HK1223571B - Implant with controlled porosity comprising a matrix covered by a bioactive glass or by a hybrid material - Google Patents

Abstract

The invention relates to an implant material for filling bone defects, for bone regeneration and for bone tissue engineering, to an implant comprising this material, and to a method for producing such an implant. The implant material of the invention comprises: a bioactive glass M based on SiO2 and CaO, optionally containing P2O5 and/or optionally doped with strontium, and a biodegradable polymer P soluble in at least one solvent S1 and chosen from among the bioresorbable polysaccharides, preferably chosen from among dextran, hyaluronic acid, agar, chitosan, alginic acid, sodium or potassium alginate, galactomannan, carrageenan, pectin; the bioresorbable polyesters, preferably polyvinyl alcohol or polylactic acid; the biodegradable synthetic polymers, preferably a polyethylene glycol or polycaprolactone; and proteins, preferably gelatin or collagen, and it consists of a matrix comprising at least the biodegradable polymer P, this matrix being covered by the bioactive glass M or by a hybrid material H formed by a biodegradable polymer identical to or different from the biodegradable polymer P and the bioactive glass M, this matrix having at least 70% by number of pores having at least one interconnection with another pore, and the shape of spheres or polyhedrons inscribed within a sphere, the diameter of the spheres being between 100 and 900 μm inclusive, with a deviation between the diameter of the smallest sphere and the largest being at most 70% with respect to the arithmetic mean diameter of all the spheres of the implant, and the interconnections between the pores having their smallest dimension between 25 μm and 250 μm inclusive. The invention has an application in the medical field in particular.

Description

The invention relates to an implant material for bone replacement, bone regeneration and bone tissue engineering, an implant comprising this material, a manufacturing process for such an implant.

The overall ageing of the population and the associated diseases of the osteoarticular system make it necessary to develop high-performance bone tissue replacement materials. 18 billion euros in healthcare costs are spent each year in France on diseases of the osteoarticular and dental systems, musculoskeletal disorders are the most common occupational diseases in industrialized countries, while osteoporosis is developing among elderly patients; these facts outline a major social and economic challenge and explain the growing demand for biomaterials, implants with a longer lifespan able to fill bone loss.

As the use of transplants is limited and animal materials may pose biocompatibility problems or risk of infection, research efforts are aimed at developing synthetic biomaterials that can promote bone regeneration.

This is called bioactive implants: the implanted material is not simply designed to passively fill a bone loss by remaining as inert as possible, but on the contrary it must stimulate and actively participate in the bone regeneration mechanism.

Currently the main bioactive materials used as bone substitutes are ceramics bioactive, such as calcium phosphates, and bioactive glasses, also known as biovers .

The first bioactive ceramics were developed by L.L. Hench (L.L. Hench et al., J. Biomed. Mater. Res. 1971, 2, 117-141; L.L. Hench et al., J. Biomed. Mater. Res. 1973, 7, 25-42).

The first bioactive glasses were prepared from SiO2, P2O5, CaO and Na2O. Silicon and phosphorus oxides are network formers that participate in the cohesion of the glass network. Alkaline and alkaline earths such as sodium and calcium do not have this ability and change the glass network by introducing chain breaks that are the cause of the low melting temperature of these glasses associated with increased structural disorder. Their presence results in greater reactivity of bioactive glasses to corrosion in a water-based environment.

The most studied biovel is a sodium-silica-phosphocalcium glass called Bioglass® or Hench Biovel. Its basic composition is 45% SiO2 - 24.5% CaO - 24.5% Na2O - 6% P2O5, in mass relative to the total mass of the composition. The remarkable bioactive properties of this material are no longer to be demonstrated. Bioglass® remains at present one of the most interesting bioactive materials (inducing a specific response of the cells).

Many developments have been made in the field of bioactive glasses since their discovery (M. Vallet-Regi et al., Eur. J. Inorg. Chem. 2003, 1029-1042), such as the incorporation of different atoms or the incorporation of active ingredients. The compositions of bioactive glasses have been optimized to promote the proliferation of osteoblasts and the formation of bone tissue (WO 02/04606).

Application WO 2009/027594 describes a bioactive glass in which strontium is introduced in quantities between 0,1 and 10% of the total weight of the bioactive glass. Bioactive glasses are used in the manufacture of bone implants (ANKE LISA METZE ET AL,BONE AND BIOMATERIALS FOR BONE Tissue ENGINEERING, vol. 541, 2013-01-01, pp. 31-39). These bioactive materials are characterised by being both biocompatible, able to bind to bone tissue spontaneously, promote bone cell adhesion and finally being bioresorbable, being gradually replaced by newly formed bone tissue as bone develops.

However, despite this very satisfactory set of characteristics, the fragility of these materials limits their applications: indeed, although their rigidity is often higher than that of bone, their lack of flexibility and toughness means that bioactive materials cannot be implanted in mechanically loaded sites.

To overcome this defect, an ingenious solution is to draw inspiration from the particular structure of bone tissue. Complex, it consists mainly of a composite frame intimately mixing an inorganic phase, the bone mineral made up of apatite crystals (resorbable calcium phosphate), with an organic phase, which is mostly collagen. Remarkably, such a composite structure combines the initial rigidity of the inorganic part with the natural toughness and flexibility of the collagen fibers.

Err1:Expecting ',' delimiter: line 1 column 466 (char 465)

Such a macroporous structure is also required for new applications in bone tissue engineering: the laboratory production of new bone tissue from cells taken from the patient, which can be re-implanted a posteriori in the patient.

In summary, although many materials and formulations have been developed for bone loss, none fully meets the specifications describing the ideal implant, namely: be biocompatible; be bioactive: spontaneously induce the formation of a strong interfacial bond with bone tissue, promote cell adhesion and activity; be bioresorbable; have an adequate morphology based on a three-dimensional matrix of interconnected macropores; have good mechanical strength; be derived from a manufacturing process that allows easy shaping and is flexible enough to accommodate many defect geometries.

Adequate morphology based on a three-dimensional matrix of interconnected macropores means that the size, shape and distribution of pores and the size of interconnections between these pores must be controlled.

The purpose of the invention is to propose a material which perfectly satisfies all these criteria and which can be manufactured by a process which enables the realization of porous architectures composed of an inorganic part and an organic part, unlike the previous state-of-the-art processes.

To this end, the invention proposes a method of manufacturing an implant for bone replacement, bone regeneration and tissue engineering in a material according to the invention, characterised by the following steps: (a) selection of a bioactive glass M based on SiO2 and CaO, optionally containing P2O5 and/or optionally strontium-doped, (b) selection of a biodegradable polymer P which is soluble in S1 and insoluble in at least S1 solvent, (c) selection of microspheres of a porogenic agent A with diameters and sizes corresponding to the desired diameters and sizes of the pores in an implant material,the material of this porogenic agent A being a polymer insoluble in solvent S1 and soluble in at least one solvent S, the at least one solvent S in which the biodegradable polymer P is insoluble and the at least one solvent S in which the porous agent A material is soluble are identical, (d) introduction of at least 60% by volume, preferably at least 70% (and less than 100%) of the total volume of the biodegradable polymer mixture P-porogenic agent A introduced into the mould, of microspheres of porogenic agent A into a mould of the desired shape and size for the implant,these microspheres forming a compact stack corresponding to the shape and size of the pores to be obtained in the implant material,e) introduction of the biodegradable polymer P into the mould,f) gelling of the mixture obtained in step e) in the mould,g) removal of the mixture obtained in step f),h) removal of the porosity by washing with at least one solvent S,i) cross-linking of the mixture obtained in step g),j) coating of the mixture obtained in step i) with bioactive glass M or with a hybrid material H formed from a biodegradable polymer identical or different from P and bioactive polymer glass M.

In a first embodiment of the process of the invention, step j) is carried out by impregnating the mixture obtained in step i) with a suspension in a solvent containing particles of bioactive glass M or hybrid material H and evaporating the solvent.

In a second embodiment of the process of the invention, step j) is a coating step of the mixture obtained in step i) with either bioactive glass M or hybrid material H, and is implemented by immersion of the mixture obtained in step i) in either a soil containing the alcohols precursors of bioactive glass M for coating with only bioactive glass M, or in a soil of the hybrid material, or in a soil of the alcohols precursors of bioactive glass M and the biodegradable polymer of hybrid material H for coating with the hybrid material H, followed by a gelling step.

In all implementations of the process of the invention, the biodegradable polymer P is chosen from: biodegradable polymers soluble in at least one S1 solvent and insoluble in at least one S solvent selected from:biodegradable polysaccharides, preferably from dextran, hyaluronic acid, agar, chitosan, alginic acid, sodium or potassium alginate, galactomannane, carrageenan, pectin,biodegradable polyesters, preferably polyvinyl alcohol or polylactic acid (PLA), synthetic biodegradable polymers, preferably polyethylene glycol or polycaprolactone, proteins, preferably gelatine or collagen, and the material of the porogenic agent A is chosen from biodegradable polymers insoluble in at least one solvent S1 and soluble in at least one solvent S, preferably from polymethyl alkyl methacrylates in C1 to C4, preferably methyl polymethylated or butyl polymethylated, polyurethane, polyglycolic acid, various forms of polylactic acids, copolymers of lactic-coglycolic acids, polycaprolactone, polypropylene styrene, paraffin, naphthalene and acrylonitrile butadiene fumarate (ABS), the material of the porous agent A is different from the biodegradable polymer P.

More preferably, biodegradable polymer P is a polymer of natural origin or a biodegradable synthetic polymer or a bioresorbable polyester.

Where the biodegradable polymer P is coated with a hybrid material, preferably, the ratio of biodegradable polymer to bioactive glass M by weight in this hybrid material shall be between 10/90 and 90/10 inclusive, preferably between 20/80 and 80/20, inclusive, preferably between 30/70 and 70/30 inclusive.

Preferably, the hybrid material shall consist of 70% by mass of biodegradable polymer and 30% by mass of bioactive glass.

The polymer of the hybrid coating material may be the same or different from the biodegradable polymer P.

Where the biodegradable polymer P is coated with bioactive glass M alone, then preferably the mass ratio of the biodegradable polymer P to the bioactive glass M is preferably between 50/50 and 90/10, preferably between 60/40 and 80/20.

Also preferably, bioactive glass M is glass based on SiO2 and CaO, the biodegradable polymer P is gelatine, the material of the porous agent A microspheres is methyl polymethacrylate and the solvent S is acetone.

However, the preferred bioactive glass is a glass consisting of 75% by mass of SiO2 and 25% by mass of CaO or a glass consisting of 75% by mass of SiO2, 20% by mass of CaO and 5% by mass of SrO.

The method of the invention may also include a step of introduction of a coupling agent, preferably an organoalkoxysilan compound, preferably 3-glycidoxypropyltrimethyloxysilan (GPTMS) and even more preferably 3-glycidoxypropyltriethyloxysilan (GPTES) at step e.

It may also include, in addition, after step (d) and before step (e), a step of enlargement of the interconnections by infiltration of a solvent from the material of porogenic agent A into the stack of porogenic agent A microspheres and/or heating of this stack.

The invention also proposes an implant material for bone replacement, bone regeneration and bone tissue engineering, obtained by the process according to the invention, characterised by the following: a bioactive glass M based on SiO2 and CaO, optionally containing P2O5 and/or optionally strontium-doped, and a biodegradable polymer P soluble in at least one S1 solvent selected from: bioresorbable polysaccharides, preferably from dextran, hyaluronic acid, agar, chitosan, alginic acid, sodium or potassium alginate, galactomannan, carrageenan, pectin,bioresorbable polyesters, preferably polyvinyl alcohol or polylactic acid,biodegradable synthetic polymers,The most commonly used is polyethylene glycol, or poly (caprolactone), and proteins, preferably gelatin or collagen. and consists of a matrix containing at least the biodegradable polymer P coated with bioactive glass M or a hybrid material H consisting of bioactive glass M and a biodegradable polymer identical or different from the biodegradable polymer P, having at least 70% pores with at least one interconnection with another pore and the shape of spheres or polyhedra in a sphere, the diameter of the spheres being between 100 and 900 μm,preferably between 200 and 800 μm, terminals included, with a difference between the diameter of the smallest or largest sphere not exceeding 70%, preferably not exceeding 50%, preferably not exceeding 30%, in relation to the arithmetic mean diameter of all the implant spheres and the interconnections between the pores having their smallest dimension between 25 μm and 250 μm, terminals included.

Finally, the invention proposes an implant for bone replacement, bone regeneration and bone tissue engineering, characterised by the fact that it comprises a material of the invention or is obtained by the manufacturing process of an implant of the invention.

The invention will be better understood and other features and advantages of the invention will be more clearly seen by reading the following explanatory description, which is made by reference to the figures in the annex in which: Figure 1 is a cut-out view of the scanning electron microscope implant of the invention obtained in Example 1 at a magnification of x70,Figure 2 is a schematic representation of the implant material according to the invention,Figure 3 is a scanning electron microscope photograph of an implant of the invention with a matrix of gelatine covered with bioactive glass at a magnification of ×100,Err1:Expecting ',' delimiter: line 1 column 274 (char 273)1, 643-52,Figure 6 is a photograph taken with the scanning electron microscope at x50 magnification of a hybrid implant material with a 70/30 mass ratio of gelatine/glass, obtained by a process involving a step of increasing the size of the interconnections between pores by infiltration with a 30% acetone-ethanol mixture by volume of acetone, relative to the total volume of the mixture for 5 minutes by infiltration with the mixture in the stacking of the porogen microspheres alone,Figure 7 is a photograph taken with the scanning electron microscope at a large x50 volume of the same implant material as depicted in Figure 6 but after infiltration with an acetone-acetone mixture by volume of 30% acetone,in relation to the total volume of the mixture, for 15 minutes, with the mixture in the stack of porogenic agent microspheres alone,Figure 8 is a scanning electron microscope photograph taken at a ×50 magnification of the material represented in Figure 6 and Figure 7 obtained by the process of the invention after increasing the size of the pore interconnections by infiltration of the acetone-ethanol mixture to 30% by volume of acetone, in relation to the total volume of the mixture, for 30 minutes, with the mixture in the stack of porogenic agent microspheres alone,Figure 9 is a curve representing the increase in the size of the pore interconnections by infiltration with an acetone-ethanol mixture at 30% by volume of acetone,in relation to the total volume of the mixture, according to this infiltration time,Figure 10 is a photograph taken with a scanning electron microscope at a magnification of ×100 of a hybrid implant material consisting of 70% gelatin and 30% glass by mass, according to the invention obtained by the process of the invention after increasing the size of the pore interconnections by heating the stacking of the porogen microspheres alone at 125°C for 15 minutes, under air,Figure 11 is a photograph taken with a scanning electron microscope at a magnification of 104 ×10 of the implant material represented by the invention but after increasing the size of the porogen interconnections at 125°C for 1 hour during the stacking of the porogen microspheres,alone, before infiltration with the hybrid material consisting of 70% gelatine and 30% glass by mass,Figure 12 is a photograph taken with a scanning electron microscope at a magnification of ×100 of the same composition of the implant material of the invention as shown in Figures 10 and 11, obtained by a process after increasing the size of the pore interconnections by heating the stacking of porogen microspheres at 125°C alone for 2 hours, andFigure 13 is the curve showing the change in the size of the interconnections between pores as a function of the heating time at 125°C of the stacking of porogen microspheres,I'm alone.

Err1:Expecting ',' delimiter: line 1 column 135 (char 134)Err1:Expecting ',' delimiter: line 1 column 51 (char 50)

Such a compact stack of porogen A microspheres can be obtained by centrifuging the porogen A-biodegradable polymer P microspheres mixture or by applying negative (void) or positive (above atmospheric pressure) pressure to the porogen A-biodegradable polymer P microspheres mixture introduced into the mould before and during the gelling of this mixture.

The implant material for bone replacement, bone regeneration and tissue engineering of the bone of the invention will be described in relation to Figures 1 and 2.

As shown in Figures 1 to 3, the implant material of the invention consists of a matrix, marked 1 in Figures 1 and 2, in a material comprising an organic part and an inorganic part.

This material is biocompatible, bioactive, bioresorbable and as shown in Figures 1 to 3, it has a very regular morphology in terms of pore distribution, as shown in Figures 1 to 3, in terms of pore shape, unlike materials of earlier art which have anarchic pore distribution, size and shape, as shown in Figures 4 and 5, which are respectively photographs taken with scanning electron microscope of implant materials obtained by a freeze-drying process (Figure 4) and a thermal induced phase separation process (Figure 5).

In particular, this material has pores in the shape of spheres, the diameter of which, as shown in Figure 2, is preferably the same at all points, such that the ratio of the smallest diameter to the largest diameter is 0,9 ± 0,1, or in the shape of polyhedra in such a sphere, the differences between the diameters at different points of the polyhedron in this sphere being not more than or equal to 15% of the diameter of the sphere in which they are inserted.

At least 70% of the pores of the implant material of the invention have these shapes.

The implant materials of the invention may have pore sizes in a very wide range from 100 μm to 900 μm, preferably 200 μm to 800 μm, terminals included, with a difference between the diameter of the smallest or largest sphere of not more than 70%, preferably more than 50%, more preferably more than 30%, relative to the arithmetic mean diameter of all the implant spheres in combination with interconnections, as shown in Figures 1 to 3, between pores with the smallest dimension between 25 μm and 250 μm, terminals included.

Err1:Expecting ',' delimiter: line 1 column 311 (char 310)

However, in the case of this article, such pore shapes had been obtained on an implant made entirely of bioactive ceramic, i.e. calcium phosphate (hydroxyapatite).

The disadvantage of such ceramics is that they do not have the flexibility required for a bone implant, and their manufacturing process cannot be applied to a material with an organic part, such as that of the invention, because it involves a sintering step at temperatures of the order of 800°C, at which the organic part disintegrates.

Such size distributions are never achieved in part organic and part inorganic implant materials from prior art processes for which pore sizes are generally well below 200 μm with interconnections of much lower sizes.

There are many implant materials from foaming processes but these then have very large, uncontrolled pores and interconnections, the porosity reaching even a millimeter, which is not favorable to the mechanical holding of the implant.

WO 2013/023064 describes two processes to obtain a composite matrix with pores whose size allows for the infiltration of cells and internal growth of bone. In the first process, a fibrous material is obtained (which therefore has no spherical pores, with size distribution controlled). In the second process which is a solvent molding process, the porosity can be increased by adding a porogenic agent.

As will be shown below, the method of manufacture of the implants of the invention makes it possible to control the dispersion of the set of pore sizes and matrix interconnections, which was not possible in the earlier techniques where the porosity generated is randomly distributed in their respective intervals.

Matrix 1 consists of an organic phase and an inorganic phase.

The inorganic phase is a bioactive glass M.

Bioactive ceramics and bioactive glasses are well known to the trade and are described in particular in L.L. Hench et al., J. Biomed. Mater. Res. 1971, 2, 117-141; L.L. Hench et al., J. Biomed. Mater. Res. 1973, 7, 25-42 for bioactive ceramics and in M. Vallet-Regi et al., Eur. J. Inorg. Chem. 2003, 1029-1042 and WO 02/04606, WO 00/76486 and WO 2009/027594, in particular.

The organic part of the implant material of the invention is a biodegradable polymer P selected from: bioresorbable polysaccharides, preferably from dextran, hyaluronic acid, agar, chitosan, alginic acid, sodium or potassium alginate, galactomannan, carrageenan, pectin,bioresorbable polyesters, preferably polyvinyl alcohol or polylactic acid,biodegradable synthetic polymers, preferably polyethylene glycol or polycaprolactone, and proteins, preferably gelatine or collagen.

Of course, when the porogenic agent A is in poly (polylactic acid) or poly (caprolactone), the biodegradable polymer P will be in a different polymer.

Matrix 1 may consist of bioactive glass M and biodegradable polymer P which form a composite material, i.e. the two phases bioactive glass M and biodegradable polymer P coexist in the matrix architecture.

Matrix 1 can also be made of bioactive glass M and biodegradable polymer P which form a hybrid material, i.e. forming a single phase. In this case, the hybrid material is obtained by forming a soil containing all the alcohol precursors of the bioactive glass, adding the biodegradable polymer desired for the hybrid material H to this soil and gelling the solution obtained by a succession of polymerization reactions (sol-gel polymerization of the inorganic phase) (condensation of the alcohols).

The hybrid material is thus distinguished from the composite material by a close integration between the two organic and inorganic phases, these two phases being indistinguishable (except at the molecular scale) in the case of a hybrid mixture.

In fact, in the invention, matrix 1 is formed from the only biodegradable polymer P, a polymer that is coated with bioactive glass M, for example by impregnating matrix 1 with biodegradable polymer P in a suspension of bioactive glass M or when matrix 1 is coated with hybrid material H by immersing matrix 1 formed only of biodegradable polymer P in a soil of the hybrid material, or in a soil of the alkoxide precursors of bioactive glass M and the biodegradable polymer of the hybrid material H.

In both cases, matrix 1 will then be dried to allow the deposition of bioactive glass particles M or soil gelling as appropriate.

The implant material of the invention is obtained by a process involving a porogenic agent A which consists of microspheres in a polymer soluble in at least one solvent S in which the biodegradable polymer P is not soluble.

Thus, the method of the invention consists in stacking microspheres of porogenic agent A in a polymer material, other than biodegradable polymer P, in a mould having the shape and size corresponding to the geometry of the bone defect to be filled or the defect where bone regeneration is desired.

These porous agent A microspheres allow the final pores to be obtained, the size and distribution of which will correspond negatively to the stacking of porous agent A microspheres initially carried out.

In addition, at least 70% of the pores formed shall be perfect spherical, i.e. have the same diameter at all points or have a ratio of the smallest diameter to the largest diameter of 0,9 ± 0,1 or, for the largest pores, shall be in the shape of a polyhedron in a sphere of the same diameter at all points, the differences between the diameters at different points of the polyhedron in that sphere being not more than or equal to 15% of the diameter of the sphere in which they are inserted.

The material intended to constitute matrix 1 will then be infiltrated into the stacking of microsphere balls of porogenic agents A, then solidified to be able to be demolded without changing the shape and size of the stacking of the desired implant.

As can be seen, this process does not use any high-temperature heat treatment to fry bioactive glass M, the only temperature required being the evaporation temperature of the solvent used.

In the process of the invention, matrix 1 is made up of only biodegradable polymer P and is then coated with either bioactive glass M or a hybrid material made up of a biodegradable polymer and bioactive glass M.

As will be apparent, the invention is based on the judicious combination of the choice of three materials: the material constituting the biodegradable polymer P, the material constituting the porogenic agent A and the solvent S of the porogenic agent A which must not dissolve the biodegradable polymer P.

The material of the biodegradable polymer P that is part of the implant material shall be a biocompatible polymer.

The material of the porous agent A must be a material, e.g. a polymer, the solvent of which is a non-solvent of the biodegradable polymer P.

Err1:Expecting ',' delimiter: line 1 column 109 (char 108)

Biodegradable polymers P shall be soluble in at least one solvent S1 and insoluble in at least one solvent S.

The S1 solvent can be water, an aqueous medium or an organic solvent.

Among the biodegradable P polymers that can be used are: bioresorbable polysaccharides, preferably from dextran, hyaluronic acid, agar, chitosan, alginic acid, sodium or potassium alginate, galactomannan, carrageenan, pectin,bioresorbable polyesters, preferably polyvinyl alcohol or polylactic acid,biodegradable synthetic polymers, preferably polyethylene glycol or polycaprolactone, and proteins, preferably gelatine or collagen.

All these polymers are soluble in at least one S1 solvent and insoluble in at least one S solvent.

In the case of polyethylene glycols, these polymers are soluble in water and in many organic solvents except diethyl ether and hexane.

The material constituting the porogenic agent A shall be soluble in at least one solvent S in which the biodegradable polymer P is insoluble.

Examples of such materials are C1 to C4 alkyl polymethacrylates, preferably methyl polymethacrylate (PMMA) or butyl polymethacrylate, polyurethane, polyglycolic acid, various forms of polylactic acids, lactic-coglycolic acid copolymers, polycaprolactone, polypropylene fumarate, paraffin and naphthalene and acrylonitrile butadiene styrene (ABS)

Examples of polymers in the composition of hybrid material H intended to coat biodegradable polymer P in the methods of implementation of the invention are the same as those cited for biodegradable polymer P. However, this polymer and biodegradable polymer P may be identical or different from each other.

The material of the porous agent A must also be different from the biodegradable polymer P.

In any case, the solvent of the material of porogenic agent A shall not be a solvent for the material chosen to be used as biodegradable polymer P.

The S solvents are in particular acetone, ethanol, chloroform, dichloromethane, hexane, cyclohexane, benzene, diethyl ether and hexafluoroisopropanol.

In the invention, preferably, the biodegradable polymer P will be gelatine, the porous agent A microspheres will be methyl polymethacrylate, the solvent S will be acetone and the solvent S1 will be water.

In the process of manufacturing the implant material of the invention, the introduction of microspheres into the mould may be made before the introduction of the biodegradable polymer P.

However, it is also possible to first introduce the biodegradable polymer P into the mold and then pour the porous agent A microspheres into it.

To obtain a material with at least 70% pores that are at least interconnected with another pore, the amount of porous agent A introduced into the biodegradable polymer P shall be at least 60% and preferably at least 70% by volume of the total volume of the biodegradable polymer mixture P-porous agent A introduced into the mould.

The size of the interconnections is related to the size of the point of contact between porous spheres in the stacking of spheres made. The increase in the size of the generated interconnections, with constant pore diameter, is possible by adding a step consisting of a partial fusion of the porous spheres in the stacking initially made, so as to increase the size of their point of contact.

This fusion may be done by infiltration of a solvent of the material of the invention on the stacking of porogenic agent A, or by heating the stacking of the microspheres of the porogenic agent, or both, so as to achieve the superficial dissolution of the spheres and allow their partial fusion.

Figures 6 to 8 show the effect of increasing the size of these interconnections by infiltration of a 30% acetone-ethanol mixture by volume of acetone, relative to the total volume of the mixture, after 15 min, 30 min and 1 hour of infiltration.

This infiltration takes place directly on the stack of porous agent microspheres, before the introduction of bioactive glass M and/or biodegradable polymer P.

Figure 9 shows this effect of increase in the form of a curve.

Figures 10 to 12 show the effect of increasing the size of these interconnections by heating at 125°C, stacking porous agent microspheres, before the introduction of bioactive glass M and/or biodegradable polymer P for 15 min, 1 hour and 2 hours.

Figure 13 shows this effect of increase in the form of a curve.

In order to give a better understanding of the invention, several examples of implementation will now be described, purely illustrative and not limitative.

Example 1: Manufacture of an implant from a gelatine composite material, as biodegradable polymer P, and a bioactive glass consisting of 75% SiO2 and 25% CaO by mass, relative to the total mass of the glass, as bioactive glass M (not part of the invention).

Gelatin was chosen as the material for the biodegradable polymer P because it is a natural, biocompatible, cheap and readily available biopolymer. Gelatin is also derived from collagen naturally found in bones. It is also already used in clinical applications, dressings, adhesives and pharmaceutical encapsulation.

Bioactive glass was chosen because of its high ability to induce mineralization, the possibility of shaping its textural and morphological properties (porosity, size and therefore specific surface) at the nanoscale, the wide range of bioactive compositions that can be formulated, for example by adding anti-inflammatory elements, or osteoprotective agents, and finally it is the combination of their bioactive and bioresorbable properties that make it the most promising biomaterials for bone regeneration, especially compared to calcium phosphates (bioactive ceramics), which are generally either less bioactive or less resorbable.

The microspheres are microspheres made of polymethyl methacrylate. This material was chosen because it can be easily dissolved by many solvents.

In addition, if residues of unremoved methyl polymethacrylate remain in the implant material, the good biocompatibility of this polymer with human tissues is a good guarantee that the implant will not present any risk of cytotoxicity.

The porogenic agent was in the form of spherical particles, namely balls of methyl polymethacrylate.

Err1:Expecting ',' delimiter: line 1 column 967 (char 966)

In this example, the biodegradable polymer and bioactive glass were used to obtain a composite matrix.

Thus, in this example, the first step was to place porous particles of polymethyl methacrylate microspheres into a mold of the size and shape required for the implant.

In a second step, bioactive glass powder was introduced.

The granulometry of bioactive glass powder plays an important role in obtaining a homogeneous composite matrix. Preferably, the granulometry of bioactive glass powder should be well below 50 μm. Ideally, the size of the powder particles should be on the order of micrometers, or even on the order of nanometers to a few hundred nanometers. Such fineness can be achieved by means of a planetary ball mill for example.

In a third step, gelatin, previously dissolved in warm water, is introduced and the composite mixture is then homogenized.

In a fourth step, the mixture obtained in the third step is gelled for several hours in the mould, the partial dehydration of the gelatin ensuring the absorption of the mixture.

This operation is carried out at a temperature between 0°C and 60°C inclusive, in order not to degrade the matrix.

In a fifth step, the porous agent microspheres in methyl polymethacrylate are removed by acetone washing.

Acetone has several advantages: firstly, the polymethyl methacrylate balls are easily dissolved in acetone and gelatine is insoluble in acetone.

Acetone allows further dehydration of the gelatin if necessary.

Finally, it is a commonly used solvent, relatively inexpensive, widely available, and poses no serious toxicity risks.

After several washing steps, the initial trace of the methyl polymethacrylate microspheres is completely removed and the final material is obtained as a bio-composite macroporous block of bioactive glass/gelatin.

The biodegradability of this implant material in the living environment and its mechanical strength can also be easily adjusted by reticulating the gelatin during a final immersion step in a solution of a reticulating agent such as genipine, carbodiimide, glutaraldehyde, formaldehyde.

However, this step is optional.

The resulting structures can be washed without any damage in ethanol baths to remove any unwanted residues such as chlorides, acetone, etc.

In this example, we obtained an implant material comprising 60% by mass of bioactive glass and 40% by mass of gelatin, relative to the total mass of the implant.

Example 2: Manufacture of an implant material according to the invention with a matrix of hybrid material (not part of the invention).

The first step was to stack the microspheres of the porous agent polymethyl methacrylate into a mold with the required geometry for the implant.

In a second step the hybrid mixture was poured into the mold containing the ball stack.

A pressure centrifugation or infiltration or vacuum infiltration can be used to help the hybrid mixture fill the gaps between the methyl polymethacrylate microspheres.

The hybrid material was obtained by a sol-gel process.

In this process, a soil containing all the alcoholic precursors of the bioactive glass is gelled by a series of polymerization reactions.

In the case of this example, gelatine (the biodegradable polymer P) was added before soil gelling to produce a hybrid mixture.

For the production of hybrid material, a major difficulty is that heat treatments at high and medium temperatures, i.e. above 150°C, are to be prohibited.

Err1:Expecting ',' delimiter: line 1 column 128 (char 127)

The use of an alcohol-oxide precursor for calcium allows the incorporation of calcium into the inorganic phase without heat treatment.

Err1:Expecting ',' delimiter: line 1 column 656 (char 655)In the example, the precursors alcohols of calcium and calcium are mixed together in a slightly acidified silicon solution. Preferably, the following alcohols are tetraoxysilane and calcium oxide. The gelatin is then dissolved in a few minutes to obtain a stable solution of this acid. This allows for a few minutes between reactions and the solvent and the solvent to be mixed.

The implant material is then obtained by applying the fourth and fifth steps as shown in example 1.

Whether in the preparation of the composite or hybrid mixture, it may be advantageous to add a coupling agent to the mixture, such as an organoalkoxysilan.

For example, it can simply be added to the aqueous solution of the biodegradable polymer P, here gelatin. The role of the coupling agent is to functionalize the gelatin, in order to allow the establishment of covalent bonds with the inorganic phase (silicate network of the bioactive glass). In the case of a composite mixture, the coupling allows to obtain bioactive glass particles bound to the gelatin surface. In the case of a hybrid mixture, a true organo-mineral copolymer is obtained. The interest is to be able to control the degradability of the implant or hybrid phase as well as its mechanical strength, simply by acting on the degree of affinity between the organic and inorganic phase composition.

An example of a coupling agent successfully used in the invention is GPTMS (3-glycidoxypropylmethoxysilane), which is soluble in an aqueous gelatine solution.

Example 3: Manufacture of an implant material according to the invention from a biodegradable polymer matrix P coated with bioactive glass

In this example, porous agent A was microplates of PMMA with a diameter of 200-400μm which accounted for 70% by volume of the total volume of the mixture introduced into the mould.

The procedure was then followed as in example 2, except that in the second step only gelatine was introduced, and after the fifth step of washing away the microspheres of methyl polymethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethylamethyllamethylamethyllamethyl

The S1 solvent here is water and the S solvent is acetone.

The biodegradable polymer matrix P is then immersed in a suspension of bioactive glass M or in a soil containing all the alcohol precursors of bioactive glass M.

In both cases, matrix 1 is then dried to allow the deposition of bioactive glass particles M or soil gelling as appropriate.

The ratio of biodegradable polymer P to bioactive glass was 70/30.

Example 4: Manufacture of a hybrid material (not part of the invention).

Products used:

The following substances are to be classified in the same category as the product:

Protocol:

Fill a 32 mm high, 9 mm diameter polyethylene tube with PMMA balls at a height of about 10 mm.2. Mix 7.80 g of TEOS and 6.39 g of ethanol in a vial.3. Shake for 15 min using a magnetic agitator. Add 1.35 mL of HCl to 2M in the Ethanol + TEOS mixture.5. Shake for 30 min.6. Weigh 6.39 g of ethanol in another vial.7. Add 1.74 g of calcium ethoxide.8. Shake for 15 min.9. Add the soil containing TEOS to the calcium ethoxide solution.10. Shake for 1 hour.4.11. Dissolve 1.26 g of B-type gelatin and at least 0.1.63g of GPTMS in 8.74g of 10mM HCl in a wet bath at 60°C.12. Remove 3g of biovalve soil and add 7g of GPTMS-grafted gelatin soil to a vial.13. Shake for a few minutes with a magnetic agitator.14. Add the hybrid soil to the PMMA balls.15. Centrifuge 1 min16. Let it gell at 0°C to 60°C for at least 24 hours.17. Dissolve the resulting hybrid block.18. Dissolve the PMMA balls in a vial filled with acetone by refilling the acetone after 24 hours. This operation is repeated 2 times.19.

Example 5: Preparation of composite porous implants with 60% biovalve (75 % SiO2- 25% CaO) and 40% gelatine (% by mass) (not part of the invention)

1) Synthesis of glass powder by sol-gel process

After 30 minutes of stirring 5,2637 g of Ca ((NO3) 2.4H2O are added. The soil is stirred for 1 hour, tested at 60°C in Teflon containers for 24 hours and then exposed to air at 125°C for 24 hours. The resulting powder is then calcined for 24 hours at 700°C (heating from 25 to 700°C in 2 hours).

The powder is then crushed for 30 minutes and then sifted to retain only the fraction below 50 μm.

2) Preparation of the composite

Pig gelatine powder (type A) is added to distilled water heated to 35°C in a ratio of 0.1 g/mL water, the mixture is stirred for 10 minutes. At the same time, a quantity of 0.025 g of glass powder is mixed with 0.2 g of PMMA balls. 0.15 mL of gelatine solution in water is then added, the resulting mixture is poured into a tube into which it is compacted.

After 1 day of air drying, the glass cylinder + beads + gelatin is removed from the mold and immersed in acetone for 6 hours under stirring, the acetone is then renewed and the dissolution is left to continue for 24 hours, still under stirring.

Example 6: In vitro evaluation of the implants obtained from examples 1 to 5.

The bioactivity of the implant materials obtained from samples 1 to 3 was assessed in vitro by immersion in a physiological solution (SBF) with an ionic composition identical to that of blood plasma (ISO-23317 test).

The high bioactivity characteristic of the bioactive lenses used in the implant materials was then verified: these implant materials were very quick to induce mineralization when in contact with the physiological medium: within 1 hour of interaction with the medium, some of the calcium ions from the vitreous matrix migrated to the surface of the composite, where phosphate ions from the physiological medium were incorporated to form a layer of calcium phosphate about 10 microns thick, which covers the surface of the pores.

This is the first step in the bioactivity process.

It has been found that this calcium phosphate layer then continues to grow to form an apatite layer similar to bone mineral.

The interlinking of the gelatin does not reduce the bioactivity of the implant, but it increases its resistance to dissolution in the physiological environment.

It has also been noted that the SBF medium is rapidly (within 1 day) depleted of phosphorus, and to a lesser extent of calcium, which is incorporated into the surface of the implants and thus cut off from the medium to form a layer of biomimetic calcium phosphate.

Gelatin cross-linking does not alter the chemical reactivity of implants, but has the advantage of allowing adjustment of their biodegradability in the living environment.

Thus, all the materials manufactured in examples 1 to 5 are prone to induce the formation of bone mineral in contact with physiological fluids.

However, there are differences between these materials.

First, the materials in which the gelatin has been cross-linked have a slowed biodegradability, which is manifested by a slower solution-making of silicon.

It is then found that the formation of calcium phosphates on the surface of the material is slower with the composite material than with the material made of the biodegradable polymer P coated with the bioactive glass M of the invention.

This is a definite advantage.

With these two materials, as expected, the formation of calcium phosphates and especially of apatite, takes place only on the surface of the material.

In contrast to the hybrid material, the formation of calcium phosphates occurs not only on the surface but also in the mass, which is a disadvantage when the bone defect to be filled requires a slower integration.

Example 7:

The same procedure as in Example 3 was followed except that the biodegradable polymer P matrix was immersed in a soil of the alcohol oxide precursors of bioactive glass M and the biodegradable polymer of hybrid material H obtained in Step 13 of Example 4.

The resulting material of the invention was evaluated in vitro as described in Example 6.

This material has the advantage of a very rapid growth of calcium phosphates not only on the surface but also in the volume of the material of the invention.

Comparative example: Manufacture of an implant according to WO 2013/023064.

Pig gelatin powder (type A) is added to distilled water heated to 35°C at a ratio of 0.1 g/mL of water, the mixture is stirred for 10 minutes. An amount of 0.75 g of glass powder is mixed with 57.38 g of NaCl particles, then 4.5 mL of gelatin solution is then added, so that the porogen volume is 90 per cent of the total volume of the mixture, as shown in WO 2013/023064.

Unfortunately, the amount of porogen (90% of the total volume) proved to be far too large for the amount of composite mixture: the dissolution of the porogen caused the composite structure to be destroyed immediately, and no implants could be obtained by this development protocol.

Characterisation of the sphericity of the obtained macropores

The proposed synthesis route allows the production of spherical pores, since, by measuring two perpendicular diameters for each pore, the ratio of the smallest diameter to the largest diameter is on average 0.9 ± 0.1.

It is thus clear that by the method of the invention, implants with all the properties of porosity, in terms of pore sizes, sphericity of these pores, distribution of this pore size in a very wide range between 100 and 900 μm, preferably between 200 and 800 μm, terminals included, with a difference between the diameter of the smallest or largest sphere being not more than 70%, preferably more than 50%, preferably more than 30%, in relation to the arithmetic mean diameter of all the spheres of the implant, can be obtained, in combination with interconnections between pores with the smallest dimension being between 25 microns and 250 micrometers, which has never been achieved before.

Claims (10)

1. Method for manufacturing an implant for filling in bone defects for bone regeneration and bone tissue engineering, characterised in that it comprises the following steps:

   a) selecting a bioactive glass M based on SiO₂ and CaO, optionally containing P₂O₅ and/or optionally doped with strontium,

   b) selecting a biodegradable polymer P which is soluble in at least one solvent S1 and insoluble in at least one solvent S,

   c) selecting microspheres of a porogen A having diameters and sizes which correspond to the desired diameters and sizes of the pores in an implant material, the material of said porogen A being a polymer which is insoluble in the at least one solvent S1 and soluble in the at least one solvent S,

   wherein the at least one solvent S in which the biodegradable polymer P is insoluble and the at least one solvent S in which the material of the porogen A is soluble are identical,

   d) introduction of at least 60% by volume, preferably at least 70% by volume with respect to the total volume of the biodegradable polymer P/porogen A mixture introduced into the mould, of microspheres of the porogen A into a mould having the desired shape and size for the implant, said microspheres forming a compact stack corresponding to the shape and size of the pores to be obtained in the implant material,

   e) introducing the biodegradable polymer P into the mould,

   f) gelling the mixture obtained in step e) in the mould,

   g) removing the mixture obtained in step f) from the mould,

   h) eliminating the porogen by washing with the at least one solvent S,

   i) crosslinking the mixture obtained in step g),

   j) coating the mixture obtained in step i) with the bioactive glass M or with a hybrid material H formed of a biodegradable polymer which is identical to or different from the biodegradable polymer P and bioactive glass M.
2. Method according to claim 1, characterised in that step j) is implemented by impregnating the mixture obtained in step i) with a suspension, in a solvent, containing particles of the bioactive glass M or the hybrid material H, and evaporating the solvent.
3. Method according to claim 1, characterised in that step j) is a step of coating the mixture obtained in step i) either with the bioactive glass M or with the hybrid material H, and is implemented by immersing the mixture obtained in step i) either in a sol containing the alkoxide precursors of the bioactive glass M for a coating only with the bioactive glass M, or in a sol of the hybrid material, or in a sol of the alkoxide precursors of the bioactive glass M and of the biodegradable polymer of the hybrid material H for a coating with the hybrid material H, followed by a gelling step.
4. Method according to any one of claims 1 to 3, characterised in that the biodegradable polymer P is selected from:

   - the biodegradable polymers which are soluble in a solvent S1 and insoluble in at least one solvent S selected from:

   - bioresorbable polysaccharides, preferably selected from dextran, hyaluronic acid, agar, chitosan, alginic acid, sodium or potassium alginate, galactomannan, carrageenan, pectin,

   - bioresorbable polyesters, preferably polyvinyl alcohol or poly(lactic acid),

   - biodegradable synthetic polymers, preferably a polyethylene glycol, or poly(caprolactone), or

   - proteins, preferably gelatine or collagen,

   and the material of the porogen A is selected from biodegradable polymers which are insoluble in the at least one solvent S1 and soluble in the at least one solvent S, preferably selected from C₁ to C₄ polyalkyl methacrylates, preferably polymethyl methacrylate or polybutyl methacrylate, polyurethane, polyglycolic acid, various forms of polylactic acids, lactic-co-glycolic acid copolymers, polycaprolactone, polypropylene fumarate, paraffin and naphthalene, and acrylonitrile butadiene styrene (ABS), wherein the material of the porogen A is different from the biodegradable polymer P.
5. Method according to any one of claims 1 to 4, characterised in that the mass ratio of biodegradable polymer P/bioactive glass M is between 90/10 and 50/50 inclusive, preferably between 80/20 and 60/40 inclusive.
6. Method according to any one of claims 1 to 5, characterised in that the bioactive glass M is a glass based on SiO₂ and CaO, the biodegradable polymer P is gelatine, the material of the microspheres of porogen A is polymethyl methacrylate, the solvent S is acetone and the solvent S1 is water.
7. Method according to any one of claims 1 to 6, characterised in that it further comprises a step of introducing a coupling agent, preferably an organoalkoxysilane compound, more preferably 3-glycidoxypropyltrimethoxysilane (GPTMS), and more preferably 3-glycidoxypropyltriethoxysilane (GPTES) in step e).
8. Method according to any one of claims 1 to 7, characterised in that it comprises, after step d) and before step e), a step of expanding the interconnections (4) by infiltration of a solvent of the material of the porogen A into the stack of microspheres of the porogen A and/or by heating this stack.
9. Implant material, for filling in bone defects, bone regeneration and bone tissue engineering, obtained by the method according to any one of claims 1 to 8, characterised in that it comprises:

   • a bioactive glass M based on SiO₂ and CaO, optionally containing P₂O₅ and/or optionally doped with strontium, and

   • a biodegradable polymer P which is soluble in at least one solvent S1 selected from:

   - bioresorbable polysaccharides, preferably selected from dextran, hyaluronic acid, agar, chitosan, alginic acid, sodium or potassium alginate, galactomannan, carrageenan, pectin,

   - bioresorbable polyesters, preferably polyvinyl alcohol or poly(lactic acid),

   - biodegradable synthetic polymers, preferably a polyethylene glycol, or poly(caprolactone), and

   - proteins, preferably gelatine or collagen,

   and in that it consists of a matrix (1) comprising at least the biodegradable polymer P covered with bioactive glass M or a hybrid material H formed of bioactive glass M and a biodegradable polymer which is identical to or different from the biodegradable polymer P, said matrix (1) having at least 70% by number of pores (2) having at least one interconnection (4) with another pore and the shape of spheres or polyhedra being embedded in a sphere, the diameter (3) of the spheres being between 100 and 900µm, preferably between 200 and 800µm inclusive, with the difference between the diameter of the smallest sphere and the largest sphere being at most 70%, preferably at most 50%, more preferably at most 30%, with respect to the arithmetic mean diameter of all the spheres of the implant and the interconnections (4) between the pores having their smallest dimension between 25µm and 250µm inclusive.
10. Implant for filling in bone defects, bone regeneration and bone tissue engineering, characterised in that it comprises a material according to claim 9 or obtained by the method according to any one of claims 1 to 8.