Biomaterial comprising a fish by-product powder and chitosan, preparation process and use as an implant for bone reconstruction

Abstract

The invention relates to a biomaterial comprising 70 to 90%, preferably 80 to 85%, by weight, of a powder of at least one fish co-product selected from the group consisting of fish scales and fish bones, and 10 to 30%, preferably 15 to 20%, by weight, of chitosan; the shaping of this biomaterial into an implant, in particular by 3D printing using fused deposition modeling; and the use of the implant for bone regrowth, bone reconstruction, and / or the treatment of osteoporosis. Figure to be published with the abstract: Fig. 1

Description

Description Title of the invention: Biomaterial comprising a fish by-product powder and chitosan, preparation and use process use as an implant for bone reconstruction

[0001] — The present invention relates to the field of biomaterials and more particularly linking biomaterials usable in the medical field, more specifically in as implants for bone regrowth, bone reconstruction and / or treatment osteoporosis.

[0002] Following accidents or operations, part of the bone reconstruction is carried out naturally through the body's ability to regenerate, however if the damaged area is If it is too large, intervention is often necessary.

[0003] — Bone grafts: autografts, xenografts and heterografts remain the methods main bone reconstruction methods. However, these methods are not always applicable particularly in cases of disease. Furthermore, xenografts and heterografts are subject to the compatibility constraints inherent to the bone donor on the one hand and to the site of the recipient of the autologous bone on the other hand.

[0004] Faced with these difficulties, bone tissue engineering constitutes a pro- bone repair device. Consequently, this area is still the subject of extensive research is being conducted to develop new biomimetic materials suitable for to form a matrix for bone regeneration.

[0005] — The process of healing bone lesions involves a series of cel- complex processes, including the aggregation of mesenchymal stem cells, the Proliferation of osteoblasts, macrophages, and the extracellular matrix. Numerous factors play a role in this process. Primarily, osteoblasts proliferate and differentiate to form hydroxyapatite, which is the sediment of minerals. bone formation, and growth factors secreted by various cells regulate angiogenesis and promote the supply of oxygen and nutrients.

[0006] — Consequently, materials for repairing bone defects must be non-cy- Toxic, with little or no immunogenicity, they must present an interface conducive to the cell adhesion growth. Furthermore, these materials must ensure the rem- placement of the (damaged) bone by the new bone (resulting from bone regrowth) in a a set time.

[0007] — There remains a need for new materials that meet these requirements, which be compatible with market prices and relatively easy to shape so that they can be used as implants.

[0008] — Biomaterials according to the present invention comprising a co-product powder Fish and chitosan meet this need. Anterior artery The main biomaterials used as bone substitutes are "bioactive ceramics" and "bioactive glasses". It is also known to use biomaterials based on synthetic hydroxyapatite or natural calcium phosphate in which the hydroxyapatite used is extracted by alkaline or enzymatic hydrolysis techniques or by calcination in order to obtain, depending on the process chosen, hydroxyapatite in powder form on the order of micrometers or nanometers, the particle size being chosen according to their final use. As an example, patent application CN112121228 A describes an implant for filling bone cavities comprising nanofibers based on a cross-linked network of chitosan and polylactic acid on the surface of which hydroxyapatite nanoparticles are formed by sol-gel process. However, it is observed that hydroxyapatite-based biomaterials do not always have sufficient mechanical properties to be used for the reconstruction of load-bearing bones. Furthermore, biomaterials formed from powders derived from fish by-products offer the advantages of natural products in terms of biocompatibility and resource availability. However, these dense biomaterials comprise both inorganic and organic components, and this complex structure makes it difficult to combine them with other components and obtain new materials that remain stable during storage prior to implantation. Unexpectedly and advantageously, the inventors have shown that the use of a particular biomaterial comprising fish by-product powder and chitosan enables the preparation of a stable implant material for filling bone defects, bone regeneration, and bone tissue engineering. Description of the invention A first object of the invention relates to a biomaterial comprising, in relation to the total weight of the biomaterial, 70 to 90%, preferably 80 to 85%, by weight of a powder of at least one fish co-product chosen from the group formed by fish scales and fish bones and 10 to 30% by weight, preferably 15 to 20% by weight of chitosan. A second object of the invention relates to a method for manufacturing the biomaterial according to the invention comprising the following steps, in this order: -to have a powder of at least one fish co-product chosen from the group formed by fish scales and fish bones, said powder having a particle size ranging from 40 to 315 microns; -have chitosan available; -prepare a mixture comprising 70 to 90%, preferably 80 to 85%, by weight of powder of at least one fish co-product and 10 to 30% by weight, preferably 15 to 20% by weight of chitosan; - stir the mixture; -dry the resulting precipitate; - to recover the biomaterial obtained. A third object of the invention relates to a composition comprising a biomaterial according to the invention and at least one agent selected from drugs, in particular antibiotics, bisphosphonates, thermoplastic polymers, in particular selected from polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), polybutylene succinate (PBS), polyhydroxyalkanoates (PHA), polycaprolactone (PCL), polybutylene-co-adipate terephthalate (PBAT). A fourth object of the invention relates to an implant comprising a biomaterial according to the invention or obtained according to the process according to the invention or the composition according to the invention, and its use for bone regrowth, bone reconstruction, and / or the treatment of osteoporosis. A fifth object of the invention relates to a method for manufacturing the implant according to the invention, advantageously comprising a 3D printing step of the biomaterial or composition. In particular, the method for manufacturing the implant according to the invention comprises the following steps, in this order: -mixture of the biomaterial according to the invention, with at least one thermoplastic material selected from polylactic acid, poly(lactic-co-glycolic acid), polybutylene succinate, polyhydroxyalkanoates, polycaprolactone, polybutylene-co-adipate terephthalate; at least one porous agent and optionally at least one agent selected from drugs in particular antibiotics, bisphosphonates; - shaping of the mixture by 3D printing using fused filament deposition, - Removal of pore-forming agents preferably by leaching, -possibly drying of the implant obtained. Fish by-products such as fish scales and fish bones contain an inorganic part comprising a particular calcium phosphate: hydroxyapatite, associated with other minor minerals and in trace amounts, as well as an organic part including collagen. Without wanting to be linked to any particular theory, it seems that combining a fish by-product powder, including hydroxyapatite as well as other minerals and collagen, with chitosan allows for the production of a biomaterial. exhibiting particularly advantageous properties for use as a bone implant material. In particular, the biomaterial displays good biocompatibility: no rejection or inflammation has been observed, it allows for the differentiation and proliferation of osteoblastic cells, and thus promotes osteogenesis. The quantities of hydroxyapatite, collagen, and minerals (trace elements) appear to be particularly beneficial for the intended applications. Indeed, the use of fish by-products according to the invention makes it possible to maintain a balance between the different organic and inorganic components. However, this balance could be disrupted in prior art applications in which hydroxyapatite extracted from a natural source is used. Benefits The biomaterial according to the invention allows for the preparation of an implant with a shape adapted to the bone defect to be repaired. The biomaterial according to the invention is non-cytotoxic and biocompatible; it can be implanted conventionally and promotes osteogenesis at the implantation site by facilitating the adhesion of bone cells. It is also bioresorbable, as it is progressively replaced by bone tissue as bone regrowth progresses. Its preparation is easy and can be carried out using readily available, commercially accessible devices at a reasonable price. In subjects, human or animal, suffering from osteoporosis, the biomaterial according to the invention makes it possible to reduce osteoporosis. Description of the figures [Fig.1] presents the structural analysis by X-ray diffraction (XRD) of the implant in Example 2 before and after implantation, compared with the diffractogram of healthy rat bone. [Fig.2] presents the structural analysis by X-ray diffraction (XRD) of the implant in Example 3 before and after implantation, compared with the diffractogram of healthy rat bone. [Fig.3] presents the results of alkaline phosphatase (ALP) assay between 15 and 90 days in subjects where the implants of examples 2 and 3 were inserted, compared to controls. [Fig.4] shows a cross-section of a femoral condyle from a healthy rat (left) and a cross-section of a femoral condyle from an ovariectomized rat (right). [Fig.5] shows sections of femoral condyles of a rat after 90 days of implantation of an implant according to example 2 (left) and of an implant according to example 3 (right), the rats having been ovariectomized 2 months earlier. [Fig. 6] presents a comparison between the 90-day histological analyses of an implant according to example 3 (right) vs. a comparative implant example 4 without chitosan (left). Other aspects, advantages, and properties of the present invention are presented in the description and examples that follow. Detailed description of the invention Biomaterial As already described, the biomaterial according to the invention comprises 70 to 90%, preferably 80 to 85%, by weight, of a powder of at least one fish co-product selected from the group formed by fish scales and fish bones and 10 to 30% by weight, preferably 15 to 20% by weight of chitosan. The main active ingredient of the biomaterial includes a powder of at least one fish co-product and chitosan. Advantageously the mass ratio between fish co-product powder and chitosan ranges from 4.5 to 5.5, preferably from 4.7 to 5.1. Fish by-product powder Usable fish by-products are selected from the group consisting of fish scales, fish bones, and mixtures thereof. Fish bones can be obtained from all species of fish. On the other hand, fish scales are generally chosen from the scales of sardines, salmon, carp, tilapia, sea bass, sea bream and preferably sardines. Advantageously, fish scales are used without processing, in particular they are not decellularized. Fish co-products can also include a mixture of fish scales and fish bones; mixtures in any proportion are usable. Advantageously, the fish co-product powder has a particle size ranging from 40 to 315 microns (106 meters). It appears that the choice of this particular particle size allows for good interaction between the different components of the mixture, namely the inorganic part, collagen and chitosan, which leads to a stable, homogeneous composite compatible with adequate shaping for use as a bone implant. The powder used according to the invention is obtained from fish by-products selected from the group formed by fish scales and fish bones. These fish by-products comprise as a major component a particular calcium phosphate: hydroxyapatite (HA) with the chemical formula [Ca,O(PO4)(OH)] which has a hexagonal network. Thus, the fish co-product used according to the invention comprises calcium and phosphorus in a calcium / phosphorus Ca / P ratio by weight ranging from 1.8 to 2.5, preferably ranging from 2.0 to 2.5. In the particular case of fish co-products used according to the invention, hydroxyapatite is associated on the one hand with other minerals, the main one being magnesium Mg, and on the other hand with collagen. The fish co-product used according to the invention comprises collagen in an amount ranging from 20% to 40% and preferably from 20% to 35% by weight relative to the total weight of the fish co-product. The fish co-product used according to the invention also includes magnesium (Mg) in an amount ranging from 0.30% to 0.60%, and preferably ranging from 0.44% to 0.46% by weight relative to the total weight of the fish co-product. The fish co-product used according to the invention also comprises the following elements by weight relative to the total weight of the fish co-product: zinc (Zn) in an amount ranging from 0.010% to 0.011%, strontium (Sr) in an amount by weight relative to the total weight of the fish co-product ranging from 0.032% to 0.033%. Fish co-product powders usable according to the invention are available on the market, they are notably marketed by the company Abyss Ingredients under the names Calcilyss and Phosphymer. According to another alternative, the fish co-product powder usable according to the invention can be prepared according to the following preparation process. Thus, the present invention also relates to a method for manufacturing fish co-product powder by implementing the following steps, in this order: -have at least one fish by-product chosen from fish scales and fish bones, - Place the fish co-product in a water bath at a temperature ranging from 50°C to 100°C for a period ranging from 5 to 120 minutes, -if necessary, drain the fish by-product, -dry the co-product, -grind the co-product to obtain a powder with a particle size ranging from 40 to 315 microns, -recover the fish co-product powder obtained. Advantageously, when fish bones are used to obtain fish co-product, then the process for manufacturing fish co-product powder may include a preliminary step of mechanically separating the fish flesh stuck to the bones. Advantageously, when fish scales are used to obtain fish co-product, then the process for manufacturing fish co-product powder may include a preliminary step of mechanically recovering the scales. Chitosan Chitosan is a natural biopolymer of marine origin consisting of bonds of B-(1—4)-2-acetamido-d-glucose and B-(1—>4)-2-amino-d-glucose units, obtained by deacylation of chitin. According to a preferred mode, the chitosan used in the present invention has a degree of deacetylation (DD) of 75% and a molecular weight between 190 and 310 kDa. Generally, chitosan is in powder form with particles smaller than 1000 microns and a density ranging from 0.45 to 0.65 g / m³*. Use The present invention relates to an implant comprising a biomaterial according to the invention or obtained according to the process according to the invention or comprising a composition according to the invention and its use for bone regrowth, bone reconstruction, bone filling and / or the treatment of osteoporosis. As shown in Figures 5 and 6, the implant acts as a matrix for bone regrowth and reconstruction. It is advantageously biodegradable and bioresorbable, as it is gradually replaced by bone tissue as bone regrowth progresses, i.e., as newly formed tissue is created. The implant therefore allows for 100% filling of the bone defect. According to a first embodiment, the implant comprises, by weight relative to the total weight of the implant, 90 to 100% of a biomaterial according to the present invention and 0 to 10% of at least one agent selected from bisphosphonates and drugs, in particular antibiotics. According to this first embodiment, the implant may be essentially composed of the biomaterial according to the present invention. According to this first variant, the implant is used in the form of a loose or compacted powder. The implant in powder form can be advantageously used in cranio-maxillofacial surgery. As shown in paragraph 2.3 below, the implant according to this first variant allows for rapid osseointegration which is particularly desirable for young subjects, and for athletes. According to a second variant, the implant comprises, by weight relative to the total weight of the implant, 30 to 50% of a biomaterial according to the present invention, 50 to 70% of a thermoplastic material selected from the group consisting of polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), polybutylene succinate (PBS), polyhydroxyalkanoates (PHA), polycaprolactone (PCL), polybutylene-co-adipate terephthalate (PBAT) and 0 to 10% of at least one agent selected from drugs, in particular antibiotics and bisphosphonates. According to this second variant, the implant is advantageously obtained by an additive manufacturing method such as 3D printing by fused filament fabrication (FFF). for "Fused Filament Fabrication." The implementation of this variant includes the initial formulation step by melt-mixing the biomaterial in powder form, a biocompatible thermoplastic matrix, and at least one porogenic agent using a twin-screw extruder. The formulation is then transformed into filaments conforming to fused filament fabrication (FFF) technology standards using a single-screw extruder equipped with a profile spinning line. The filament, generally 1.75 mm or 2.80 mm in diameter, is then shaped by 3D printing and subsequently undergoes a porogenic agent removal treatment, including aqueous leaching, possibly followed by oven drying. During the implementation of the additive manufacturing method, 20 to 40% by weight, and preferably 25 to 35% by weight, of thermoplastic material, 30% to 65% by weight, and preferably 42 to 57%, of at least one porogenic agent, and 15 to 25% by weight of the co-produced biomaterial according to the present invention are used. Generally, two to three organic or inorganic porogenic agents are used. The water-soluble porogenic agents create a porous structure after their removal from the formulation, generally through a leaching step in an aqueous medium. Generally, organic porogen agents are water-soluble polymers chosen from polyvinyl alcohol (PVA) and polyethylene glycol (PEG) with molecular weights between 1000 g / mol and 15,000 g / mol and preferably between 4000 g / mol and 10,000 g / mol. As an inorganic pore-forming agent, sodium chloride (NaCl) is generally used in powder form with a particle size ranging from 10 to 700 µm and preferably from 100 to 400 µm. As shown in paragraph 2.3 below, this second variant of the implant allows for slower osseointegration, still incomplete 3 months after implantation, a duration corresponding to the end of the study. The following examples are intended to illustrate the invention without limiting its scope. Examples - Preparation of co-product powders of poi P of scales d î The collected sardine scales are placed in a water bath at 90°C for 30 minutes, then drained before being frozen. After thawing, 100 g of scales are placed in an oven for 60 hours at 94°C. The dried product is then ground and sieved to obtain a sardine scale powder with a particle size of less than 315 microns. This yields 46 g of powder. 2. White fish bone powder The bones of white fish from the cod family (Gadidae) are collected and then subjected to a process designed to separate the flesh from the bones: mechanical separation followed by immersion in a hot water bath at 90°C for 30 minutes and then centrifugation. The treated bones are then drained on a conveyor belt and frozen. After thawing, 100 g of bones are placed in an oven for 60 hours at 94°C. The dried product is then ground and sieved to obtain a sardine scale powder with a particle size ranging from 40 to 315 microns. This yields 43 g of powder. Table 1 presents the chemical compositions of the scale and edge powders obtained above. The quantities of the different elements are obtained by inductively coupled plasma atomic emission spectrometry (ICP-OES) analysis using an iCAP 7000 instrument (ThermoFisher Scientific), with the exception of the collagen protein (collagen) content, which is obtained by nitrogen determination. [Table 1] Hydroxyapatite [Synthetic mature bone (rat)] Scale powder, bone powder of sardines and white fish, 29.3 24.2 Jo [Not measured] 29.3) | 5.18 279 “35.98 20.01 [1227 [16 16.86 9.46 2.05 2.13 2.12 [Not measured -Jo,17 Jos (0.011 [Not measured [0.004 [0.033 0.032 [Not measured - [0.014 0.004 [Not measured |0.48 -|0475 The scale powder used according to the invention has a Ca / P ratio close to that of mature healthy bone. Furthermore, the scale powder comprises the main minerals present in mature bone in significant quantities. B. Preparation and characterization of biomaterials Example: preparation of an implant in powder form 24.9 g (83 wt%) of sardine scale powder was dispersed in 50 mL of acetic acid. The co-product powder solution was then added dropwise to a previously prepared chitosan solution (5.1 g chitosan (17 wt%) in 200 mL of acetic acid). The resulting mixture was stirred for 24 hours using a magnetic stirrer at 1200 rpm. After removing the supernatant, the mixture was frozen and then solidified in liquid nitrogen for 10 minutes. It was then lyophilized at -60°C for 24 hours to remove solvents. The resulting biomaterial was subsequently immersed in 0.2 M sodium hydroxide for 2 hours and then washed with distilled water to completely neutralize any acetic acid radicals. The resulting biomaterial was mixed at room temperature with 20% by weight of polyvinyl alcohol, then refrozen and freeze-dried for 24 hours to completely remove the water.This yields 0.068 g of an implant in powder form. Example 2: Preparing an implant The biomaterial obtained in the example was inserted into a brass mold with two compartments and two pistons. The powder was compacted by applying a force of 15 kg using the pistons to obtain cylinders measuring 3 mm by 3 mm. A 0.068 g implant in the form of a compacted powder, 3 mm in diameter and 3 mm in height, was obtained. This implant was then used for implantation in femoral condyles for in vivo study. È =; inn of a 1 manufacturing itiv A thermoplastic biocomposite formulation is prepared by melt mixing using a TSA (Luisago, Italy) FSCM21 co-rotating twin-screw extruder equipped with 21 mm diameter screws with an L:D ratio of 40 and a 3-rod extrusion die with a diameter of 4 mm. All formulation components are fed into the main hopper of the twin-screw extruder using two Brabender (Duisburg, Germany) gravimetric feeders: DDW-MD3-DDSR20 and DDW-M-DSR28. The first feeder is dedicated to dosing a premix of powdered components, including fish by-products, chitosan, and pore-forming agents consisting of a water-soluble PEG 8000 polymer and a water-soluble NaCl salt. The second dosing unit is dedicated to dosing the polylactic acid (PLA) biopolymer in granular form. The combined flow rate of these dosing units is set at 2.4 kg / h.The temperature profile across the eight heating zones is as follows: Z1.7 = 160°C, Z2 = 175°C, and Z4 = 180°C. The screw rotation speed is set at 400 RPM. The rods are cooled in ambient air on a conveyor belt as they exit the die, then granulated using a granulator. with rotating knives. The composition of the formulation used is given in Table 2 below. [Tables 2] |PLA |PEG 8000 |NaCl |Co-Product |Chitosan 1082 — [2154 = [1476 3.94 (Density (g / L) |L25 L20 2.16 L82 L40 Ratio 30 8 2 166 34 Mass Ratio 38.04 1082 3154 Jiaz6 3.04 Volume The formulation is then transformed into 1.75 mm diameter filaments using a SCAMEX (Saumur, France) single-screw extruder with a 20 mm diameter and a length-to-diameter ratio of 20, equipped with a 2 mm diameter annular die and a drawing line, dimensional control, and winding. The temperature profile across the four heating zones is as follows: Z1 = 180°C, Z34 = 170°C. The screw rotation speed is set at 15 RPM and the drawing line speed is set at 3.8 m / min. The produced filament is then used in FFF 3D printing with an Emotiontech u-Delta Reworks printer (Toulouse, France) equipped with a 0.8 mm diameter nozzle. The implant consists of a cylinder 3 mm in diameter and 3 mm high. The 3D geometry of the implant is modeled using Solidworks software from Dassault Systèmes. The output 3D file format is STL. The printing process is programmed from the STL file using Repetier Host software from Hot-World GmbH & Co (Willich, Germany) with the Slic3r slicing algorithm (open source). The printing is performed at a nozzle temperature of 225°C and a build plate temperature of 75°C. The layer height is set to 0.6 mm and the layer width to 0.9 mm. The printing speed is set to 6 mm / s. The implant obtained by FFF 3D printing is then post-treated by aqueous leaching to remove porogenic agents. To do this, the implant is immersed in demineralized water and subjected to an ultrasonic bath at 45°C for 100 minutes. It is then rinsed with demineralized water. The implant undergoes five cycles of this post-treatment before being vacuum-cured at 45°C for 96 hours. im A chitosan-free implant is obtained using the same process as shown in Example 3, based on the formulation in Table 3 below. [Tables 3] |BLA |PEG 8000 (NaCl of 10.92 31846 — [85 [Density (e / L) |L25 Mass Ratio 30 Volumetric Ratio 39.30 C. Biological tests _ ity Cytotoxicity was measured by MTT. MTT is a yellow tetrazolium salt, 3-[4,5-di-methylthiazol-2-γ1]-2,5-diphenyl tetrazolium bromide (Sigma-Aldrich Corporation, St. Louis, USA). In the presence of metabolically active live cells, MTT is cleaved by succinate dehydrogenases of the mitochondrial respiratory chain to form purple formazan crystals. After solubilization of the crystals with DMSO (dimethyl sulfoxide), the medium changes from yellow to purple, and the absorbance of the resulting colored solution is measured at 570 nm by spectrophotometry. The intensity of the color is proportional to the number of live cells and provides an indication of the percentage of cell viability. The MTT assay allows for the measurement of the metabolic activity of cells and thus the quantification of the cytotoxic effect of co-products after 72 hours of contact. The cytotoxicity of the biomaterials in Examples 2 and 3 was assessed in vitro on three cell types involved in bone regeneration: fibroblasts (L929), SaOS2 osteoblasts (human osteosarcoma cells), and bone marrow cells (BMC). Both biomaterials showed good tolerance and no toxicity. The biomaterials according to the invention are biocompatible. 2. Tests in vi To evaluate bone formation after implantation of the biomaterial according to the invention, the following protocol was implemented: PB the experiment Six-month-old Wistar rats with an average weight of 230 ± 20 g were randomly assigned to the six groups (af) below for physicochemical and biological evaluations: - group a: control (-): 8 healthy rats (no treatment); - group b: control (+): 6 ovariectomized rats; - group c: gap empty: 6 ovariectomized rats with creation of a bone defect without insertion of an implant; - group d: insertion of the implant from example 2 into the gap created in the 2 femurs by rats: 12 rats implanted; - group e: insertion of the implant from example 3 into the gap created in the 2 femurs by rats: 16 rats implanted; - group A: comparative: insertion of the comparative implant of example 4 obtained from fish co-product powder by additive manufacturing in the gap created in the 2 femurs by rats: 16 rats implanted. The conditions for raising animals in pet shops that complied with ethical recommendations were as follows: - Temperature: ambient temperature varies between 23 and 30 °C; - light: the light intensity cycle required for laboratory animals is 12h / 12h (day / night cycle); - the animals had free access to water and food that was balanced from an energy point of view. After acclimating the rats to the conditions of the animal facility, the implants were inserted into the 2 femoral condyles of each rat (groups df) and then samples of the implanted femurs were taken after 15 days, 30 days, 60 days and 90 days. The following physicochemical analyses and biological evaluations were carried out. 2.) XRD Analysis Figure 1 shows the structural analysis by X-ray diffraction (XRD) of the implant from example 2 (group d) before and after implantation. A PA-Nalytical X'Pert PRO diffractometer was used. Measurements were performed over a range of 5 to 70° (20). A 2-hour acquisition was performed per sample. The resulting XRD patterns were then analyzed and compared to a healthy rat bone. The numbering of the following curves corresponds to the curves from bottom to top: the curve | corresponds to the lines of the sardine scale powder prepared in paragraph A (reference); curve 2 corresponds to the lines of the implant after 15 days (D15); Curve 3 corresponds to the lines of the implant after 30 days (D30); Curve 4 corresponds to the lines of the implant after 60 days (D60); curve 5 corresponds to the lines of the implant after 90 days (D90); Curve 6 corresponds to the lines of the bone of a healthy rat. On curve 2 (J15), two lines at 10° and 20°, characteristic of chitosan, are observed. These lines indicate the incorporation of the fish by-product powder into the chitosan matrix. These lines are no longer clearly visible on curves 3 to 5, leading to the conclusion that the chitosan does not remain at the implantation site. The chitosan thus released can contribute to the local treatment of osteoporosis caused by ovariectomy. Figure 2 shows the structural analysis by X-ray diffraction (XRD) of the biomaterial from Example 3 (Group e) before and after implantation. The following curves are numbered from bottom to top: the curve | corresponds to the lines of the implant after 15 days (D15); curve 2 corresponds to the lines of the implant after 30 days (D30); Curve 3 corresponds to the lines of the implant after 60 days (D60); Curve 4 corresponds to the lines of the implant after 90 days (D90); Curve 5 corresponds to the lines of the bone of a healthy rat. Figures 1 and 2 show the growth of biological apatite closely resembling the composition of healthy bone: indeed, the implant growth curves at days 60 and 90 are very close to those of healthy bone, reflecting progressive bone maturation. The evolution of the minerals in the implants of examples 2 and 3 was also determined by inductively coupled plasma atomic emission spectrometry (ICP-OES) using an iCAP 7000 instrument (ThermoFisher Scientific). These values ​​are presented in Table 4. [Tables 4] f (% |P (% wt) |Ratio # (% 5 (% " (% wt) Ca / P(% |wt) wt) wt wt) pre Jo 2151 — |966 223 —Jo3o oo: = |o.005 healthy (390 [2283 [1001 |208 029 0.033 |0.005 Rat bone Lo [2133 9.53 223 —Jo31 029 = |o.005 Re 190 |20.93 [9.34 224 [032 |o029 = |0.007 o bet po po [es Joss 247 031 joois l0.027 ex2 [ro [2088 [9.14 228 —|jo3s Jo026 |0.004 i Jo |1L6 34 341 0.126 |o.003 0.014 ex.3 [100 [1397 6272 223 Jo227 |o007 Jo.008 Table 4 shows that the mass Ca / P ratio evolves over time to approach a value corresponding to that of healthy rat bone: the phos- equilibrium The phocalcic system is not disturbed and the mineral profiles are not altered. 2.2 ALP Assay Figure 3 presents the results of the alkaline phosphatase (ALP) enzyme assay between 15 and 90 days in the groups where the implants from examples 2 and 3 were inserted, groups d and e respectively, compared to group a (negative control) and group b (positive control). Alkaline phosphatase (ALP) is a marker of bone remodeling. This enzyme, present in osteoblasts (bone cells), ensures the deposition of hydroxyapatite crystals and is also associated with increased osteoblast activity. ALP levels were measured using the kinetic method with a Beckman-Coulter analyzer. Alkaline phosphatase enzymes, in the presence of diethanolamine at pH 10.3, a transphorylant, hydrolyze nitrophenyl-4-phosphate. The catalytic activity of ALP is determined by measuring the rate of appearance of one of the reaction products, nitro-4-phenol, which exhibits a maximum absorption at 410 nm. Values ​​are obtained in IU / L. Figure 3 shows that the results obtained for groups d and e are significantly higher than the controls a and b, confirming activation of bone formation. 2.3 the end of 90 days “implantation In [Fig. 4], the photograph on the left shows a section of a femoral condyle from a healthy rat, and the photograph on the right shows a section of a femoral condyle from an ovariectomized rat. The histological sections ([Fig. 4]) were taken at 50x magnification. In the photograph on the left, the femoral condyle of the healthy rat shows a dense network of trabeculae, while in the photograph on the right, the femoral condyle of the ovariectomized rat shows a network of enlarged trabeculae. These photographs demonstrate the presence of osteoporosis in the femur of the ovariectomized rat. In [Fig. 5], the photograph on the left shows a cross-section of a femoral condyle from an ovariectomized rat into which the implant from Example 2 has been inserted for 90 days, and the photograph on the right shows a cross-section of a femoral condyle from an ovariectomized rat into which the implant from Example 3 has been inserted for 90 days. Macroscopically, all implants are well tolerated by the bone tissue; there is no evidence of tissue necrosis. After 90 days, the implant in Example 2 had practically disappeared: it had been degraded, allowing new bone to form. This implant exhibits excellent osseointegration and promotes the formation of mineralized tissues characteristic of mature bone. This implant could be advantageous for young individuals with highly active bone metabolism and relatively rapid bone remodeling. At the end of 90 days, that is, at the end of the study, we still clearly observe the implant in example 3. The formation of the new bone continues more slowly. Figure 6 shows a comparison between the histological analyses of an implant according to example 3 (right), i.e., group e, versus a comparative implant according to example 4 without chitosan (left), i.e., group f. These photographs correspond to a magnification of 50x. It can be observed that the integration of the implant according to the invention into the bone is greater than the integration of the comparative implant into the bone. 2.4 Conclusions The implants according to the invention are biocompatible and well tolerated by the body. Furthermore, it has been observed that the fish by-product powder and chitosan bind instantly upon implantation, with the chitosan subsequently being released locally. The chitosan is thus available to contribute to the treatment of osteoporosis, particularly osteoporosis induced by oophorectomy. It has also been observed that the implants according to the invention allow the growth of apatite with a composition close to that of healthy bone as well as progressive bone maturation.

Claims

Demands

1. Biomaterial characterized in cc that it comprises, relative to the total weight of biomaterial, 70 to 90%, preferably 80 to 85%, by weight of a powder made from at least one fish co-product chosen from the group formed by fish scales and fish bones and 10 to 30%, preferably 15 to 20%, by weight of chitosan.

2. Biomaterial according to claim 1 wherein the co-product of fish contain calcium and phosphorus according to a report calcium / phosphorus ratio by weight ranging from 1.8 to 2.5 and preferably ranging from 2.032.,5.

3. Biomaterial according to any one of the preceding claims in which the fish co-product includes collagen in a quantity ranging from 20% to 40%, and preferably ranging from 20% to 35%, in weight relative to the total weight of the fish co-product.

4. Biomaterial according to any one of the preceding claims in which the fish co-product includes magnesium in one quantity ranging from 0.30% to 0.60%, and preferably ranging from 0.44% to 0.46%, by weight relative to the total weight of the fish co-product.

5. Biomaterial according to any one of the preceding claims. previous in which the fish co-product includes the elements following in weight relative to the total weight of the fish co-product: zinc in an amount ranging from 0.010% to 0.011%; strontium in a quantity ranging from 0.032% to 0.033%.

6. Biomaterial according to any one of the preceding claims previous in which the fish co-product powder presents a particle size ranging from 40 to 315 microns.

7. A method for manufacturing the biomaterial according to any one of the re- previous claims characterized in that it includes the steps following, in this order: -have a powder made from at least one chosen fish co-product in the group formed by fish scales and bones fish, said powder having a particle size ranging from 40 to 315 microns; -have chitosan available; -prepare a mixture comprising 70 to 90%, preferably 80 to 85%, by weight of powder, of at least one fish co-product and 10 at 30% by weight, preferably 15 to 20% by weight of chitosan, - stir the mixture; -dry the resulting precipitate; - to recover the biomaterial obtained.

8. Method for manufacturing the biomaterial according to the preceding claim in which the fish co-product powder is obtained by putting implement the following steps, in this order: -have at least one fish by-product chosen from the scales of fish and fish bones; - place the fish co-product in a water bath at a temperature ranging from 50°C to 100°C for a duration ranging from 5 to 120 minutes; -dry the co-product; -grind the co-product in a manner suitable for obtaining a granular powder- measurement ranging from 40 to 315 microns; -recover the fish co-product powder obtained.

9. Composition comprising a biomaterial according to any one of the re- claims | to 6 or obtained according to one of claims 7 or 8 and to minus one agent chosen from among the drugs, in particular the anti- biotics, bisphosphonates, thermoplastic polymers in part- particular ones chosen from polylactic acid, poly (lactic-co-glycolic), polybutylene succinate, polyhydroxyal- canoates, polycaprolactone, polybutylene-co-adipate terephthalate.

10. Implant comprising a biomaterial according to any one of the claims indications 1 to 6 or obtained by the process according to any of the re- claims 7 to 8 or comprising the composition according to claim 9

11. — Implant according to claim 10 comprising, by weight relative to the total weight of the implant, from 90 to 100% of a biomaterial according to one any of claims 1 to 6 or obtained by the process according any one of claims 7 to 8 and from 0 to 10% of at least one agent chosen from among bisphosphonates and drugs, in par- antibiotics in particular.

12. Implant according to claim 10 comprising by weight relative to the total weight of the implant, from 30 to 50% of a biomaterial depending on one any of claims 1 to 6 or obtained by the process according any one of claims 7 to 8, from 50 to 70% of a material thermoplastic chosen from the group formed by polylactic acid, poly(lactic-co-glycolic acid), polybutylene succinate, the po- lyhydroxyalkanoates, polycaprolactone, polybutylene-co-adipate terephthalate and 0 to 10% of at least one agent chosen from the bis- phosphonates and drugs, in particular antibiotics.

13. Implant according to any one of claims 10 to 12 for its use- lization for bone regrowth, bone reconstruction, and / or the treatment of osteoporosis.

14. Method for manufacturing the implant according to claim 12 including the following steps, in this order, -mixture of the biomaterial according to any one of claims 1 to 6, with at least one thermoplastic material selected from poly- acid lactic acid, poly(lactic-co-glycolic acid), polybutylene succinate , polyhydroxyalkanoates, polycaprolactone, polybutylene- co-adipate terephthalate; at least one porogenous agent and possibly at least one agent chosen from among the drugs, in particular the anti- biotics, bisphosphonates; - shaping of the mixture by 3D printing using fused filament deposition, - Removal of pore-forming agents preferably by leaching, -possibly drying of the implant obtained.