Bone substitute material based on calcium silicate and calcium phosphate

Abstract

The present invention relates to the field of bone substitute materials and bone scaffolds. Especially, the present invention refers to a bone substitute material comprising: - at least one porous calcium silicate-coated granule comprising: • a core which is a porous granule having both microporosity and macroporosity; and made of at least one calcium phosphate-based compound; and • a calcium silicate coating embedding totally or partially said core; said calcium silicate coating comprising from 60% to 100% wt. of at least one calcium silicate compound to the total weight of said calcium silicate coating; and optionally, at least one uncoated porous granule having both microporosity and macroporosity and made of at least one calcium phosphate-based compound. The present invention also relates to a bone scaffold resulting from the hydration of a bone substitute material of the invention; and to a process for manufacturing said bone substitute material.

Description

BONE SUBSTITUTE MATERIAE BASED ON CAECIUM SIEICATE AND CAECIUM PHOSPHATEFIEED OF INVENTION

[0001] The present invention relates to the field of bone scaffolds. Especially, the present invention refers to a bone substitute material comprising at least one porous calcium phosphate-based granule, totally or partially coated with a calcium silicate coating. The present invention also refers to a process for manufacturing said bone substitute material. The bone substitute material of the invention can advantageously be hydrated in order to provide a bone scaffold.BACKGROUND OF INVENTION

[0002] Bone defects can develop from different origins like infection, tumor, trauma, surgery, congenital etiology and so on. For centuries, bone replacement has been practiced with various materials of natural origin. However, since the 19thcentury, new materials have emerged as bone substitutes and are either derived from biological products such as demineralized bone matrix, platelet-rich plasma, hydroxyapatite, adjunction of growth factors (like bone morphogenetic protein) or from synthetic material such as calcium sulfate, tri-calcium phosphate ceramics, bioactive glasses, or polymer- based substitutes.

[0003] In the dental field, generally used bone substitutes are based on hydroxyapatite (HA) and / or [3-TCP (beta-tricalcium phosphate). These materials come in the form of porous granules, applied using a spatula, a pre-filled syringe or combined with a collagen matrix acting as a binder between the granules. However, once placed in the bone defect, and in contact with biological fluids, they lose their mechanical properties and their cohesion. Consequently, this no longer makes it possible to form a "scaffold" or stable support for the migration, proliferation and differentiation of cells within the defective site, thus disrupting the process of angiogenesis (essential for good bone regeneration). Cement based bone substitutes also exist to overcome these drawbacks thanks to theirsetting properties. However, these systems present a low porosity so that cell migration inside these structures is poor.

[0004] Thus, there is still a need for providing alternative bone scaffolds. Especially, there is still a need for providing a bone scaffold featuring both a porous network suitable for cell migration and good mechanical properties, especially featuring mechanical properties similar to those of cancellous bone. Consequently, there is a need to provide bone scaffolds having suitable tensile strength, compressive strength, and elastic modulus. Especially, there is a need to provide bone scaffolds having a compressive strength and an elastic modulus in the range of those of healthy cancellous bone. There is also still a need for providing a bone scaffold that, after implantation and contact with biological fluids, does not disintegrate, while maintaining a single structure in which the constitutive granules retain good cohesion between them.

[0005] In addition, there is also a need to provide bone scaffold which fulfills ethic requirements, which is safer and features a better traceability and a lower biological risk. Advantageously, the bone scaffold of the invention is free of animal origin and enables to solve this technical problem. Furthermore, the process for providing a bone substitute material or bone scaffold free of animal origin enables to reduce the number of procedure steps required to provide said bone material or scaffold; and is more reproducible than a process involving the use of materials of animal origin. Thus, the process for manufacturing the bone substitute material and / or bone scaffold of the invention is less time-consuming and more economic and safer than a process using animal sources.

[0006] In the field of bone regeneration, there is also a need to provide biomaterials able to induce new bone formation within bone defects in a subject in need thereof, while providing the highest amount of mature bone. Thus, the aim of the present invention is to guarantee a high quality in the new bone formation for the patient.

[0007] In the field of dental implant screw fixation procedures, there is also a need to provide biomaterial with improved tensile strength in order to ensure efficient mechanical holding of screws.

[0008] Surprisingly, the Applicant has evidenced that a bone substitute material comprising calcium phosphate-based granules totally or partially embedded with a calcium silicate coating, provides after hydration of said granules, a single unified bone scaffold overcoming the drawbacks mentioned herein above. Indeed, after hydration, the calcium- silicate coated granules advantageously lead to a bone substitute material having an improved tensile strength compared to commercially available bone substitute materials. Furthermore, the calcium- silicate coated granules feature a good cohesion between them after being hydrated. Thus, the bone substitute material comprising said calcium- silicate coated granules not only features both good mechanical properties but is also in the form of a bone scaffold with a single unified structure in which said granules are aggregated together. Furthermore, the Applicant evidences that the new bone formation obtained thanks to the bone substitute material of the invention includes at least 80%, preferably at least 90% of mature bone.

[0009] The Applicant has also evidenced that a bone substitute material comprising both calcium phosphate-based granules totally or partially embedded with a calcium silicate coating, and uncoated calcium phosphate-based granules, provides, after hydration of said granules, a single unified bone scaffold featuring an optimized residual porosity allowing improving cell migration inside said scaffold.

[0010] Advantageously, the bone substitute material and / or the bone scaffold of the present invention features higher tensile strength than commercial bone substitutes.SUMMARY

[0011] This invention thus relates to a bone substitute material comprising:- at least one porous calcium silicate-coated granule comprising:• a core which is a porous granule having both microporosity and macroporosity; and made of at least one calcium phosphate-based compound; and• a calcium silicate coating embedding totally or partially said core; and- optionally, at least one uncoated porous granule having both microporosity and macroporosity and made of at least one calcium phosphate-based compound.

[0012] According to one embodiment, the bone substitute material comprises or consists of:- at least one porous calcium silicate-coated granule comprising or consisting of:• a core corresponding to a porous granule made of at least one calcium phosphate-based compound having both micropores and macropores; the longest dimension of the inner volume of said micropores ranging from 0.1 pm to 100 pm and the longest dimension of the inner volume of said macropores being equal or higher than 100 pm; said dimension being measured by porosimetric analysis technique, preferably by mercury intrusion porosimetry; and• a calcium silicate coating totally or partially embedding said core; said calcium silicate coating comprising from 60% to 100% wt. of at least one calcium silicate compound to the total weight of said calcium silicate coating; and- optionally, at least one uncoated porous granule having both microporosity and macroporosity and made of at least one calcium phosphate-based compound.

[0013] According to one embodiment, the calcium silicate coating does not comprise silica (SiOi). According to one embodiment, the calcium silicate-coated granule does not comprise silica (SiC ). According to one embodiment, the bone substitute material does not comprise silica (SiCh). According to one embodiment, the bone scaffold does not comprise silica (SiC ).

[0014] According to one embodiment, the bone substitute material further comprises at least one uncoated porous granule made of at least one calcium phosphate-based compound having both micropores and macropores; the longest dimension of the inner volume of said micropores ranging from 0.1 pm to 100 pm and the longest dimension of the inner volume of said macropores being equal or higher than 100 pm; said dimension being measured by porosimetric analysis technique, preferably by mercury intrusion porosimetry.

[0015] According to one embodiment, the porous calcium silicate-coated granule has a size ranging from 50 pm to 50000 pm; preferably from 50 pm to 3000 pm.

[0016] According to one embodiment, the porous calcium silicate-coated granule has a size corresponding to the longest dimension of said granule ranging from 50 pm to 50000 pm; preferably from 50 pm to 3000 pm.

[0017] According to one embodiment, the calcium phosphate-based compound is selected from natural bone such as human bone or bovine bone, tricalcium phosphate (TCP), octacalcium phosphate (OCP), dicalcium phosphate anhydrous (DCPA), dicalcium phosphate dihydrated (DCPD), apatite, hydroxyapatite (HA), whitlockite and mixtures thereof; preferably from tricalcium phosphate (TCP), hydroxyapatite (HA) and mixtures thereof.

[0018] According to one embodiment, the tricalcium phosphate is alpha-tricalcium phosphate or beta-tricalcium phosphate or mixture thereof; preferably is beta-tricalcium phosphate.

[0019] According to one embodiment, the at least one porous calcium silicate-coated granule further comprises at least one biocompatible polymer; preferably selected from polyesters, polysaccharides, polyvinylpyrrolidones and mixtures thereof; more preferably is selected from poly(lactic-co-glycolic acid) (PLGA), polyvinylpyrrolidone (PVP), ethyl cellulose (EC) and mixtures thereof; even more preferably is PVP.

[0020] According to one embodiment, the at least one porous calcium silicate-coated granule further comprises at least one additive; preferably selected from setting accelerators, radio-opacifiers, silica-based compounds and mixtures thereof; more preferably comprising at least one setting accelerator.

[0021] According to one embodiment, the weight ratio r2 between the amount of the at least one porous calcium silicate-coated granule and the amount of the at least one uncoated porous granule ranges from 0.05 to 2, preferably from 0.1 to 1.5.

[0022] The present invention also refers to a process for manufacturing a bone substitute material of the invention; said process comprising or consisting of the following steps:i. Providing at least one porous granule having both microporosity and macroporosity; and made of at least one calcium phosphate-based compound; and ii. mixing said at least one porous granule with a solution of at least one calcium silicate-based compound in a non-aqueous solvent; and iii. evaporating the non-aqueous solvent to provide at least one porous calcium phosphate-based granule coated, partially or totally, with at least one calcium silicate-based compound; and iv. optionally, mixing the at least one coated porous calcium phosphate-based granule obtained at step (iii) with at least one uncoated porous granule having both microporosity and macroporosity and made of at least one calcium phosphate- based compound.

[0023] According to one embodiment, the process for manufacturing a bone substitute material comprises or consists of the following steps:(i) Providing at least one porous granule having both micropores having the longest dimension of the pore ranging from 0.1 pm to 100 pm and macropores having the longest dimension of the pore being equal or higher than 100 pm; and made of at least one calcium phosphate-based compound; and(ii) mixing said at least one porous calcium phosphate based-granule with at least one calcium silicate-based compound in a non-aqueous solvent; and(iii) evaporating the non-aqueous solvent to provide at least one porous calcium phosphate-based granule , partially or totally coated with at least one calcium silicate-based compound; and(iv) optionally, mixing the at least one coated porous calcium phosphate-based granule obtained at step (iii) with at least one uncoated porous granule having both micropores having the longest dimension of the pore ranging from 0.1 pm to 100 pm and macropores having the longest dimension of the pore being equal or higher than 100 pm and made of at least one calcium phosphate-based compound.

[0024] According to one embodiment, steps (ii) and (iii) are simultaneously implemented.

[0025] According to one embodiment, step (ii) and / or (iii) is(are) implemented under reduced pressure.

[0026] According to one embodiment, step (ii) and / or (iii) is(are) implemented under reduced pressure ranging from 2.10'3hPa et 1013 hPa.

[0027] According to one embodiment, the non-aqueous solvent comprises or consists of acetone, acetamide, acetonitrile, isopropanol, ethanol, dimethyl sulfoxide (DMSO), propanol, methanol, ethyl acetate or any mixtures thereof; preferably is ethanol.

[0028] According to one embodiment, step (iii) is carried out at a temperature ranging from 20°C to 250°C, preferably from 50°C to 100°C, more preferably at a temperature of about 60°C.

[0029] Another object of the present invention refers to a bone scaffold resulting from the hydration of the bone substitute material of the invention.

[0030] According to one embodiment, the hydration is implemented by contacting the bone substitute material of the invention, with a body fluid, preferably when the bone substitute material is placed in a bone defect of a patient. According to one embodiment, the hydration is implemented by contacting the bone substitute material of the invention, with a body fluid in vitro.

[0031] According to one embodiment, the bone scaffold is porous; preferably the bone scaffold comprises interconnected pores with both microporosity and macroporosity.

[0032] According to one embodiment, the bone scaffold is porous; preferably the bone scaffold comprises interconnected pores with both micropores having the longest dimension of the pore ranging from 0.1 pm to 100 pm and macropores having the longest dimension of the pore being equal or higher than 100 pm.DEFINITIONS

[0033] In the present invention, the following terms have the following meanings:

[0034] “About” preceding a figure means more or less 10%, preferably more or less 5%, more preferably more or less 1%, of the value of said figure.

[0035] “Apatite”: refers to hexagonal phosphates of formula Cas PC ; preferably of formula Cas(Mg, Fe PC MOH).

[0036] “Biocompatible polymer”: refers to any polymer eliciting little or no immune response in a given organism, or to any polymer that is able to integrate with a particular cell type or tissue.

[0037] “Body fluid”: refers to any liquids within the human body. In one embodiment, the body fluid is saliva and / or blood.

[0038] “Bone”: refers to rigid organs that constitute part of the endoskeleton of vertebrates. For instance, the term “bone” encompasses bone, mandibular bone, spongious bone and cortical bone.

[0039] “Calcium silicate compound” refers to a family of compounds comprising both calcium and silicate ions. In one embodiment, the calcium silicate compound is selected from tricalcium silicate, dicalcium silicate and their mixtures; preferably the calcium silicate compound is tricalcium silicate.

[0040] “Calcium silicate coating” refers to a coating comprising or consisting of at least one calcium silicate compound, preferably selected from tricalcium silicate, dicalcium silicate and their mixtures; more preferably the calcium silicate compound is tricalcium silicate. In one embodiment, the expression “calcium silicate coating” means that the coating is made of calcium silicate. In one embodiment, the calcium silicate coating contains from 60% wt. to 100% wt., preferably from 70% wt. to 100% wt., more preferably from 80% wt. to 100% wt., even more preferably from 90% wt. to 100% wt., of at least one calcium silicate compound to the total weight of said calcium silicate coating. In one embodiment, the calcium silicate coating contains 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% wt. of at least one calcium silicate compound,preferably selected from tricalcium silicate, dicalcium silicate and their mixtures; to the total weight of said calcium silicate coating. In one embodiment, the calcium silicate coating does not comprise silica (of formula SiCh).

[0041] “Calcium phosphate” refers to a family of compounds comprising both calcium and phosphate ions.

[0042] “Coated” refers to any compound or material covered, partially or totally, by a coating.

[0043] “Derivates” indicates a relationship between a first and a second molecule. It generally refers to structural similarity between the first molecule and the second molecule and does not connote or include a process or source limitation on a first molecule that is derived from a second molecule. For example, in the case of the derivates of cellulose, the expression “cellulose derivates” refers to cellulose scaffolded by one or more chemical substituents such as a methyl group (methylcellulose) or an ethyl group (ethylcellulose) for example.

[0044] “Granule” refers to any material having an elongated shape with a size corresponding to the longest dimension of said material ranging from 50 pm to 50 000 pm; preferably ranging from 50 pm to 3 000 pm, even more preferably from 450 pm to 2,5 mm. In one embodiment, the term “granule” refers to a material having a cylindrical or parallelepipedal shape. In one embodiment, the term “granule” does not refer to a particle (i.e. a spherical shape) or to a grain. In one embodiment, the size (or the longest dimension) of the granule is measured by the technique of laser granulometry.

[0045] “Hydroxyapatite”: refers to the chemical compound of formula Cas(PO4)3(OH).

[0046] “Interconnected pores”: refers to a network of pores. In one embodiment, the terms “interconnected pores” refers to open pores.

[0047] “Macropores”: refers to pores having a mean size, preferably a mean pore diameter, equal or higher than 100 pm. According to one embodiment, the longest dimension of the inner volume of the macropore is equal or higher than 100 pm; preferably said dimension being measured by porosimetric analysis technique, morepreferably by mercury intrusion porosimetry. In one embodiment, if the macropore is spherical or spheroid, the mean size of the macropore refers to its mean pore diameter being equal or higher than 100 pm; preferably said dimension being measured by porosimetric analysis technique, more preferably by mercury intrusion porosimetry.

[0048] “Mature bone”: refers to a kind of bone tissue in which the cells (osteoblasts and osteocytes) are organized in concentric lamellae in the Haversian system. Especially, the mature bone is highly mineralized and rich in endogenous collagen, preferably in endogenous type I collagen. To the contrary, an “immature bone” refers to a bone tissue in which the cells (osteoblasts and osteocytes) are randomly arranged so that the bone structure appears disorganized and is poorly mineralized.

[0049] “Micropores”: refers to pores having a mean size, preferably a mean pore diameter, lower than 100 pm; preferably ranging from 0.1 pm to lower than 100 pm. According to one embodiment, the longest dimension of the inner volume of the micropore ranges from 0.1 pm to 100 pm; preferably said dimension being measured by porosimetric analysis technique, more preferably by mercury intrusion porosimetry. In one embodiment, if the micropore is spherical or spheroid, the mean size of the micropore refers to its mean pore diameter ranging from 0.1 pm to 100 pm; preferably said dimension being measured by porosimetric analysis technique, more preferably by mercury intrusion porosimetry.

[0050] “Patient”: refers to any warm-blooded animal, preferably human, who / which is waiting for, or is receiving medical care or is / will be the object of a medical procedure.

[0051] “Polyester”: refers to any polymer comprising a repeating unit with at least one ester function. In the present invention, the term “polyester” includes any polymer resulting from a polycondensation reaction between a diacid compound and a dialcohol compound or any polymer resulting from the transesterification of a polyester.

[0052] “Poly(glycolic acid)”, “poly(glycolide)”or “PGA”: refers to any polymer obtained from the polymerization of either glycolic acid or the cyclic diester glycolide.

[0053] “Poly(lactic acid)”, “polylactide” or ‘ ‘PLA”: refers to any polymer obtained from the polymerization of either lactic acid or the cyclic diester lactide.

[0054] “Polymer”: refers to any chain or material resulting from the multiple repetition of a repeating unit (monomer), said monomers being covalently linked to each other.

[0055] “Polysaccharide”: refers to any polymeric carbohydrate molecule composed of long chains of monosaccharide units bound together by glycosidic linkages; which may be linear or branched. Examples include starch, glycogen, cellulose and chitin.

[0056] “Porous”: refers to any compound having pores. In one embodiment, the pores may be micropores and / or macropores. In the present invention, the pore diameter or the porosity of a porous compound is measured by the well-known techniques of the skilled artisan such as for example, Brunauer-Emmett-Teller Method (BET), microtomography, Scanning Electron Microscopy (SEM) or Mercury Intrusion Porosimetry (MIP). In one embodiment, the pore diameter is preferably measured by Mercury Intrusion Porosimetry (MIP).

[0057] “Residual porosity (rp) or final porosity”: refers to the porosity of the bone substitute material after hydration that-is-to-say the porosity of the bone scaffold. According to one embodiment, the residual porosity (or the final porosity) of the bone scaffold is different from the porosity of each granule aggregated inside said bone scaffold. According to one embodiment, the residual porosity rp (or the final porosity) of the bone scaffold is the same as the porosity before hydration (noted pi or p ), of each granule coated or not, composing said bone scaffold.

[0058] “Setting accelerator”: refers to any agent which reduces the setting time of a material when added to said material compared to the setting time of the same material without said agent.

[0059] “Treatment” or “Treating”: refers to therapeutic treatment wherein the object is to cure or slow down (lessen) the targeted pathologic condition or disorder. A subject or mammal is successfully “treated” for the condition or disorder if, after receiving the coated calcium phosphate granules, the bone substitute material or the bone scaffold of the present invention, the patient shows observable and / or measurable reduction in one or more of the symptoms associated with the specific disease or condition; and improvement in quality-of-life issues. The above parameters for assessing successfultreatment and improvement in the disease or conditions are readily measurable by routine procedures familiar to a physician.

[0060] “Tricalcium phosphate” refers to the chemical compound of formula Ca3(PO4)2. In one embodiment, the term “tricalcium phosphate” refers to the alpha-tricalcium phosphate and / or the beta-tricalcium phosphate.

[0061] “Tricalcium silicate”: refers to the chemical compound of formula CagSiOg.

[0062] “Whitlockite”: refers to the chemical compound of formula Cag(Mg, Fe2+)(PO4)6(PO3OH).DETAILED DESCRIPTION

[0063] Porous calcium silicate-coated granule

[0064] This invention relates to a calcium silicate-coated granule, preferably a porous calcium silicate-coated granule.

[0065] In one embodiment, the porous calcium silicate-coated granule comprises or consists of:- a core which is a porous granule, preferably is a porous calcium phosphate-based granule, preferably having both micoporosity and macroporosity; and- a calcium silicate coating embedding totally or partially said core.

[0066] Core

[0067] In one embodiment, the core is a porous granule having both microporosity and macroporosity, preferably is a porous calcium phosphate-based granule having both microporosity and macroporosity. In one embodiment, the core is a porous granule having interconnected pores.

[0068] In one embodiment, the core comprises or consists of a calcium phosphate-based granule, preferably an uncoated calcium phosphate-based granule. In one embodiment, the calcium phosphate-based granule is a biocompatible material.

[0069] In one embodiment, the core comprises or is made of at least one calcium phosphate-based compound; preferably selected from natural bone such as human boneor bovine bone, tricalcium phosphate (TCP), octacalcium phosphate (OCP), dicalcium phosphate anhydrous (DCPA), dicalcium phosphate dihydrated (DCPD), apatite, hydroxyapatite (HA), whitlockite and mixtures thereof; more preferably selected from tricalcium phosphate (TCP), hydroxyapatite (HA) and mixtures thereof. In one embodiment, the core comprises or is made of at least two calcium phosphate-based compounds, preferably the core comprises or is made of only two calcium phosphate- based compounds; preferably selected from natural bone such as human bone or bovine bone, tricalcium phosphate, apatite, hydroxyapatite, whitlockite and mixtures thereof. More preferably the core comprises or is made of both tricalcium phosphate (alphatricalcium phosphate (a-TCP), or beta-tricalcium phosphate (P-TCP)) and hydroxyapatite; even more preferably of both beta-tricalcium phosphate (P-TCP) and hydroxyapatite (HA).

[0070] In one embodiment, the core comprises from at least 10% to 100%, preferably from 15% to 50%, preferably from 20% to 45% of tricalcium phosphate. In one embodiment, the core comprises at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% wt. of tricalcium phosphate, preferably selected from alpha-tricalcium phosphate or beta-tricalcium phosphate or mixture thereof; more preferably is beta-tricalcium phosphate; to the total weight of said core.

[0071] In one embodiment, the tricalcium phosphate is alpha-tricalcium phosphate or beta-tricalcium phosphate or mixture thereof; preferably is beta-tricalcium phosphate.

[0072] In one embodiment, the core is a calcium phosphate-based granule comprising or consisting of: from 50% to 80% by weight of hydroxyapatite (HA) and from 20% to 50% by weight of tricalcium phosphate, preferably of beta-tricalcium phosphate (P-TCP) relative to the total weight of said granule.

[0073] In one embodiment, the core is a calcium phosphate-based granule comprising 70% by weight of hydroxyapatite (HA) and 30% by weight of tricalcium phosphate, preferably of beta-tricalcium phosphate (P-TCP), relative to the total weight of said granule. In one embodiment, the core is a calcium phosphate-based granule comprising60% by weight of hydroxyapatite (HA) and 40% by weight of tricalcium phosphate, preferably of beta-tricalcium phosphate (P-TCP), relative to the total weight of said granule.

[0074] In one embodiment, the calcium phosphate-based granule is a commercially available calcium phosphate-based granule; preferably is purchased from MEDICAL GROUP or EPRUI BIOTECH firms.

[0075] In one embodiment, the core, which is preferably a calcium phosphate granule, comprises at least two distinct mean sizes of pores. In one embodiment, the mean size of the pores of the core ranges from 0.1 pm to 550 pm, preferably from 0.4 pm to 530 pm, preferably measured by Mercury Intrusion Porosimetry (MlP).In one embodiment, the core is a calcium phosphate-based granule comprising interconnected pores.

[0076] In one embodiment, the porosity pl of the core, which is preferably a calcium phosphate-based granule, may vary, preferably the porosity pl is greater than or equal to 60%, more preferably greater than or equal to 65%, even more preferably ranges from 70 to 90% relative to the core.

[0077] In the present invention, the porosity is expressed by the percentage of the volume occupied by the pores relative to the total volume of the porous material. For example, the porosity pl of the core is expressed by the percentage of the volume occupied by the pores relative to the total volume of the core.

[0078] In one embodiment, the core, which is preferably a calcium phosphate-based granule, has a size ranging from 0.05 mm to 3.00 mm; preferably from 0.2 mm to 2.5 mm; more preferably from 0.4 mm to 2.1 mm. In one embodiment, the core, which is preferably a calcium phosphate granule, has a size of about 0.5; 0.6; 0.7; 0.8; 0.9; 1.0; 1.1; 1.2; 1.3; 1.4; 1.5; 1.6; 1.7; 1.8; 1.9 or 2.0 mm.

[0079] In one embodiment, the core, which is preferably a calcium phosphate-based granule, has its longest dimension ranging from 0.05 mm to 3.00 mm; preferably from 0.2 mm to 2.5 mm; more preferably from 0.4 mm to 2.1 mm. In one embodiment, thecore, which is preferably a calcium phosphate granule, has its longest dimension of about 0.5; 0.6; 0.7; 0.8; 0.9; 1.0; 1.1; 1.2; 1.3; 1.4; 1.5; 1.6; 1.7; 1.8; 1.9 or 2.0 mm.

[0080] In one embodiment, the weight of the core, which is preferably a calcium phosphate-based granule, may vary, preferably the weight of the core ranges from 1 mg to 5 mg, preferably from 2 mg to 5 mg, more preferably from 3 mg to 4 mg.

[0081] In one embodiment, the uncoated calcium phosphate-based granule further comprises at least one additive; preferably selected from doping agents such as zinc ion, magnesium ion, strontium ion and their mixtures; stabilizers; and radioopacifiers such as bismuth oxide, strontium carbonate, strontium phosphate, barium sulfate, tantalum oxide, cerium oxide, tin oxide, zirconium oxide compounds and radioopaque glasses containing tantalum, barium and strontium, and mixtures thereof.

[0082] Coating

[0083] In the present invention, the coating covers at least part of the external surface of the calcium phosphate-based granules. In some embodiments, the coating covers at least 50% of the external surface of the calcium phosphate granules, preferably at least 80%, in particular at least 95%. In some embodiments, the coating covers the whole external surface of the calcium phosphate granules (100%).

[0084] In one embodiment, the coating is a mineral coating, preferably is a non-hydrated mineral coating, even more preferably is a non-hydrated calcium silicate-based coating, even more preferably is a non-hydrated tricalcium silicate coating.

[0085] In one embodiment, the coating is a dry mineral coating, preferably is a dry non- hydrated mineral coating, even more preferably is a dry non-hydrated calcium silicate- based coating, even more preferably is a dry non-hydrated tricalcium silicate coating. In one embodiment, the coating is a non-hydrated mineral coating comprising or consisting of at least one calcium silicate compound, preferably selected from dicalcium silicate, tricalcium silicate and their mixtures thereof, more preferably is tricalcium silicate. In one embodiment, the coating is a non-hydrated mineral coating comprising or consisting of at least one non-hydrated calcium silicate compound, preferably selected from non-hydrated dicalcium silicate, non-hydrated tricalcium silicate and their mixtures thereof, more preferably is non-hydrated tricalcium silicate. In one embodiment, the mineral coating is not a silica solution (or silica sol).

[0086] In one embodiment, the calcium silicate coating comprises or consists of one or more layers of calcium silicate particles, preferably non-hydrated calcium silicate particles.

[0087] In one embodiment, the coating comprises or consists of at least one mineral compound and optionally, at least one organic compound. In one embodiment, the at least one mineral compound is calcium silicate, preferably is selected from dicalcium silicate, tricalcium silicate and their mixtures thereof, more preferably is tricalcium silicate. In one embodiment, the at least one mineral compound comprises at least one additional calcium silicate compound which is not tricalcium silicate.

[0088] In one embodiment, the at least one organic compound comprises or consists of at least one polymer; preferably at least one biocompatible polymer and / or biologically inert polymer; more preferably selected from the group consisting of: polyesters such as poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL), poly(lactic acid) (PLA), poly(glycolic acid) (PGA), polyhydroxybutyrate (PHB), poly(3-hydroxy valerate), poly(ethylene succinate) (PESu), poly(butylene succinate) (PBSu) and any combinations or copolymers thereof; polysaccharides such as chitosan, collagen, alginate, cellulose, carboxymethyl cellulose (CMC), ethyl cellulose (EC) and cellulosic derivates; polyvinyl alcohols and polyvinylpyrrolidones (PVP); even more preferably is selected from poly(lactic-co-glycolic acid) (PLGA), polyvinylpyrrolidone (PVP), ethyl cellulose (EC) and their mixtures.

[0089] According to one embodiment, the coating further comprises at least one additive selected from: setting accelerators such as calcium chloride; silica and radioopacifiers such as bismuth oxide, strontium carbonate, strontium phosphate, barium sulfate, tantalum oxide, cerium oxide, tin oxide, zirconium oxide compounds and radioopaque glasses containing tantalum, barium and strontium, and mixtures thereof.

[0090] In one embodiment, after being coated with the calcium silicate coating of the invention, the size of calcium silicate-coated granule is at least 20% higher / larger, preferably at least 30% higher / larger, more preferably is 37% higher / larger, than the size of the corresponding uncoated granule.

[0091] In one embodiment, the porosity p2 of the calcium silicate-coated granule is of the same magnitude as the porosity pi of the uncoated granule and the pore size ranges from 40% to 90%, preferably from 50% to 80%, preferably ranges from 60% to 70%. The porosity p2 of the calcium silicate-coated granule is expressed by the percentage of the volume occupied by the pores relative to the total volume of the calcium silicate- coated granule.

[0092] Advantageously, the calcium silicate coating of the present invention does not clog the pores of the granule, i.e. the pores of the core.

[0093] In one embodiment, the weight ratio rl (calcium silicate compound / calcium phosphate granules) of the calcium silicate-coated granule ranges from more than 0 to 1; preferably ranging from 0.1 to 0.8; more preferably ranging from 0.2 to 0.7. In one embodiment, the weight ratio rl is about 0.1; 0.2; 0.3; 0.4; 0.5; 0.6; 0.7; 0.8; 0.9 or 1.

[0094] In one embodiment, the calcium phosphate-based granule is coated by one layer of at least one calcium silicate compound. In one embodiment, the calcium phosphate- based granule is not coated by two or more different coatings. In other words, according to this embodiment, the calcium phosphate-based granule is coated by only one coating.

[0095] In one embodiment, the calcium phosphate-based granule is not coated by a coating comprising or consisting of both a calcium silicate compound and a calcium phosphate compound. In one embodiment, the calcium phosphate-based granule may be coated by two or more different coatings provided that it does not comprise both a calcium silicate coating and a coating comprising or consisting of silica. In one embodiment, the calcium phosphate-based granule may be coated by two or more different coatings provided that it does not comprise silica.

[0096] Bone substitute material

[0097] The invention also relates to a bone substitute material comprising or consisting of at least one porous calcium silicate-coated granule as defined above.

[0098] In one embodiment, the bone substitute material further comprises at least one uncoated porous granule having both microporosity and macroporosity and made of at least one calcium phosphate-based compound as defined above.

[0099] In one embodiment, the bone substitute material comprises or consists of a plurality of porous calcium silicate-coated granules as defined above and a plurality of uncoated porous granules having both microporosity and macroporosity and made of at least one calcium phosphate-based compound as defined above.

[0100] In one embodiment, the weight ratio r2 between the amount of the at least one porous calcium silicate-coated granule and the amount of the at least one uncoated porous granule ranges from more than 0 to 2, preferably from 0.1 to 1.5; more preferably is 1.0.

[0101] In one embodiment, the bone substitute material is non-hydrated. In one embodiment, the bone substitute material does not comprise any calcium silicate hydrate.

[0102] Bone scaffold

[0103] The invention also relates to a bone scaffold. Especially, the present invention refers to a bone scaffold resulting from the hydration of a plurality of porous calcium silicate-coated granules, i.e. of porous calcium phosphate granules coated with a calcium silicate coating as defined above (also called “coated calcium phosphate granules”).

[0104] Hydration may be performed by any suitable technique known in the art. Hydration may be performed for instance by contacting the plurality of porous calcium silicate-coated granules with a fluid comprising water, preferably with a biological fluid such as blood or saliva. The duration of the contacting step may vary in a wide range. In some embodiments, the contacting step is implemented for a duration suitable for hydrating at least 50% of the porous calcium silicate-coated granules, preferably at least 80%, more preferably 100%, relative to the total number of porous calcium silicate-coated granules. In some embodiments, the porous calcium silicate-coated granules remain incontact with the fluid comprising water after hydration is complete, that is after 100% of the porous coated calcium phosphate granules are hydrated.

[0105] In one embodiment, the coating of the bone scaffold is a mineral coating, preferably is a hydrated mineral coating, more preferably is a hydrated calcium silicate- based coating, even more preferably is a hydrated tricalcium silicate coating. In one embodiment, the coating of the bone scaffold is a mineral coating comprising or consisting of a mixture of non-hydrated calcium silicate particles and hydrated calcium silicate particles (also called calcium silicate hydrates (CSH)). In one embodiment, the mineral coating of the bone scaffold comprises or consists of at least 80% by weight, preferably at least 90% by weight, more preferably at least 95% by weight of calcium silicate hydrates, relative to the total weight of said mineral coating.

[0106] In one embodiment, the hydration is implemented by contacting the plurality of porous calcium silicate-coated granules as defined above with a body fluid, preferably when the plurality of porous calcium silicate-coated granules is placed in a bone defect of a patient.

[0107] In the present invention, the hydration of tricalcium silicate (C3S) occurs according to the following reaction:2Ca3SiOs+ 6H2O - 3CaO-2SiO2-3H2O + 3Ca(OH)2C3S CSH

[0108] Thus, the bone scaffold of the invention comprises calcium silicate hydrates (CSH).

[0109] In one embodiment, the body fluid is blood and / or saliva. In one embodiment, when the bone scaffold is used for jaws and / or teeth, the body fluid is saliva. In one embodiment, when the bone scaffold is used for bone tissues other than teeth, the body fluid is blood.

[0110] In one embodiment, the bone scaffold of the invention comprises or consists of a porous calcium phosphate scaffold comprising porous calcium phosphate based- granules, at least one calcium silicate hydrate and calcium hydroxide.

[0111] In one embodiment, the bone scaffold of the invention comprises or consists of a porous calcium phosphate scaffold made of at least one calcium phosphate compound as defined above, said scaffold being partially or totally embedded by a coating comprising calcium silicate hydrates and optionally non-hydrated calcium silicate particles.

[0112] In one embodiment, the bone scaffold is porous. In one embodiment, the bone scaffold comprises micropores and / or macropores; preferably the bone scaffold comprises micropores and macropores. In one embodiment, the bone scaffold of the invention comprises at least two pore sizes. In one embodiment, the pores of the bone scaffold are micropores ranging from 1 pm to lower than 100 pm and / or macropores ranging from 100 pm to 400 pm, preferably measured by Mercury Intrusion Porosimetry (MIP). In one embodiment, the bone scaffold has interconnected pores. In one embodiment, the pores of the bone scaffold are micropores ranging from 2 pm to 8 pm and / or macropores ranging from 250 pm to 350 pm, preferably measured by Mercury Intrusion Porosimetry (MIP).

[0113] In one embodiment, the porosity inside the bone scaffold (also called residual porosity (rp)) ranges from more than 0% to 90% vol.; preferably from 30% vol. to 80% vol.; more preferably from 40% vol. to 70% vol., even more preferably from 60% to 70% vol., even more preferably from 60% to 65% vol. relative to the total volume of said bone scaffold. In one embodiment, the porosity inside the bone scaffold is about 60%, 61%, 62%, 63%, 64% or 65% vol. relative to the total volume of said bone scaffold.

[0114] Advantageously, the pores of the bone scaffold of the invention favor cell migration and thus, bone regeneration. Advantageously, the bone scaffold has mechanical properties similar to those of natural bone (cortical bone and / or spongious bone). Advantageously, the bone scaffold has a homogenous and unitary structure. That means that, 24h after hydration of the porous coated calcium phosphate granules placed in a bone defect, the bone scaffold features a single unified structure in which all the coated granules are linked to each other.

[0115] In one embodiment, the bone scaffold of the invention has a compressive strength ranging from 1 MPa to 10 MPa, preferably from 1.5 MPa to 10 MPa, more preferably from 1.9 MPa to 10 MPa, more preferably from 1.9 MPa to 5 MPa.

[0116] In one embodiment, the bone scaffold of the invention has an elastic modulus ranging from 50 MPa to 500 MPa, preferably from 50 MPa to 300 MPa.

[0117] Process for manufacturing the coated calcium phosphate granules

[0118] The invention also relates to a process for manufacturing a plurality of porous calcium silicate-coated granules as defined above, said process comprising or consisting of the following steps: i. Providing at least one porous granule made of at least one calcium phosphate- based compound as defined above; and ii. mixing said at least one calcium phosphate based porous granule with at least one calcium silicate-based compound as defined above in a non-aqueous solvent.

[0119] The invention also relates to a process for manufacturing a plurality of porous calcium silicate-coated granules as defined above, said process comprising or consisting of the following steps: i. Providing at least one porous granule having both microporosity and macroporosity; and made of at least one calcium phosphate-based compound as defined above; and ii. mixing said at least one porous granule with a solution of at least one calcium silicate-based compound as defined above in a non-aqueous solvent.

[0120] In one embodiment, the process of the invention further comprises a step (iii) of removing the non-aqueous solvent to provide at least one porous calcium phosphate- based granule coated, partially or totally, with at least one calcium silicate-based compound, preferably with tricalcium silicate. In one embodiment, the non-aqueous solvent is removed by evaporation.

[0121] In one embodiment, in step (i) the porous non-coated calcium phosphate granules are commercially available non-coated calcium phosphate granules. In one embodiment,in step (i) the porous non-coated calcium phosphate granules are manufactured in a preliminary step.

[0122] In one embodiment, steps (ii) and (iii) are simultaneously implemented. In one embodiment, step (iii) is implemented after step (ii).

[0123] In one embodiment, steps (ii) and / or (iii) are implemented at a temperature ranging from 10°C to 180°C, preferably from 20°C to 85°C, more preferably from 25°C to 50°C. In one embodiment, steps (ii) and / or (iii) are implemented at a temperature ranging from 30°C to 180°C, preferably from 50°C to 85°C, more preferably at a temperature about 60°C.

[0124] In one embodiment, steps (ii) and / or (iii) are implemented at a pressure ranging from 25 hPa to about 1013 hPa; preferably at a pressure ranging from 100 hPa to about 1013 hPa; more preferably ranging from 100 hPa to 500 hPa. In one embodiment, steps (ii) and / or (iii) are implemented at a pressure about 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 hPa. In one embodiment, steps (ii) and / or (iii) are implemented at a pressure about 175 hPa.

[0125] In one embodiment, steps (ii) and / or (iii) are implemented under reduced pressure; preferably at a pressure ranging from 2.10'3hPa et 1013 hPa.

[0126] In one embodiment, the non-aqueous solvent is selected from acetone, acetamide, acetonitrile, alcohols such as isopropanol, ethanol, and any mixtures thereof; preferably is ethanol.

[0127] In one embodiment, step (iii) is implemented by evaporation of the non-aqueous solvent. In one embodiment, step (iii) is implemented by any suitable techniques well- known by the skilled artisan such as for example by using a rotary evaporator, or by heating the mixture obtained at step (ii), preferably while stirring. In one embodiment, stirring is implemented during the whole heating period of step (iii). In one embodiment, stirring is implemented during the heating period of step (iii) at predetermined times.

[0128] In one embodiment, step (ii) comprises or consists of mixing the non-coated calcium phosphate granules with a calcium silicate compound, preferably tricalciumsilicate, in a weight ratio rl (calcium silicate compound / calcium phosphate granules) ranging from more than 0 to 1; preferably ranging from 0.1 to 0.8; more preferably ranging from 0.2 to 0.7. In one embodiment, the weight ratio r is about 0.1; 0.2; 0.3; 0.4; 0.5; 0.6; 0.7; 0.8; 0.9 or 1.

[0129] Advantageously, the process for manufacturing a plurality of porous calcium silicate-coated granules as defined above, does not require any sintering step. Advantageously, the process for manufacturing a plurality of porous calcium silicate- coated granules as defined above, does not require elevated temperatures (as for example ranging from 1100°C to 1300°C).

[0130] Process for manufacturing the bone substitute material

[0131] The invention also relates to a process for manufacturing the bone substitute material as defined above, said process comprising or consisting of the following steps:- providing a plurality of porous calcium silicate-coated granules as defined above; and- optionally, mixing the plurality of porous calcium silicate-coated granules with at least one uncoated porous calcium phosphate-based granule as defined above.

[0132] In one embodiment, the process for manufacturing the bone substitute material as defined above comprises or consists of implementing the process for providing at least coated porous calcium phosphate-based granule as defined above and then, implementing a step (iv) for mixing the at least one coated porous calcium phosphate-based granule obtained at step (iii) with at least one uncoated porous granule having both microporosity and macroporosity and made of at least one calcium phosphate-based compound.

[0133] In one embodiment, step (iv) comprises or consists of mixing the coated porous calcium phosphate-based granules with the uncoated porous granules having both microporosity and macroporosity and made of at least one calcium phosphate-based compound, in a weight ratio r2 (coated / uncoated calcium phosphate-based granules) ranging from more than 0 to 2; preferably ranging from 0.1 to 1.5; more preferably is 1.0.

[0134] Process for manufacturing the bone scaffold

[0135] The invention also relates to a process for manufacturing the bone scaffold as defined above, said process comprising or consisting of the following steps:- Providing the bone substitute material as defined above; and then- Contacting said bone substitute material with an aqueous fluid in order to provide a single unified structure comprising or consisting of a porous calcium phosphate scaffold partially or totally embedded by a coating comprising or consisting of calcium silicate hydrates.

[0136] In one embodiment, the aqueous fluid is selected from water and biological fluids such as blood or saliva.

[0137] In one embodiment, the process for manufacturing the bone scaffold is implemented in a bone defect of the body of a patient. In one embodiment, the process for manufacturing the bone scaffold is implemented before to be implanted in the body of a patient.

[0138] Uses

[0139] The present invention also refers to the use of the porous calcium silicate-coated granules of the invention, of the bone substitute material of the invention and / or of the bone scaffold of the invention as defined above.

[0140] In one embodiment, the present invention also refers to a method for treating a bone defect in a patient by using the porous calcium silicate-coated granules of the invention or the bone substitute material of the invention or the bone scaffold of the invention as defined above. In one embodiment, the present invention also refers to a method for treating an alveolar bone defect in a patient by using the porous calcium silicate-coated granules of the invention or the bone substitute material of the invention or the bone scaffold of the invention as defined above.

[0141] In one embodiment, the porous calcium silicate-coated granules of the invention or the bone substitute material of the invention or the bone scaffold of the invention asdefined above are for use in dental and / or orthopedic fields, preferably in the treatment of a bone defect such as an alveolar bone defect, more preferably in anchor implant procedures. In one embodiment, the invention relates to a method for treating dental and / or orthopedics diseases in a subject in need thereof, preferably for treating a bone defect such as an alveolar bone defect, more preferably for anchoring implant in a subject in need thereof by using the porous calcium silicate-coated granules of the invention or the bone substitute material of the invention or the bone scaffold of the invention as defined above.

[0142] In one embodiment, the porous calcium silicate-coated granules of the invention or the bone substitute material of the invention or the bone scaffold of the invention as defined above are useful as support for dental and / or orthopedical anchor. Advantageously, the bone scaffold of the invention as defined above features a good mechanical strength (especially, a good tensile strength) so that to efficiently maintain a dental and / or orthopedical screw inserted into said bone scaffold of the invention. In one embodiment, the tensile strength of the bone scaffold of the invention ranges from 10 from 70 N. In one embodiment, the tensile strength of the bone scaffold of the invention ranges from 20N to 60N, preferably from 30N to 55N, more preferably is 50N. In one embodiment, the tensile strength is measured by an electromechanical testing apparatus, preferably by the apparatus MTS CRITERION C.43. In one embodiment, the tensile strength is measured by the apparatus MTS CRITERION C.43.

[0143] In one embodiment, the porous calcium silicate-coated granules of the invention or the bone substitute material of the invention or the bone scaffold of the invention as defined above are for use in formation of mature bone in a bone defect of a subject. In one embodiment, when the porous calcium silicate-coated granules of the invention or the bone substitute material of the invention or the bone scaffold of the invention as defined above are placed in a bone defect in a subject in need thereof, the area of new bone formation after 90 days is higher than 0.2 mm2, preferably ranges from more than 0.2 mm2to 1 mm2, more preferably ranges from 0.5 mm2to 1 mm2, even more preferably ranges from 0.75 mm2to 0.95 mm2, even more preferably is 0.8 mm2. In one embodiment, the area of new bone formation within a bone defect after 90 days is measured by usinga light microscope (preferably with the apparatus Keyence VHX2000) and an image analysis system (preferably Image J software).

[0144] In one embodiment, the present invention also refers to an injection device such as a syringe comprising the porous calcium silicate-coated granules of the invention or the bone substitute material of the invention. In one embodiment, the present invention also refers to a kit comprising the porous calcium silicate-coated granules of the invention or the bone substitute material of the invention. In one embodiment, the kit comprises a first container with the porous calcium silicate-coated granules of the invention and a second container with the uncoated porous granules having both microporosity and macroporosity and made of at least one calcium phosphate-based compound.BRIEF DESCRIPTION OF THE DRAWINGS

[0145] Figure 1 is a set of microscopy cliches featuring uncoated beta-tricalcium phosphate granules (Figure 1A) and beta-tricalcium phosphate granules coated with tricalcium silicate (Figure IB).

[0146] Figure 2 is a set of photographs showing a bone scaffold obtained from a bone substitute material consisting of: uncoated beta-tricalcium phosphate granules (Figure 2A), beta-tricalcium phosphate granules coated with a tricalcium silicate coating without any polymer (Figure 2B) or consisting of beta-tricalcium phosphate granules coated with a tricalcium silicate coating including polyvinylpyrrolidone (Figure 2C).

[0147] Figure 3 is a set of photographs showing bone scaffolds obtained after setting for 24 hours in 150pL PBS, from bone substitute materials consisting of a mixture of calcium silicate-coated granules and uncoated beta-tricalcium phosphate granules (Formulations BSM1 (Fig.3A), BSM2 (Fig. 3B), BSM3 (Fig. 3C) and BSM4 (Fig. 3D) of example 2).

[0148] Figure 4 is a photograph of a complex “mold + bone scaffold of the invention (obtained from the hydration of formulation R41) in which is inserted a screw”; said complex being located on a mechanical test bench for a pull-out test.

[0149] Figure 5 is a set of histograms showing the tensile strength (expression in Newton (N)) obtained during a pull-out test of a screw inserted in a bone scaffold of the invention (obtained from formulation R41), in the commercial bone material Bio-Oss® (purchased from GEISTLICH) or in a bone material consisting of uncoated beta-TCP granules.

[0150] Figure 6 is a set of histograms showing the compressive strength (expressed in megapascal (MPa)) measured for bone scaffolds of the invention obtained from either formulation R41, formulation BSM3, or from formulation R33.

[0151] Figure 7 is a set of histograms showing the elastic modulus (expressed in megapascal (MPa)) measured for bone scaffolds of the invention obtained from either formulation R41, formulation BSM3, or from formulation R33.

[0152] Figure 8 is a set of histograms showing the areas of new bone formation within a bone defect of rat calvaria bone defect of 6 mm after the implantation of the bone substitute material of the invention (formulation R41) and compared to an empty bone defect (negative control) and to a bone defect filled with uncoated [3- tricalcium phosphate (P-TCP) granules.EXAMPLES

[0153] The present invention is further illustrated by the following examples.

[0154] Example 1: General process for manufacturing calcium silicate-coated granules of the invention

[0155] For providing porous coated calcium phosphate-based granules of the invention, the following general process has been implemented from porous uncoated granules of beta-tricalcium phosphate (P-TCP), optionally including hydroxyapatite (P-TCP / HA); said granules having a size ranging from 500 pm to 2 mm.

[0156] First, the porous uncoated granules of P-TCP or of P-TCP / HA (in a weight ratio of 30 / 70 or 40 / 60), and particles of the calcium silicate compound such as tricalcium silicate, is mixed in a non-aqueous solvent selected from acetone or ethanol. Then, thenon-aqueous solvent is evaporated at a temperature of about 60°C in order to provide porous granules of P-TCP or of P-TCP / HA embedded by a non-hydrated calcium silicate coating. In this way, the calcium phosphate-based granule is partially or totally covered by a layer of non-hydrated calcium silicate particles.

[0157] The process of the invention may further comprise the use of a polymer such as for example, polyvinylpyrrolidone (PVP), poly(lactic-co-glycolic) acid (PLGA) or ethyl methylcellulose (EC). In this case, the polymer is added in the solvent during the mixing step of the general process as described above. Advantageously the use of said polymer improves the adhesion of the calcium silicate coating on the calcium phosphate-based granules during the coating step. In this way, the calcium phosphate-based granule is partially or totally covered by a layer including both non-hydrated calcium silicate particles and the polymer.

[0158] The process of the invention has been implemented either by using a rotary evaporator (“R” formulations), by using a magnetic stirrer (“S” formulations), by using a manual method consisting of using a beaker and a spatula for stirring the reactional mixture (“M” formulations), or by using a spray coating method (“SC” formulations).

[0159] Several batches of coated calcium phosphate granules have been manufactured by the process of the invention and are shown in the following Table:PVP: polyvinylpyrrolidone; PLGA: poly(lactic-co-glycolic) acid and EC: ethyl cellulose. * Dicalcium silicate C2S ( instead of tricalcium silicate CSS ); ** granule ofbeta-TCP / HA (in a weight ratio of 30 / 70); *** Biodentine® powder (instead of CSS). All formulations have been done from granules having a size of 1.0 - 2.0 mm from provider MEDICAL GROUP except for formulation R41**** using granules having a size of 0.5 - 1.0 mm from provider EPRUI and ***** formulation R42 using granules of beta-TCP having a size of 1.0 - 2.0 mm from provider EPRUI.

[0160] Figure 1 shows an example of granules of beta-TCP before (Figure 1A) and after (Figure 2A) being coated with a tricalcium silicate coating (formulation R5).

[0161] Example 2: General process for manufacturing a bone substitute material comprising both calcium silicate-coated calcium phosphate-based granules and uncoated calcium phosphate-based granules

[0162] For providing a bone substitute material of the invention comprising a mixture of porous uncoated and calcium silicate coated calcium phosphate-based granules, the following general process has been implemented.

[0163] First, porous calcium phosphate-based granules have been coated according to the process described in example 1.

[0164] Then, the obtained porous calcium silicate coated- granules are mixed with porous uncoated calcium phosphate-based granules with a weight ratio r2 between calcium silicate coated-granules and uncoated calcium phosphate-based granules of 1.

[0165] Several bone substitute materials have been manufactured by the process of the invention and are shown in the following Table:

[0166] The Applicant has observed a single unified structure for the bone scaffolds resulting from the hydration of the bone substitute materials BSM1, BSM2, BSM3 and BSM4 (see Figure 3).

[0167] Example 3: Porosity and mechanical properties of bone scaffolds of the invention

[0168] Process for manufacturing the bone scaffolds in vitro

[0169] For manufacturing the bone scaffolds in vitro, the granules of the bone substitute material of the invention as manufactured in example 1 or 2, are located first in a mold acting as a bone defect. Then, the granules of the bone substitute material were soaked in 150 pL of phosphate buffer solution (PBS) or in a calcium chloride solution (CaCh). After that, the mold was heated in a bain-marie at 37°C during Ih, 2h, 24h or 48h.

[0170] Figure 2 shows the state of the resulting bone scaffolds, when the initial bone material consists of: uncoated granules of P-TCP (Figure 2A), of P-TCP granules coated with a tricalcium silicate coating without any polymer (Formulation R9 of example 1, Figure 2B), and of P-TCP granules coated with a tricalcium silicate coating with polyvinylpyrrolidone (Formulation R14 of example 1, Figure 2C).

[0171] Figure 3 shows the state of the resulting bone scaffolds when the initial bone material consists of a mixture of P-TCP granules coated with a tricalcium silicate coating and uncoated P -TCP granules (corresponding to Formulations BSM1 to BSM4 of example 2).

[0172] It can be noticed from Figure 2 that when the bone substitute material consists of uncoated P-TCP granules, the hydration of said granules does not provide any bone scaffold. Especially, the P-TCP granules do not aggregate together and do not form a single unified structure. To the contrary, when the bone substitute material comprises P- TCP granules coated with a tricalcium silicate coating with or without polymer, the hydration of said coated granules leads to a bone scaffold in which all granules stick together to provide a unified structure.

[0173] It can also be noticed that bone scaffolds of Figures 2B and 2C have pores, which are useful for a bone scaffold to help body fluids permeate the material and cells to colonize the porous network as if it were real bone tissue.

[0174] It can also be noticed from Figure 3 that when the bone substitute material consists of a mixture of coated and uncoated P-TCP granules, the hydration of coated granules also leads to a bone scaffold having a unified structure, ensuring good adhesion between all granules, coated or not.

[0175] Porosity

[0176] The porosity of the resulting bone scaffolds has been also determined by Mercury Intrusion Porosimetry (MIP) with the apparatus MicroActive AutoPore V 9600.

[0177] The final porosity of the bone scaffold (“residual porosity rp”) has been compared to the porosity of the corresponding bone substitute material before hydration (“porosity ps”) for several formulations. The results are presented in the following table.

[0178] All bone scaffolds of the invention feature both a high residual porosity and the association of macroporosity with microporosity. This is a key factor in bone regeneration. Indeed, macroporosity plays an important role in osteoconduction, in particular promoting vascularization and local oxygenation. The microporosity provides a larger contact surface for protein adsorption as well as osteoblast adhesion.

[0179] Furthermore, the results show that for bone scaffolds obtained from a mixture of calcium silicate-coated granules with uncoated calcium phosphate-based granules have a porosity rate ranging from 60.4% to 62.2% after 24h of hydration and setting in a PBS solution at 37 °C.

[0180] By comparison, the porosity rate for the corresponding bone substitute materials before hydration, ranges from 60.7% to 68.8%.

[0181] Thus, all the bone scaffolds of the invention maintain a high porosity even after setting and hydration of the calcium silicate coating. Their porosity is suitable for osteoconduction and oesteoblast adhesion. In addition, the bone scaffolds obtained from the mixture of coated and uncoated calcium phosphate-based granules feature the highest residual porosity and thus, are the best candidates for cell migration.

[0182] Tensile strength - Pull-out test of a screw inserted in a material of the invention

[0183] The aim of this experiment is to show the ability of the bone scaffold of the invention to be used in dental anchor implant procedure. For this goal, first, a dental implant anchor model was designed and then a pull-out test from this model has been implemented.

[0184] Design of a dental implant anchor model

[0185] First, a circular mold, representing a clinical size alveolar defect, has been synthetized. Then, the inner volume of this mold has been filled with either (1) the granules of the invention corresponding to formulation R41 as defined above; (2) with a commercially available product Bio-Oss® or (3) with uncoated beta-TCP granules. A metal insert with thread (diameter: 3.0 mm; height: 5.5 mm) was introduced into the material filling the mold until the screw is completely anchored in said material.

[0186] 250pL of a phosphate buffer solution (PBS) were then added to the material inside the mold (i.e. formulation R41 of the invention, Bio-Oss® or uncoated beta-TCP granules) allowing its complete hydration within the circular mold.

[0187] The resulting complexes “mold + Formulation R41 + screw”, “mold + uncoated- P-TCP granules + screw”, “mold + Bio-Oss® + screw” were then placed in a water bath at 37°C, humidity 95% for 24 hours.

[0188] Pull-out test from the dental implant anchor models

[0189] In a second step, a pull-out test has been implemented on a mechanical test bench (MTS CRITERION C.43) with the three complexes “mold + Formulation R41 + screw”, “mold + uncoated-P-TCP granules + screw”, “mold + Bio-Oss® + screw” obtained herein above (see Figure 4 regarding the position of the complex on the mechanical test bench).

[0190] The tensile strength to anchor the screw to the complex has been measured for each bone scaffold. The results are presented in Figure 5.

[0191] Surprisingly, the bone scaffold of the invention features a tensile strength four times higher than the commercially available product Bio-Oss®.

[0192] Bone substitute material from uncoated B-TCP granules shows no mechanical strength after 24 hours of hydration in PBS, hence its tensile strength is close to 0.

[0193] Compressive strength and elastic modulus

[0194] The aim of this experiment is to show that the mechanical properties of the bone scaffold of the invention is similar to those of human cancellous bone. For this goal, thecompressive strength and the elastic modulus have been measured for bone scaffold of the invention obtained from formulation 41, formulation BSM3 or formulation R33 as defined above.

[0195] The compressive strength of human cancellous bone ranges from 1.5 to 10 MPa and the elastic modulus of human cancellous bone ranges from 50 MPa to 500 MPa.

[0196] The results, presented in Figures 6 and 7, show that all formulations of the invention enable to exceed the minimum values of compressive strength and elastic modulus of human cancellous bone. Consequently, the bone scaffolds of the invention are suitable mechanical properties to be used as bone substitute materials of human cancellous bone.

[0197] No mechanical setting was obtained from uncoated beta-TCP granules or from the product Bio-Oss®.

[0198] Example 4: In vivo bone scaffolds

[0199] Process for manufacturing the bone scaffolds in vivo

[0200] For manufacturing the bone scaffolds in vivo, the granules of the bone substitute material of formulation R41 as previously detailed, were used to fill a 6-mm bone defect of rat calvaria. Thirty-one rats were used and underwent 6 mm circular calvarial defect procedures as described below.

[0201] Method

[0202] First, each animal was anesthetized by the inhalation of 2% isoflurane and oxygen and then subcutaneously injected with a mixture of Ketamine (IMALGENE1000®: 40 mg / kg weight) and Medetomidine (DOMITOR®: 0.4 mg / kg animal weight). Then, a L-shaped incision was made. The skin and the periosteum were elevated and the parietal and frontal calvarium bones were exposed. The 6-mm-diameter defect, made using trephine, was centered on the intersection of the sagittal and coronal sutures. The 6 mm circular critical bone defect was rinsed using sterile saline and was either filled with the bone substitute material of formulation R41 or filled with [3-TCP(comparative control) or left unfilled (negative control). Finally, the periosteum and skin of the animals were closed with absorbable sutures. After surgery, all animals were given subcutaneous injections to relieve pain during the post-operative period. After 90 days post-implantation, the animals were euthanized. The calvarial bone was then excised, defleshed, and prepared for histological analyses.

[0203] In vivo CT-scans

[0204] For each animal, a longitudinal analysis of the radiopacity into the circular calvarial defects filled was performed immediately after surgery on day 0, and then at days 15, 30, 60 and 90 using a high-resolution, non-invasive, in vivo micro- scanner (micro-CT, Skyscan 1176, Brucker, Aartselaar, Belgium). The animals were anesthetized using inhaled isoflurane 2%.Histology and histomorphometryThe excised calvarial specimens were prepared for non-demineralized histological analysis. Each bone specimen was fixed, rinsed with water, cleared with xylene, and then embedded in methyl methacrylate. After polymerization, the calvarial bone blocks were oriented to radiography to obtain cross-sections cut parallel to the sagittal plane.Each section was then stained with Stevenel’s blue and Van Gieson's picro fuschin before visualization and acquisition using standard light microscopy. Images of histological sections were obtained using a light microscope (Keyence VHX2000) with the objective VH-Z20R / W / T and image analysis system (Image J software). The images that corresponded to the three successive cross-sections of each defect center were analyzed. To overcome the problem arising from the similarity of bone and material pixels on the digitized and calibrated images, the newly formed bone area and residual particles were delineated manually into the defined region of interest. The total area of newly formed bone was calculated by adding the areas measured and reported in mm2.

[0205] Results

[0206] The areas of new bone formation achieved within the bone defects are presented in Figure 8. The results show that the bone substitute material of the invention enables to achieve the highest area of new bone formation (0.8 mm2) within the bone defectcompared to an empty defect (negative control - 0.20 mm2) and compared to a bone substitute material consisting of P-TCP granules (i.e. without any calcium silicate coating - area of new bone formation less than 0.20 mm2).

[0207] This experiment evidences that the presence of the calcium silicate coating comprising tricalcium silicate, enables to provide a bone substitute material / scaffold with a higher osteoconductive potential compared to a bone substitute material made of P-TCP granules.

[0208] Furthermore, the analysis and quantification of the new bone area with the bone defects show that 95.4% of the new bone is mature bone when starting from the bone substitute material of the invention, whereas when starting from the bone substitute material made of P-TCP granules (with no calcium silicate coating), the amount of mature bone is only of 71.3%.

[0209] Thus, the bone substitute material of the invention enables to increase the amount of mature bone present in newly formed bones of 33.8% compared to the bone substitute material made of P-TCP granules (with no calcium silicate coating).

[0210] In conclusion, using the bone substitute material / scaffold of the invention, it is possible to both (1) generate a high amount of new bone within the bone defect and (2) to provide a new bone of which more than 90% is mature bone, that-is-to say bone having the same physiological, biological and mechanical properties of healthy bone.

Claims

CLAIMS1. A bone substitute material comprising:- at least one porous calcium silicate-coated granule comprising or consisting of:• a core corresponding to a porous granule made of at least one calcium phosphate-based compound having both micropores and macropores; the longest dimension of the inner volume of said micropores ranging from 0.1 pm to 100 pm and the longest dimension of the inner volume of said macropores being equal or higher than 100 pm; said dimension being measured by porosimetric analysis technique, preferably by mercury intrusion porosimetry; and• a calcium silicate coating embedding totally or partially said core; said calcium silicate coating comprising from 60% to 100% wt. of at least one calcium silicate compound to the total weight of said calcium silicate coating; and- optionally, at least one uncoated porous granule having both microporosity and macroporosity and made of at least one calcium phosphate-based compound.

2. The bone substitute material according to claim 1, wherein the porous calcium silicate-coated granule has a size corresponding to the longest dimension of said granule ranging from 50 pm to 50 000 pm; preferably from 50 pm to 3000 pm.

3. The bone substitute material according to claim 1 or claim 2, wherein the calcium phosphate-based compound is selected from natural bone, tricalcium phosphate (TCP), octacalcium phosphate (OCP), dicalcium phosphate anhydrous (DCPA), dicalcium phosphate dihydrated (DCPD), apatite, hydroxyapatite (HA), whitlockite and mixtures thereof; preferably from tricalcium phosphate (TCP), hydroxyapatite (HA) and mixtures thereof.

4. The bone substitute material according to claim 3, wherein the tricalcium phosphate is alpha-tricalcium phosphate or beta-tricalcium phosphate or mixture thereof; preferably is beta-tricalcium phosphate.

5. The bone substitute material according to any one of claims 1 to 4, wherein the at least one porous calcium silicate-coated granule further comprises at least one biocompatible polymer; preferably selected from polyesters, polysaccharides, polyvinylpyrrolidones and mixtures thereof; more preferably is selected from poly(lactic-co-glycolic acid) (PLGA), polyvinylpyrrolidone (PVP), ethyl cellulose (EC) and mixtures thereof; even more preferably is PVP.

6. The bone substitute material according to any one of claims 1 to 5, wherein the at least one porous calcium silicate-coated granule further comprises at least one additive; preferably selected from setting accelerators, radio-opacifiers, silica- based compounds and mixtures thereof; more preferably comprising at least one setting accelerator.

7. The bone substitute material according to any one of claims 1 to 6, wherein the weight ratio r2 between the amount of the at least one porous calcium silicate- coated granule and the amount of the at least one uncoated porous granule ranges from 0.05 to 2, preferably from 0.1 to 1.5.

8. A process for manufacturing a bone substitute material according to any one of claims 1 to 7; said process comprising or consisting of the following steps: i. Providing at least one porous granule having both micropores having the longest dimension of the inner volume of the pore ranging from 0.1 pm to 100 pm and macropores having the longest dimension of the inner volume of the pore being equal or higher than 100 pm; and made of at least one calcium phosphate-based compound; and ii. mixing said at least one porous calcium phosphate based-granule with at least one calcium silicate-based compound in a non-aqueous solvent; andiii. evaporating the non-aqueous solvent to provide at least one porous calcium phosphate-based granule coated, partially or totally, with at least one calcium silicate-based compound; and iv. optionally, mixing the at least one coated porous calcium phosphate-based granule obtained at step (iii) with at least one uncoated porous granule having both micropores having the longest dimension of the inner volume of the pore ranging from 0.1 pm to 100 pm and macropores having the longest dimension of the inner volume of the pore being equal or higher than 100 pm and made of at least one calcium phosphate-based compound.

9. The process according to claim 8, wherein steps (ii) and (iii) are simultaneously implemented.

10. The process according to claim 8 or claim 9, wherein step (ii) and / or (iii) is(are) implemented under reduced pressure ranging from 2.10'3hPa et 1013 hPa.

11. The process according to any one of claims 8 to 10, wherein the non-aqueous solvent comprises or consists of acetone, acetamide, acetonitrile, isopropanol, ethanol, dimethyl sulfoxide (DMSO), propanol, methanol, ethyl acetate or any mixtures thereof; preferably is ethanol.

12. The process according to any one of claims 8 to 11, wherein step (iii) is carried out at a temperature ranging from 20°C to 250°C, preferably from 50°C to 100°C, more preferably at a temperature of about 60°C.

13. A bone scaffold resulting from the hydration of the bone substitute material according to any one of claims 1 to 7.

14. The bone scaffold according to claim 13, wherein the hydration is implemented by contacting the bone substitute material according to any one of claims 1 to 7, with a body fluid, preferably when the bone substitute material is placed in a bone defect of a patient.

15. The bone scaffold according to claim 13 or claim 14, wherein the bone scaffold is porous; preferably the bone scaffold comprises interconnected pores with both microporosity and macroporosity.

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