IMPLANTABLE MEDICAL DEVICE FOR BONE REPAIR

Description

TECHNICAL FIELD OF THE INVENTION

[0001] The present invention relates to the field of implantable medical devices. It finds a particularly advantageous application in the field of bone repair following a loss of bone substance. The invention applies particularly to large volume bone defects of 2 cm 3 < to 15 cm 3 < . STATE OF THE ART

[0002] To date, autologous bone grafts remain the primary clinical solution for treating extensive bone loss and trauma, but they are hampered by several drawbacks, including limited availability, patient pain, additional healing time, and donor site morbidity.

[0003] Tissue engineering using synthetic scaffolds, bioactive factors and / or stem cells offers alternative therapeutic strategies and holds promise for bone regeneration.

[0004] However, these techniques are still not suitable for the repair of large bone defects (approximately 5 cm 3 < ), which remains difficult. In particular, for large bone defects, a structural synthetic scaffold may not be sufficient to allow complete regeneration.

[0005] Ceramics, particularly hydroxyapatite (HAP) and tricalcium phosphate (TCP) composites, are the most biomimetic scaffolds, but are brittle and exhibit variable biodegradability. In addition, they induce a basic level of bone formation. Metals such as titanium are interesting for their mechanical properties, but their high stiffness, greater than that of bone, generates stresses and they are not biodegradable.

[0006] The use of polymers has grown due to their versatility, tunable mechanical properties, and biodegradability. To date, polycaprolactone (PCL) and poly(lactic acid) (PLA) derivatives are the most widely used in bone tissue engineering.

[0007] Interestingly, the recent development of additive manufacturing allows for the design of custom-made 3D architectural scaffolds, adaptable to the size of the defect and easier to implement from a regulatory perspective. Polymers are particularly well-suited for the additive manufacturing of scaffolds. They can be manufactured in filament form and 3D printed using several techniques, including fused deposition modeling (FDM). The 3D architectural scaffold acts as a space filler that should be mechanically stable to allow bone growth inside the scaffold pores.

[0008] However, for large defect areas, a structural scaffold may not be sufficient to allow complete regeneration.

[0009] In these cases, stem cells or exogenous factors can be added to the scaffold to enhance regeneration. The use of stem cells in combination with scaffolds appears to have potential given their secretion of factors, but is more complicated to implement, as different steps are required to harvest the cells from patients, expand them in culture, and finally reimplant them into the patient.

[0010] As an alternative to stem cell implantation, the use of growth factors aims to recruit stem cells directly to the implantation site. To date, BMP-2 has been the most studied clinically approved protein due to its ability to directly target BMP receptors on the cell surface and trigger stem cell differentiation into bone cells.

[0011] The publication MacDonald ML et al. "Tissue integration of growth factor eluting layer by layer polyelectrolyte multilayer coated implants" Biomaterials. 32 (2011) 1446-1453 describes an implant based on β-TCP type ceramic, i.e. non-inert, covered with a film which can include BMP-2 in quantities of the order of 10 µg / mm 3< .

[0012] A previous study, Bouyer M, Guillot R, Lavaud J, Plettinx C, Olivier C, Curry V, et al. Surface delivery of tunable doses of BMP-2 from an adaptable polymeric scaffold induces volumetric bone regeneration. Biomaterials. 104 (2016) 168-81 showed that it is possible to repair a critical-sized femoral bone defect in rats smaller than 2 cm 3< by combining a hollow polymeric tube with an osteoinductive surface coating using a polyelectrolyte film as a carrier for BMP-2. An object of the present invention is therefore to provide an optimized implantable medical implant for the repair of a large volumetric bone defect.

[0013] Other objects, features, and advantages of the present invention will become apparent from the following description and accompanying drawings. It is understood that other advantages may be incorporated. SUMMARY OF THE INVENTION

[0014] To achieve this objective, according to one embodiment, an implantable medical device is proposed for bone repair of a loss of bone substance according to claim 1 comprising: a scaffold having a three-dimensional structure and advantageously comprising at least one polymer, a film comprising at least one protein of the Bone Morphogenetic Proteins (BMP) family, characterized in that the scaffold defines an interior volume comprising a three-dimensional mesh delimiting pores, advantageously the pores being open and interconnected, each pore having a largest dimension greater than 200µm, preferably of the order of 2000µm, the scaffold has a minimum porosity of 80%, and that the film coats the three-dimensional mesh and comprises polyelectrolytes.

[0015] This arrangement ensures homogeneous regrowth of the bone within the scaffold. Said scaffold ensures circulation of fluids, in particular blood, which will arrive within the scaffold when it is implanted within the bone defect. The porosity of the scaffold ensures that it can be advantageously covered homogeneously by the film. The film advantageously perfectly matches the three-dimensional mesh of the scaffold. The three-dimensional mesh is laminated by the film.

[0016] The scaffold is also advantageously mechanically strong enough to stand alone within the bone volume defect. In addition, its 3-dimensional architecture allows it to withstand mechanical stresses, particularly compression.

[0017] The porosity of the scaffolding is preferably such that the scaffolding can be homogeneously covered by the film. Advantageously, the film is deposited using an automated process. Thus, the film perfectly matches the surface of the three-dimensional structure of the scaffolding.

[0018] This optimized cooperation of the scaffold thanks to its 3-dimensional architecture and its porosity with the film ensures effective bone repair of bone volume defects and in particular those of critical size and large volume, for example between 2 cm 3 and 15 cm 3.

[0019] The use of the film as a coating allows the decoupling of the 3D scaffold architecture from the film which is osteoinductive.

[0020] The invention makes it possible to independently control the porosity of the scaffold and the loading of osteoinductive factors so as to modulate the release of said factors, ultimately ensuring homogeneous and high-quality bone growth. BRIEF DESCRIPTION OF THE FIGURES

[0021] The aims, objects, as well as the characteristics and advantages of the invention will emerge more clearly from the detailed description of one embodiment thereof which is illustrated by the following accompanying drawings in which: There figure 1 represents a 3D printed PLA scaffold according to the invention. The Figure 2A represents a macromagnifying phase contrast imaging of a device according to the invention comprising a 3D printed PLA scaffold covered with a film according to the invention. The Figure 2B represents a confocal phase contrast image of a section of a device according to the Figure 2Ato visualize the film-coated filaments of the scaffold. The Figure 2C represents a scanning electron microscopy (SEM) image of the scaffold struts. The 2D figure represents a scanning electron microscopy (SEM) image of a scratch made using a needle to assess the presence of the film on the scaffold. The Figure 2E represents the quantification, using a µBCA test, of BMP-2 loading in a 24-bilayer (PLL / HA) film, for two crosslinking levels EDC30 and EDC70, as a function of the initial BMP-2 concentration in the loading solution. Figure 2F represents the quantification of BMP-2 release by a 24-bilayer (PLL / HA) film as a function of the initially loaded BMP-2 concentration, for two levels of crosslinking EDC30 and EDC70. Figures 3A to 3Crepresent the kinetic quantification of bone formation using CT sections obtained with two levels of film crosslinking and two doses of BMP-2. EDC30 and EDC70 films loaded with BMP-2 doses (20 and 110 µg / cm 3 of scaffold) were compared. Two negative controls were added: a film-coated implant without BMP and loss of substance without complement. CT-scores calculated from CT sections as a function of time and corresponding exponential fits to the data for the EDC30 groups ( Figure 3A ) and EDC70 ( Figure 3B ). The plateau value (Bmax), the characteristic time (T) deduced from the adjustments and the quality of adjustment R 2< are given in the corresponding tables Table 2 and Table 3. The Figure 3Crepresents the amount of total bone volume as a function of time (D29, D50 and D90) and as a function of BMP-2 dose, for the two cross-linked films EDC30 and EDC70. Linear fits are also shown. The figure 4 represents 3D reconstructions obtained from CT sections showing the kinetics of bone regeneration for four representative conditions: negative control (Ctrl -) (film-coated scaffold without BMP-2 in the film), film-coated scaffold with a low dose of BMP-2 (BMP50, LD) and a high dose of BMP-2 (BMP110, HD) and bone graft (BG). The total length of the implant, corresponding to the distance from the lower edge of the mandible to the level of bone loss, is 4 cm, X direction in the diagram. Figures 5A to 5Eillustrate the quantitative analysis of bone formation kinetics monitored on CT sections for EDC30 films loaded with two doses of BMP-2. The film-coated scaffolds were loaded with BMP-2 at 50 (LD, n = 6) and 110 µg / cm 3< (HD, n = 5) and their bone regeneration capacity was compared to BG bone autograft, (n = 4). Figures 5A to 5C represent the box plot data of total bone volume ( Figure 5A ), poorly mineralized bone volumes ( Figure 5B ) and highly mineralized ( Figure 5C ) as a function of BMP-2 LD dose versus HD versus BG. * p < 0.05; ** p < 0.01. The Figure 5D represents CT scores as a function of time and corresponding exponential fits to the data for EDC30 films; the corresponding plateau value (Bmax), characteristic time (T) derived from the fits and the goodness of fit R 2< are given in Table 4. The Figure 5Erepresents the percentage of bone outside the implant (called "ectopic bone") as a function of time for LD and HD. The Figures 6A to 6E illustrate the quantitative analysis by micro-computed tomography of bone formation at 3 months (D91), after explantation. Figure 6A represents the 3D reconstructions of the negative control (ctrl-, EDC30 film-coated scaffold without BMP-2), the film-coated scaffolds containing low-dose LD and high-dose HD BMP-2, and the bone graft BG. Figure 6B illustrates a box plot representation of total bone volume. ** p <0.01. The Figure 6C is a box plot representation of the bone volume formed as a function of the total dose of BMP-2 per implant. Figure 6D is a box plot of bone mineral density (BMD) as a function of BMP-2 dose. * p <0.05 The Figure 6Eis a box plot representation of the homogeneity score measured for LD and HD.

[0022] In each box of the Figures 6B to 6E , the coefficient of variation of the data is given in %. * p <0.05; ** p <0.01.

[0023] THE Figures 7A and 7B illustrate the histomorphometric analysis of the histological sections taken after explantation.

[0024] There Figure 7A is a box plot representation of the amount of bone present on a histological section (BA) over the total area of ​​the section (TA).

[0025] There Figure 7B is a box plot representation of the percentage of bone within each histological section of the implant (S1, S2, S3).

[0026] The drawings are given as examples and are not limiting of the invention. They constitute representations intended to facilitate the understanding of the invention. DETAILED DESCRIPTION OF THE INVENTION

[0027] Before beginning a detailed review of embodiments of the invention, optional features that may optionally be used in combination or alternatively are set out below: According to one example, the scaffold is inert, also called bioinert. It does not have the ability to induce bone regrowth, i.e., it is not osteoinductive. The scaffold plays a role in supporting the film and passively supporting bone regrowth. The scaffold has an osteoconductive role in guiding bone regrowth.

[0028] According to one example, the scaffold comprises at least one polymer selected from the group consisting of polylactic acid (PLA) or polycaprolactone (PCL) or poly(glycolic) acid (PGA) or copolymers thereof. Preferably, the scaffold does not comprise a ceramic, in particular such as β-TCP (beta-tricalcium phosphate).

[0029] According to one example, the three-dimensional mesh has orientation angles between 0° and 120°, preferably -120° / +120°, preferably the three-dimensional mesh has a pore orientation of -45° / +45°C.

[0030] According to one example, the film comprises an amount of BMPs protein of between 0.01 mg / cm 3< and 0.2 mg / cm 3<, more precisely from 0.01 mg / cm 3< to 0.15 mg / cm 3< or even from 0.01 mg / cm 3< to 0.12 mg / cm 3<, for example from 0.017 mg / cm 3< to 0.072 mg / cm 3<.

[0031] Preferably, the BMP protein is the BMP-2 protein. This selection of the BMP dose is particularly interesting, because it is very significantly lower than the quantities of the state of the art, which is surprising compared to the idea that a higher dose would be beneficial for bone repair. The selected dose of BMP surprisingly made it possible to avoid possible side effects such as inflammation around the implant and the production of bone outside the implant (ectopic bone). This dose of BMP, significantly lower than the state of the art, is notably possible due to the characteristics specific to the scaffold such as its porosity and pore size.

[0032] The film includes polyelectrolytes.

[0033] In one example, the film is a crosslinked multilayer polyelectrolyte film.

[0034] According to one example, the film is crosslinked by a crosslinking agent, preferably 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), preferably at a concentration of between 30 mg / mL (EDC30) and 70 mg / ml (EDC70).

[0035] According to one example, the film is crosslinked at a concentration of between 30 mg / mL and 70 mg / mL of 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC). The crosslinking ensures the formation of covalent bonds within the film. According to one example, the device is suitable for bone repair of a volume of between 2 cm 3< and 15 cm 3< . Preferably, greater than 6 cm 3< , more preferably greater than 10 cm 3< .

[0036] According to one aspect, the invention relates to a method of manufacturing an implantable medical device as described above, characterized in that the scaffold is manufactured by 3D printing.

[0037] According to one example, the manufacturing method comprises a step of sterilizing the implantable medical device.

[0038] The implantable medical device according to the invention is intended for bone repair.

[0039] The medical device according to the invention is intended to be implanted in the human or animal body, more precisely in a bone defect. In particular, the device is particularly effective when the bone defect is a volume defect. According to one possibility, the defect is of critical size. Critical means that the bone defect cannot be filled spontaneously by the usual healing mechanisms alone. Advantageously, the device according to the invention is particularly suitable for large bone defects. A large bone defect means that the bone volume defect is greater than 2 cm 3< , more precisely greater than 6 cm 3< , more preferably greater than 10 cm 3< and advantageously up to 15 cm 3< .

[0040] The implantable device according to the invention comprises a scaffold and a film covering said scaffold and advantageously comprising a protein from the Bone Morphogenetic Protein (BMP) family. The implantable medical device according to the invention is also called an activated device or bioactive medical device or active or bioactive implant when it comprises BMP proteins.

[0041] The scaffold has a three-dimensional structure advantageously intended to be inserted into the bone defect to be repaired. The scaffold is intended to provide at least partial filling of the bone defect and preferably total filling.

[0042] A three-dimensional structure is defined as a scaffolding having a three-dimensional organization, also known as a 3-dimensional conformation. The 3-dimensional conformation goes beyond defining the perimeter of the scaffolding and also defines the structure of the scaffolding's internal volume.

[0043] The scaffold is advantageously complementary in shape to the bone defect to be repaired. The scaffold also provides support for the film and serves as a guide for bone regrowth.

[0044] Scaffolding is a solid. It is a geometric shape with 3 dimensions: height (Z), width (X), depth (Y). According to one possibility, the aspect ratio, that is, the ratio of one of the dimensions to the other, is less than 100.

[0045] For example, the scaffolding is a paving stone, for example, with a width (X) of 4 cm, a height (Z) of 3 cm, a depth (Y) of 1 cm, i.e. a volume of 12 cm 3< .

[0046] The scaffolding defines an interior volume. The scaffolding comprises a mesh delimiting pores. Advantageously, the mesh extends throughout the entire volume of the scaffolding. The mesh extends within the scaffolding in the interior volume. The mesh is three-dimensional. It extends in the interior volume along the three dimensions of the scaffolding.

[0047] The scaffolding is said to be porous and has a porosity of at least 80%. Preferably around 85%, more preferably around 90%. This porosity is due in particular to the mesh.

[0048] The porosity of the scaffolding is understood as the empty volume of the interior volume of the scaffolding.

[0049] This high porosity of the scaffold ensures optimal colonization of the scaffold, for example by progenitor cells and then by the bone over time, while providing a mechanical support role.

[0050] The scaffolding includes pores that are advantageously open, meaning that the pores are connected to each other. In this case, the pores form channels. The pores are then also referred to as being interconnected. This type of porosity allows the circulation of fluids within the scaffolding.

[0051] The pores of the scaffold have at least one dimension greater than 200 µm, preferably of the order of 2000 µm, plus or minus 10%. Preferably, the dimension is the largest dimension of the pore. According to one possibility, the largest dimension of the pore is understood to be in a plane parallel to the plane in which the filaments or trabeculae forming the mesh described below extend.

[0052] The scaffold according to the invention provides temporary mechanical support for bone repair while the bone forms and grows within the pores.

[0053] According to one possibility, the scaffold is biodegradable. According to one possibility, the scaffold is bioresorbable.

[0054] The scaffolding advantageously has interesting mechanical properties. The scaffolding is called polymeric scaffolding.

[0055] The scaffold is architected in 3 dimensions, for example, by 3D printing, particularly using fused deposition modeling (FDM). 3D printed scaffolds are particularly suitable for repairing large bone defects. The scaffold can adapt to the complex geometry of the bone defect to be filled. The term "architectural" means that the scaffold is manufactured, constructed, or arranged as an organized whole to give it a defined architectural character.

[0056] For example, the scaffold mesh is formed by trabeculae or filaments. The filaments or trabeculae are arranged in different orientations, in particular to control the porosity. The orientation can be 0° / 90°, 0° / 15°, 0° / 30°, 0° / 45°, 0° / 60° or -15° / +15°, -30° / +30°, -45° / +45°, -60° / +60°, 60° / 120°. The arrangement of the crossing filaments or trabeculae defines spacers and advantageously pores.

[0057] The filaments or trabeculae form the mesh of the scaffold. For example, the filaments or trabeculae are made of an inert material such as PLA, advantageously having a diameter of 400 µm in the scaffold. Preferably, the filaments are spaced apart, thus defining a filament spacing, i.e. a spacer of between 200 µm and 2.5 mm, more specifically between 1 and 2.5 mm.

[0058] The scaffold is particularly suitable for being covered with a film, preferably a polyelectrolyte film, preferably a multilayer film of polyelectrolytes.

[0059] Polyelectrolyte multilayer films are formed by crosslinking by condensation reaction of complementary groups located on the adjacent layer. EP 1 535 952 describes a practical method for producing crosslinked polyelectrolyte multilayer films.

[0060] Document EP 1 535 952 describes a crosslinked polyelectrolyte multilayer film, the manufacturing process of which comprises reacting pairs of layers of an anionic polymer with carboxylic groups and a cationic polymer with amino groups in the presence of a carbodiimide coupling agent.

[0061] The polyelectrolyte film is more preferably biocompatible. In particular, such biocompatible films can make any coated surface biocompatible. Therefore, such biocompatible materials when applied to biological tissues, especially inside the body, have the advantage of not irritating the surrounding tissues, not causing an abnormal inflammatory response, and not causing an allergic or immunological reaction.

[0062] The polyelectrolyte film advantageously comprises two or more layers, the film is therefore called a multilayer polyelectrolyte film.

[0063] Preferably, each additional layer has the opposite charge of the previous layer. The film architecture is precisely designed and can be controlled with an accuracy of 1 nm with a range of 1 to 50,000 nm, preferably from 100 nm to 30 µm and with precise knowledge of its molecular composition.

[0064] The number of layer pairs in a prepared polyelectrolyte multilayer film can vary within a wide range and depends on the desired thickness. In particular, the number of layer pairs can vary from 5 to 2000, preferably from 5 to 1000, more preferably from 5 to 100, preferably from 20 to 30, preferably 24.

[0065] As indicated above, the thickness of the film can generally vary from 1 nm to 50,000 nm. A film is considered a thick film when its thickness is greater than 300 nm. According to the invention and in a particular embodiment, the thickness of the film is from 500 nm to 20 µm, more preferably from 1 to 10 µm.

[0066] In a particular aspect of the invention, the cationic polymer of the film comprising an amino group is poly (L-lysine) (or PLL).

[0067] In a particular aspect of the invention, the anionic polymer comprising an amino group is hyaluronic acid or a salt thereof, such as sodium hyaluronan (also generally referred to as HA), or a mixture thereof.

[0068] The polyelectrolyte multilayer film is more preferably a PLL / HA film.

[0069] The polyelectrolyte multilayer film can advantageously be loaded with a protein such as an osteoinductive factor. A loaded film is of particular interest because it acts as a biomimetic reservoir for the storage and release of the protein. For example, controlled delivery of growth factor from a biomaterial surface can be achieved under very satisfactory conditions, as it allows the growth factor to be conveniently concentrated and released locally, and to be protected from degradation by enzymes in tissue fluids, particularly proteases.

[0070] The incorporation of proteins may take place by adsorption or diffusion or by coupling of said materials to at least one of the polyelectrolytes and adsorption of said polyelectrolyte.

[0071] The film forms a coating on the scaffolding. Preferably, the film covers the mesh. The mesh is coated with the film. The film will thus cover the mesh, more precisely, the mesh spacers are covered. Preferably, the entire mesh, therefore the scaffolding, is covered and therefore each spacer is covered. The mesh is said to be film-coated. The film covers the mesh evenly.

[0072] The film contains proteins from the bone morphogenetic protein (BMP) family, which are osteoinductive factors. As such, the film plays a biomimetic role by delivering BMP proteins and promoting bone regrowth. Biomimicry is the application of knowledge from biological models. The film loaded with BMP proteins advantageously exhibits an osteoinductive action. The homogeneous presence of the film on the mesh ensures homogeneous bone regrowth.

[0073] Osteoinductive factors are proteins of the Bone Morphogenetic Proteins (BMP) family, more preferably, BMP-2, BMP-7, BMP-4, BMP-9, BMP-6 or a mixture of BMPs or a heterodimer of BMPs or fragments of BMPs or peptides derived from these BMPs.

[0074] The following description is made with reference to BMPs but also applies to other osteoinductive factors.

[0075] The film forms a reservoir for BMPs, advantageously corresponding to a biomimetic reservoir. The BMP proteins are advantageously adsorbed within the film in a non-covalent manner, which allows in vivo release after implantation of the device.

[0076] The amount of BMP in the film is selected so that the amount of BMP within the device is between 0.01 mg / cm 3< and 0.2 mg / cm 3< , more precisely between 0.017 mg / cm 3< and 0.072 mg / cm 3< , more precisely between 0.02 mg / cm 3< and 0.08 mg / cm 3< . For example, the amount of BMP within a 12 cm 3< bone defect is between 240 µg and 1000 µg. The mass of BMP is given per unit volume of the scaffold. The amount of BMP is thus reduced by 1 / 20 to 1 / 75 compared to commercial collagen sponges.

[0077] Advantageously, the device according to the invention allows controlled spatial delivery of BMP proteins. The delivery of BMP proteins is perfectly controlled via the 3D architecture, i.e. the mesh of the scaffold covered by the film. This controlled spatial delivery ensures spatially localized bone regrowth within the volume of the scaffold. Bone regrowth in the bone defect to be repaired is very advantageously homogeneous.

[0078] Advantageously, this selection of the quantity of BMP ensures the absence of local inflammation, swelling, bone pseudocyst or bone resorption. Similarly, the quantity of BMP helps to limit ossification outside the implant.

[0079] The film is advantageously loaded with BMP proteins by soaking the scaffold in a solution having a defined concentration of BMP proteins.

[0080] According to one possibility, the amount of BMP proteins in the film can be modulated depending on the concentration of BMPs in the solution in which the entire scaffold is soaked.

[0081] According to one possibility, the amount of BMP proteins in the film can be modulated depending on the physicochemical properties of the film, including its thickness and degree of crosslinking.

[0082] According to one embodiment, the film is a multilayer polyelectrolyte film. Advantageously, the film is formed by alternating layers of poly(L-Lysine) (PLL) and hyaluronic acid (HA). The film is formed by a stack of pairs of layers, each pair comprising polymers of poly(L-Lysine) (PLL) and hyaluronic acid (HA). A pair is also referred to as a bilayer. For example, the number of bilayers is 24.

[0083] According to one embodiment, the film is crosslinked before loading with osteoinductive factors.

[0084] The implantable medical device according to the invention is advantageously sterilizable according to the requirements in force for this type of device while retaining its properties and structure. Preferably, the activated implantable medical device is sterilized while retaining its properties and structure. Sterilization can be carried out, for example, by gamma irradiation.

[0085] According to one aspect, the invention relates to a method for manufacturing an implantable medical device as described above comprising a step of architecting the scaffold. The architecting corresponds to the manufacturing of the scaffold. The manufacturing of the scaffold is done by 3-dimensional printing. Manufacturing by polymer printing makes it possible to produce an implant whose geometry (size, shape, porosity) is controlled in 3 dimensions.

[0086] According to one example, the manufacturing step comprises depositing PLA filaments, for example of approximately 400 µm in diameter in a predefined pattern such as for example -45° / 45° with a height of 200µm and a spacing distance of ±2 mm.

[0087] According to one example, the film is manufactured according to example 1. The film is manufactured by a step of successive layer deposition of, for example, PLL and HA on the scaffold.

[0088] Preferably, the step of manufacturing the film comprises a crosslinking step and a step of loading the film with osteoinductive factors. According to one example, the crosslinking step is carried out before the step of loading the film with osteoinductive factors.

[0089] According to one possibility, the manufacturing process comprises a step of sterilizing the implantable medical device obtained. Examples Example 1 : Preparation of PLA scaffolds, polyelectrolyte multilayer film (PEM) coating

[0090] Scaffolds, advantageously biodegradable and bioresorbable, 10 x 30 x 40 mm parallelepiped in medical grade poly(lactic) acid (Poly-Med, Inc, Lactoprene ®< 100M Monofilament 1.75 mm) manufactured by fused deposition (3DXP - One) were produced. figure 1illustrates a device according to the invention. PLA filaments of approximately 400 µm diameter were deposited in a -45° / 45° pattern with a height of 200 µm and a spacing distance of ± 2 mm. The scaffold has a porosity of 85% with fully interconnected pores. After fabrication and before coating with polyelectrolyte multilayer films, the scaffolds were stored in a moisture-free desiccator with silica gel.

[0091] Polyelectrolyte multilayer films were deposited using 0.5 mg / mL poly(L-lysine) (PLL, Sigma, France) and 1 mg / mL hyaluronic acid (HA, Lifecore, USA) and the DR3 dip-coating robot (Kirstein and Riegler GmbH) after hand-deposition of 5 mg / mL polyethyleneimine (Aldrich). The extent of film crosslinking was controlled by incubating the coated scaffolds in 30 or 70 mg / mL 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, Sigma, France). After UV sterilization of the implants, BMP-2 (InductOs, Medtronic) was post-loaded into the polyelectrolyte multilayer films at initial BMP-2 concentrations in the loading solution of 17, 46, or 92 µg / mL as previously described in Bouyer M, Guillot R, Lavaud J, Plettinx C, Olivier C, Curry V, et al. Surface delivery of tunable doses of BMP-2 from an adaptable polymeric scaffold induces volumetric bone regeneration. Biomaterials.104 (2016) 168-81 and Crouzier T, Ren K, Nicolas C, Roy C, Picart C. Layer-by-Layer films as a biomimetic reservoir for rhBMP-2 delivery: controlled differentiation of myoblasts to osteoblasts. Small. 5 (2009) 598-608. Finally, the osteoinductive coated scaffolds were rinsed, dried and stored away from moisture in a desiccator with silica gel until implantation. Example 2: Characterization of polyelectrolyte multilayer films and quantification of the amount of loaded BMP-2.

[0092] Fluorescence microscopy and scanning electron microscopy were used for the characterization of the film coating on the scaffold. The air-dried polyelectrolyte multilayer films coated on PLA scaffolds were imaged by scanning electron microscopy (SEM) using a FEI-Quanta 250 SEM-FEG under high vacuum at 15 keV using the Everhart-Thornley detector according to the publications Bouyer M, Guillot R, Lavaud J, Plettinx C, Olivier C, Curry V, et al. Surface delivery of tunable doses of BMP-2 from an adaptable polymeric scaffold induces volumetric bone regeneration. Biomaterials. 104 (2016) 168-81 and Crouzier T, Ren K, Nicolas C, Roy C, Picart C. Layer-by-Layer films as a biomimetic reservoir for rhBMP-2 delivery: controlled differentiation of myoblasts to osteoblasts. Small. 5 (2009) 598-608.For fluorescence observations, the film-coated scaffolds were labeled with PLL FITC< according to the publication Crouzier T, Sailhan F, Becquart P, Guillot R, Logeart-Avramoglou D, Picart C. The performance of BMP-2 loaded TCP / HAP porous ceramics with a polyelectrolyte multilayer film coating. Biomaterials. 32 (2011) 7543-54. imaged using a Leica Macrofluo fluorescence system (Z16 Apo) using a 0.8X objective and a Zeiss LSM 700 confocal microscope with a 10X objective.

[0093] The results obtained are visible in Figures 2A to 2D and allow visualization of a homogeneous coating of the scaffold. The scaffold mesh is coated with the film. The amount of BMP-2 increases with the initial concentration of BMP-2 in the loading solution, but it reaches a plateau more quickly for the EDC70 film ( Figure 2E). Regarding the release of BMP-2, it is higher for the EDC30 film, with the maximum percentage of BMP-2 released from the film being around 50% for EDC30 films compared to around 20% for EDC70 films ( Figure 2F ).

[0094] Quantification of BMP-2 initially loaded in the polyelectrolyte film was performed using a micro bicinchoninine acid (µBCA) assay and the percentage of in vitro release after several washes with physiological buffer (HEPES-NaCl), was determined by fluorescence spectrometry using BMP-2 CF< . The results obtained are visible at Figures 2E And 2FFor the quantification of BMP-2 loaded in the film-coated scaffolds, a µBCA assay was used for a low dose of BMP-2 (BMP20) while the Nanodrop (Thermofisher) was used for high concentrations. The concentration of BMP-2 in the loading solution was measured initially and after incubation with the film-coated scaffold. The loaded amount corresponds to the difference between these two values. It is also expressed as µg of protein per volume of scaffold (µg / cm 3< ).

[0095] Table 1. Quantification of the amount of BMP-2 loaded in the film-coated architectural scaffold. The total volume of the scaffold was 12 cm 3< and its surface area estimated from the design was 144 cm 2< . We targeted a BMP-2 dose (unit mass per unit volume of scaffold in µg / cm 3< ). The total amount of BMP-2 loaded was calculated for each implant and reported as “mass per volume of implant” (µg / cm 3< ). Table 1 Target BMP-2 dose (µg / cm 3< ) Total dose of loaded BMP-2 (µg / implant) Volumetric dose of loaded BMP-2 (µg / cm 3< ) Low dose (n = 2) 20 240 µg 20 High dose (n = 2) 110 870 µg 72,5 Low dose (n = 6) 50 326 ± 80 µg 27 High dose (n = 5) 110 1000 ± 64 83 Example 3: Repair of a critical-sized mandibular defect in vivo in a mini-pig.

[0096] To optimize bone regeneration, we performed a preliminary experiment on mini-pig mandibles. We initially screened under 4 different conditions (n ​​= 1), corresponding to two film crosslinking levels (EDC30 and EDC70) and two BMP-2 doses (BMP20 and BMP110). These doses expressed in µg of BMP-2 per cm 3< of scaffold are the targeted “volume” doses of BMP-2. Knowing the scaffold surface area (144 cm 2< ) and the amount of BMP-2 loaded in the polyelectrolyte film, we defined the BMP-2 concentration in the loading solution (in µg / mL) in which the scaffold was soaked. The amounts of BMP-2 actually loaded in the film-coated 3D scaffolds were also quantified (Table 1). Two negative controls were added: an empty defect without any implant and a defect with the film-coated implant crosslinked to EDC70, but without BMP-2.

[0097] The animals were healthy. There was no postoperative infection, implant rupture, or evidence of blood disorders. All surgeries were uneventful, and there were no surgical complications. For one implant, the anterior edge of the bone defect had to be recut to improve the fit with the implant. All titanium plates were stable and fixed to the native bone (no loosening or loss of screw adhesion). At explantation, it was completely impossible to macroscopically identify the active implant (loaded with BMP) from the native bone from bone reconstruction or scar tissue. Complete blood count, haptoglobin, and protein electrophoresis were measured to assess inflammation and hemostasis.Aspartate aminotransferases (AST), alanine aminotransferases (ALT), alkaline phosphatase (ALP), gamma-glutamine transferase (GGT), and bilirubin levels were measured to assess liver function. Serum creatinine and urea were measured to assess kidney function. Blood sample analysis revealed no abnormalities. We concluded that the scaffold with or without the film and / or BMP-2 did not cause general inflammation, nor any specific liver or kidney reaction due to this experiment regardless of the condition.

[0098] CT scans were performed during the follow-up period. For each scan, a CT scan score was given blindly by four clinicians. The results are illustrated in Figures 3A and 3B. Negative controls did not show bone formation throughout the follow-up period. All groups with BMP-2 showed bone regeneration, regardless of the extent of film crosslinking and BMP-2 concentration.

[0099] The CT-scan score was used to calculate a plateau value (Bmax) and a characteristic plateau time (T) by fitting an exponential function to the experimental data. Table 2 BMP (µg / cm3) B max (A.U.) τ (Days) R 2< 20 1.4 ± 0.0 21 ± 2 0.999 110 4.9 ± 0.7 60 ± 14 0.999

[0100] For EDC30 films, Table 2 above shows the calculated values, the scores increased steadily before reaching a plateau Bmax value which was higher for BMP110 than for BMP20 (4.9 ± 0.7 vs. 1.4 ± 0.0 respectively). The time to reach the plateau (τ) was approximately 3 times faster for the low dose than for the high dose (21 ± 2 vs. 60 ± 14 days). Table 3 BMP (µg / cm3) B max (A.U.) τ (Days) R 2< 20 3.3 ± 0.4 30 ± 12 0.995 110 4.2 ± 3.0 88 ± 111 0.971

[0101] In contrast, for the EDC70 films, whose calculated values ​​are shown in Table 3 above, the exponential fit to the data was poor for the highest BMP-2 concentration and there was no clear dose dependence. For the low dose, Bmax was 3.3 ± 0.4 and τ was 30 ± 12 days.

[0102] Quantification of total regenerated bone volumes from CT images for the different time points (D29, D50, D90) confirmed the dependence of bone repair on BMP-2 dose for EDC30 films but not for EDC70 films. The results are illustrated in Figure 3C. The amount of low-mineralized bone and high-mineralized bone was also plotted. The slopes of the linear fits were higher for the high-mineralized portion, suggesting that this type of bone has more influence on total bone volume than the low-mineralized one.

[0103] After 3 months, µCT acquisitions performed after scaffold explantation were used to calculate total bone volume (BV) and bone mineral density (BMD) for all samples. BV also showed a clear BMP-2 dose dependence, independent of the crosslinking level, while BMD was rather stable. µCT imaging of EDC70 films confirmed that there was no visible BMP-2 dose dependence for this film condition.

[0104] Overall, these data established the critical size of the mandibular bone defect, the difference in bone repair kinetics as a function of BMP-2 dose, and the influence of the film crosslinking level on the amount of newly formed bone. A clear BMP-2 dose dependence was demonstrated for EDC30 films. Example 4: The influence of BMP-2 dose on bone repair kinetics and the amount of highly mineralized bone.

[0105] Experiments were repeated with more minipigs per condition to quantitatively assess the effect of BMP-2 doses. We focused only on EDC30 films and selected two doses of BMP-2. We kept the highest dose BMP110 and increased the low dose of BMP-2 to BMP50 (n = 5 for BMP110 and n = 6 for BMP50). We added a bone graft as a positive control (n = 4) and a film-coated scaffold as a negative control (EDC30 film without BMP-2). In addition, an additional earlier time point (D16) was added for CT acquisitions.

[0106] Again, there were no surgical complications. In two cases, the bone graft was in two pieces due to the small size of the iliac bone (donor site), but in all cases the defect was completely filled. In three cases (two cases of bone grafting and one case of high-dose BMP-2), a small serous collection was found around the implant, and in one case a suppurative collection with a cutaneous fistula appeared at D86.

[0107] There figure 4shows three-dimensional CT representations over time for bone autograft, synthetic implants with both doses of BMP-2, and the negative control. At first glance, we observed that the bone autograft mineralized over time, but its total amount did not change. In contrast, the film-coated and BMP-2-loaded scaffolds induced bone repair over time, and mineralization was also visible. Total bone volume, low-mineralized bone volume, and high-mineralized bone volume were quantified from the CT scans ( Figures 5A , B, C). Total bone volume increased over time to reach a plateau, with this increase being significantly greater for the high BMP-2 dose condition compared to the low dose ( Figure 5A). The amount of bone graft volume remained constant. The amount of poorly mineralized bone increased rapidly over time for implants loaded with low and high doses of BMP-2, but with no statistical difference between the two doses ( Figure 5B ). In contrast, the amount of highly mineralized bone increased over time, being maximal at D51 ( Figure 5C ). It was significantly higher for the high dose than for the low dose of BMP-2.

[0108] CT-scan scores were plotted versus time and the data fitted with an exponential function ( Figure 5D). The score increased for both doses of BMP-2, but again with different characteristics: Bmax was lower for the scaffold with the low dose of BMP-2 than that of the high dose of BMP-2 (2.9 ± 1.2 vs. 5.2 ± 2.5). T was approximately two times lower for the low dose of BMP-2 (71 ± 40 days) than for the high dose of BMP-2 (142 ± 96 days). Table 4 BMP (µg / implant) B max (A.U.) τ (Days) R 2< 326 2.9 ± 1.2 71 ± 40 0.978 1000 5.2 ± 2.5 142 ± 96 0.977

[0109] We then analyzed the part of the regenerated bone that formed outside the implant ( Figure 5E ), what we here call “ectopic bone.” Initially high, the fraction of bone growing outside the implant quickly reached a plateau value around 28–35%, regardless of the BMP-2 dose. We noted that the dispersion of values ​​was slightly higher for the high BMP-2 dose, and also slightly higher at the D91 endpoint.

[0110] µCT analysis performed on day 90 after minipig sacrifice was used to further analyze the newly formed bone. The negative control confirms the critical size of the mandibular bone defect, and the bone grafts provide a positive reference value. For the low dose of BMP-2, bone formation was rare. The amount of bone increased gradually, and the newly formed bone completely filled the scaffold pores homogeneously. There was no evidence of excessive ectopic bone formation, even at the highest doses ( Figure 6A ). Bone volume was significantly higher for the high BMP-2 dose (mean value of 7.3 cm 3< ) than for the low BMP-2 dose (mean value of 4.9 cm 3< ) ( Figure 6B) and was also higher than for the reference bone graft. In addition, bone regeneration was more scattered with a low dose, with a coefficient of variation of 34% compared to 13% for the high dose. When plotting all newly formed bone volumes as a function of BMP-2 dose per implant, a linear correlation was found ( Figure 6C ). BMD was not significantly different for the different conditions ( Figure 6D ) and it was very homogeneous with less than 3.5% variation for each experimental condition. Moreover, the amount of bone developed outside the implant did not depend on the BMP-2 dose. Finally, the bone homogeneity score inside the scaffold was similar for low and high doses of BMP-2 ( Figure 6E ).

[0111] Overall, these data show that bone formation within the 3D architected scaffold is homogeneous, that there is a significantly dose-dependent bone formation of BMP-2. BMP-2 mainly influences the formation of mineralized bone and does not induce the formation of ectopic bone. Example 5: Bone homogeneity score.

[0112] The implant of defined dimensions (3 cm x 4 cm x 1 cm, total TV volume of 12 cm 3< ) was taken as region of interest (ROI). It was separated into ten slices of equal thickness along each axis (X, Y, Z). For each slice, the bone volume ratio (BVr = BV / TV) was calculated as the bone volume in a slice (BVs) divided by the volume of the slice of interest (corresponding to TV / 10). This quantification was done for each axis: the standard deviation (SD) of BVr was calculated and the homogeneity score was defined as the sum of the three SDs on the X, Y and Z axes.

[0113] Histological examination revealed the presence of mature bone with a characteristic Haversian structure in the BMP-2-loaded implants. The interface between native bone (HB) and newly formed bone (NB) was visible. Imaging under unpolarized and polarized light allowed better visualization of the Haversian canals and connections between osteocytes. In addition, the interface between native and newly formed bone was visible, as native bone had a more lamellar structure than newly formed bone. Some bridges were visible between the two types of bone, which may contribute to increasing the mechanical strength of newly formed bone. In some cases, especially for HD (high dose), the difference between native and new bone was not even visible. In the case of autologous bone graft (BG), native and grafted bone were in direct contact or separated by mesenchymal tissue.In the absence of BMP-2, only mesenchymal tissue (m) was formed. With a low dose (LD) of BMP-2, the amount of new bone was low and mesenchymal tissue was visible. The amount of bone per histological section (BA / TA in %) was quantified based on these images (. Figure 7A ). In agreement with CT and µCT quantifications, more bone was formed for HD, whose median value was similar to that of BG. Finally, the homogeneity of the newly formed bone within the 3D architecture was quantified ( Figure 7B ). Again, bone formation was similar for all three histological sections of each sample, proving the homogeneity of bone formation.

[0114] In this application, as an example, statistical analyses are performed as described below.

[0115] OriginPro (OriginLab), Excel (Microsoft Office), and R for Mac OS X (R Foundation for Statistical Computing, CRAN) were used for all analyses. Data were expressed as mean ± standard deviation. Nonparametric data were presented as median and interquartile range. Between-group differences were assessed by analysis of variance (ANOVA) and Bonferroni post-hoc analysis or Student's t-test. Between-group differences at p < 0.05 (*) and p < 0.01 (**) were considered significant.

[0116] The invention is not limited to the embodiments previously described and extends to all embodiments covered by the claims.

Claims

1. An implantable medical device for bone repair following a loss of bone substance comprising: - a scaffold having a three-dimensional structure and comprising at least one polymer, - a film comprising at least one protein from the Bone Morphogenetic Proteins (BMP) family, characterised in that the scaffold defines an internal volume comprising a three-dimensional mesh delimiting pores, the pores being open and interconnected, each pore having a largest dimension greater than 200 µm, the scaffold having a minimum porosity of 80%, and in that the film is a film comprising polyelectrolytes and coats the three-dimensional mesh.

2. The device according to the preceding claim, wherein the scaffold is inert.

3. The device according to any one of the preceding claims, wherein the scaffold comprises at least one polymer selected from a group consisting of polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL), or copolymers thereof.

4. The device according to the preceding claim, wherein the three-dimensional mesh has orientation angles between -120° and +120°.

5. The device according to any one of the preceding claims, comprising an amount of BMPs ranging from 0.01 mg / cm3 to 0.2 mg / cm3.

6. The device according to any one of the preceding claims, comprising an amount of BMPs ranging from 0.017 mg / cm3 to 0.072 mg / cm3.

7. The device according to any one of the preceding claims, wherein the film is a crosslinked multilayer polyelectrolyte film.

8. The device according to any one of the preceding claims, wherein the crosslinked film has a concentration ranging from 30 mg / mL to 70mg / mL of 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC).

9. The device according to any one of the preceding claims for bone repair of a volume between 2 cm3 and 15 cm3.

10. The device according to any one of the preceding claims, wherein the three-dimensional mesh comprises filaments with a diameter of 400 µm.

11. The device according to the preceding claim, wherein the filaments are spaced apart defining a spacing of the filaments between 200 µm and 2.5 mm.

12. A method for manufacturing an implantable medical device according to any one of the preceding claims, characterised in that the scaffold is manufactured by 3D printing.

13. The manufacturing method according to the preceding claim, comprising a step of sterilising the implantable medical device according to any one of claims 1 to 11.