) (6.99 + 2.09 mm¹) at 3 months show that the bone produced is undergoing remodeling to achieve a more compact structure.

[0232] 5.6. Histology

[0233] All data obtained by microtomography are correlated with local information obtained by histological analysis and performed successively on the same sample. The harvested femurs are fixed in a Burckhardt solution and embedded in an MMA (methyl methacrylate) resin suitable for hard tissues.

[0234] Thick sections (to prevent the biomaterial from tearing) were then stained with stains specific to the different cell types: - Masson's Trichrome (TM) stains mineralized tissue blue, highlighting the cells responsible for bone formation (osteoblasts) and bone remodeling (osteoclasts), as well as osteoid mineralization zones (in pink) and blood vessels. - Sudan Black (NS) colors the poly(caprolactone-urea-urethane) elastomer matrix black. - Sirius Red (RS) stains collagen fibers red under a light microscope, and yellow / orange under polarized light. Allows visualization of the level of tissue structure.

[0235] 5.7, Batch Results

[0236] 5.7.1. "Control" lot

[0237] The results obtained are presented in Figure 16.

[0238] The bone defect remained empty; no reconstruction was observed within the defect during the 3 months of the study (Figure 16). Bone formed only at the edges, and a rudimentary form of bone synthesis was observed in some animals under the fixator (Figure 16). The defect was filled with fibrous tissue. This confirms the critical size of the bone defect.

[0239] 5.7.2. Batch “Positive Control”

[0240] The size of the bone defect is not critical here and is representative of a simple fracture. After 3 months, virtually complete reconstruction is observed when This size is approximately 2 mm (Figure 17A). Highly active mineralization zones, indicated by the arrows in Figure 17C, are found at the bony edges after one month, as well as areas of endochondral ossification in the center of the defect (Figure 17D), probably related to a certain "elasticity" of the system. Indeed, rats are active and dynamic, and this area is subjected to significant mechanical stress.

[0241] We can observe in figure 17B a complete repair with restoration of bone continuity after 3 months when the size of the defect is less than 1 mm (saw cut only).

[0242] The results obtained are presented in Figures 17A to 17D.

[0243] 5.7.3. Batch “poly(caprolactone-urea-urethane) elastomer matrix only”

[0244] The level of reconstruction is low one month post-implantation (Figure 18A) and localized only at the bone margins. However, Sudan black staining allows visualization of the poly(caprolactone-urea-urethane) elastomer matrix alone, located in the center of the defect and partially integrated into newly synthesized bone (Figure 18C).

[0245] We can observe that the poly(caprolactone-urea-urethane) elastomer matrix alone underwent a macroscopic structural change: the sponge "flattened, unrolled" as we had observed one month later in a cavity defect during previous studies. Masson's trichrome staining reveals numerous osteoid zones—corresponding to areas undergoing mineralization by osteoblasts—around this part, which is intimately integrated into the bone (Figure 18D, indicated by the arrows). Furthermore, biological material is visible within the poly(caprolactone-urea-urethane) elastomer matrix alone, demonstrating its preserved porosity.

[0246] The 3D reconstruction obtained after 3 months (Figure 18B) reveals a partial reconstruction of the defect, with virtually restored continuity. We unambiguously confirm the osteoinductive nature of the poly(caprolactone-urea-urethane) elastomer matrix, previously demonstrated on a model of a cavity bone defect.

[0247] However, we observed in some samples a particular trabecular structure of the newly formed bone within the defect in the presence of the poly(caprolactone-urea-urethane) elastomer matrix alone (Figure 19). Furthermore, slight bone synthesis is observed at the ends of the fixator, appearing to be "attached" to it, as indicated by an arrow in Figure 19.

[0248] 5.7.4. Batch “Decellularized Bone”

[0249] In order to verify the biocompatibility and osteoconduction properties, the bone defect was filled with a cylindrical rod of Allodyn® with a diameter of 2 mm. The implant was carefully trimmed during placement to ensure it completely filled the defect and was in contact with both bone edges. No bone regeneration was observed after 3 months (Figure 20). A preliminary resorption was noted for the biomaterial. Moderate bone synthesis around the ends of the fixator (outside the area to be reconstructed) was observed in some samples, at a level similar to that observed with the poly(caprolactone-urea-urethane) elastomeric matrix alone. No rejection or infection reactions were recorded.

[0250] 5.7.5. Batch “bone substitute material according to the invention”

[0251] The incorporation of decellularized bone in granule form into the poly(caprolactone-urea-urethane) elastomeric matrix modifies the reconstruction process. Indeed, after one month, bone forms on the bone margins, detectable by Masson's trichrome staining, with numerous osteoid zones at this level (Figures 21C and 21D). However, the area of ​​the bone defect still appears empty; the decellularized bone granules are clearly visible and are grouped more or less eccentrically with respect to this area (Figure 21A). Nevertheless, intense "delocalized" bone synthesis is observed, with the fixators becoming covered by a mineralized layer. This is even more pronounced after 3 months (Figure 21B), with the fixators being completely covered with bone at their distal and proximal ends.Bone synthesis is intense at this stage, and the defect area is almost repaired: continuity is restored, and the defect is practically filled with mineralized tissue in half of the animals. Histological analysis will allow verification of the structure of this newly formed bone and its level of remodeling.

[0252] In some samples, mineralized granules are found completely outside the area to be reconstructed, trapped between the bone tissue and the surrounding muscle. Here too, histological analysis will determine whether these are residual granules of decellularized bone that have migrated outside the defect area without being resorbed, or whether they are external foci of mineralization.

[0253] Indeed, on certain histological section planes, one can see from one month the presence of more or less diffuse zones undergoing mineralization located outside the bone defect (figures 21E and 21F). Conclusions:

[0254] Bone formation generally occurs around biomaterials and, to a lesser extent, within them. The multi-scale porosity of the porous bone substitute material according to the invention appears to be a positive factor since several ossification foci are detected within it, highlighting the fact that differentiated and active cells have been able to migrate into this area, probably accompanied by blood vessels.

[0255] These histological analyses also allow visualization of the porous bone substitute material according to the invention within the bone defect: it appears fragmented and partially embedded in the mineralized tissue, demonstrating degradation and biointegration consistent with the repair kinetics. The estimated in vivo lifespan of the porous bone substitute material according to the invention is 13 to 42 months. This lifespan is compatible with clinical use.

[0256] Histological analyses confirm the osteoconductive nature and degradation of the porous bone substitute material according to the invention, visible from the first month of implantation, as well as the formation of bone.

[0257] The overall results indicate that the porous bone substitute material according to the invention possesses good mechanical properties, good biocompatibility, and degradation suitable for bone tissue reconstruction. It exhibits promising in vivo performance by inducing intense bone synthesis, even restoring tissue continuity between two bone fragments. The presence of decellularized bone in the porous bone substitute material according to the invention increases the production of mineralized tissue, which is three times greater than that obtained with the poly(caprolactone-urea-urethane) elastomer matrix alone after three months. This intense production is not, however, localized solely within the bone defect but also occurs around the fixators. Several mineralization zones were detected between the bone tissue and the surrounding muscle.

Claims

Demands

1. Porous bone substitute material comprising: - at least one elastomeric matrix, and - decellularized bone particles.

2. Porous bone substitute material according to claim 1, characterized in that at least one elastomeric matrix comprises a poly(urea-urethane ester) based elastomer, the ester being selected from caprolactone oligomers (PCL), lactic acid oligomers (PLA), glycolic acid oligomers (PGA), hydroxybutyrate oligomers (PHB), hydroxyvalerate oligomers (PVB), dioxanone oligomers (PDO), poly(ethylene adipate) oligomers (PEA), poly(butylene adipate) oligomers (PBA) or combinations thereof.

3. Porous bone substitute material according to any one of claims 1 to 2, characterized in that decellularized bone particles can be obtained from natural bone.

4. Porous bone substitute material according to any one of claims 1 to 3, characterized in that the decellularized bone particles have a diameter between Inm and 1mm.

5. Porous bone substitute material according to any one of claims 1 to 4, characterized in that the decellularized bone particles represent at least 10% by weight of the porous bone substitute material.

6. Porous bone substitute material according to any one of claims 1 to 5, characterized in that said porous bone substitute material has a multi-scale pore size between 50 pm and 2000 pm.

7. Porous bone substitute material according to any one of claims 1 to 6, characterized in that said porous bone substitute material has a total porosity greater than or equal to 60%.

8. Porous bone substitute material according to any one of claims 1 to 7, characterized in that said porous bone substitute material has a volume between 0.1 and 400 cm3.

9. Porous bone substitute material according to any one of claims 1 to 8, for use in bone repair, preferably the repair of a cavity bone defect and / or the repair of a segmental bone defect.

10. Porous bone substitute material for its use according to the re- claim 9, in which the bone repair is greater than or equal to 5% by volume of the volume of bone to be repaired.

11. Bone repair kit comprising the porous bone substitute material according to any one of claims 1 to 8 and a fixative.

12. A process for preparing a porous bone substitute material comprising the following steps: a) preparing an organic phase comprising the compounds necessary for the synthesis of poly(ester-urea-urethane), b) adding water and decellularized bone particles to the organic phase of step a) to form an emulsion, c) polymerizing / crosslinking the emulsion obtained in step b) to obtain said porous bone substitute material, d) washing said porous bone substitute material obtained in step c), and e) drying said porous bone substitute material obtained in step d).

Images