HK1247859B - Semi-synthetic powder material, obtained by modifying the composition of a natural marine biomaterial, method for producing same, and applications thereof - Google Patents

Description

Field of the invention

The present invention relates to a pulverulent semi-synthetic, bio-resorbable material derived from a natural marine biomaterial, originating from the shell of bivalve mollusks such as Pinctadines in general, and notably Pinctada maxima, margaritifera, and Tridacnas: Tridacna gigas, maxima, derasa, tevaroa, squamosa, crocea, Hippopus hippopus, and Hippopus porcelanus.

Invention Context

WO9014111 describes a bone structure replacement product made from the nacreous layer of aquatic mollusk shells.

Generally, the materials used for filling bone substance losses caused by traumatic, tumoral, dystrophic, or degenerative origins are calcium phosphate cements, biopolymers, or materials of animal or human origin.

Regarding the fixation of prostheses, only polymethylmethacrylate (PMMA) is used, optionally associated with antibiotics, an initiator, an activator, a pigment or a coloring agent. Endoprostheses are generally fixed using PMMA cements, whose disadvantages are well known, especially the exothermic reaction produced during cement polymerization, the resulting bone cell necrosis, the cement shrinkage over time and its aging, which cause prosthesis loosening and the need for revision within 10 to 15 years after surgery, in most cases.

All these materials are biocompatible; some of them, such as phosphate cements, claim osteoconductive properties; few are bioactive, the majority being inert.

Injectable cements consist of a mineral phase and a liquid phase which can be phosphoric acid, an aqueous solution or HPMC gel, stoichiometric water of 0.1 mole, sulfuric acid, or citric acid.

Biomaterials, synthetic or of bovine origin, used as bone substitutes, are essentially endowed with osteoconductive properties and are generally not fully bioresorbable.

For some of them, particularly polymers, the release of degradation products is observed, which can have harmful effects on surrounding tissues or systemic morbidity in the long term. This biodegradation is patient-dependent.

Moreover, the vast majority of bone substitutes are not bioactive; this requires them to be combined with animal-derived collagen or other substances that, in order to be bioresorbable, induce a major inflammatory reaction in the host, which is more significant and different from the physiological reaction.

The major disadvantage of bone substitutes in powder or granule form lies in the fact that when they are applied, whether with autologous blood, physiological serum or any other liquid carrier, they do not form a "clot" with adhesive and plastic properties that promote their cohesion and retention on and within the site.

It is known that human bone is composed of 43% inorganic components, 32% organic components, and 25% water. The organic component consists of 90% collagenous proteins—of which 97% are types I, III, IV, and V collagen—along with 10% non-collagenous proteins, including osteocalcin, osteonectin, osteopontin, sialoprotein, proteoglycans, fibronectin, growth factors, and morphogenetic proteins. These non-collagenous proteins play an essential role in the processes of osteogenesis and tissue repair.

The inorganic fraction is mainly composed of hydroxyapatite in the form of calcium phosphate crystals; this fraction also contains other minerals such as sodium, potassium, copper, zinc, strontium, fluorine, aluminum, and silicon in very small quantities. All these elements play an important role in cellular metabolism as well as in bone healing and regeneration.

The study of the architecture and composition of the shells of bivalve mollusks, such as pinctadines in general, particularly Pinctada maxima, margaritifera, and tridacnas, notably Tridacna gigas, maxima, derasa, tevaroa, squamosa, crocea, Hippopus hippopus, and Hippopus porcelanus, has shown that they consist of an inner nacreous layer composed of 3 to 5% organic fraction, which itself is made up of collagenous and non-collagenous proteins, essentially insoluble and soluble biopolymers. The inner nacreous layer also contains an inorganic fraction representing 95 to 97%, mainly composed of calcium carbonate, minerals, and metal ions, as well as 3% water. This study of the shell architecture of the mollusks concerned by the invention also shows that it consists of an outer calcitic layer, structurally different from the inner aragonitic layer, but also containing an organic fraction composed of insoluble and soluble biopolymers.

Many publications have highlighted the osteoinductive and osteoconductive properties of the natural biomaterial derived from the aragonitic layer of the marine mollusks mentioned above.

These properties arise from the presence of biopolymers within the organic fraction, in which structural proteins similar to those contributing to the architecture of organs such as teeth, bones, skin, muscles, mucous membranes, etc., have been identified. Functional proteins similar to those involved in metabolic and biochemical processes (enzymology, immunology, membrane receptors, signaling molecules, etc.) are also present. Among these structural proteins, collagens are particularly represented: thus, collagens of types I, II, III, and related types have been identified.

In addition to free amino acids, proteoglycans (carbohydrates linked to small peptides) and glycoproteins (association of collagen and carbohydrates) have been identified, including low molecular weight glycoproteins, generally considered to be growth factors related to BMP, TNF β, TGF β, PGF, etc.

Moreover, the fundamental role of certain non-collagenous molecules in the physiological healing process and in cellular and tissue regeneration is known.

In vitro and in vivo, the wound-healing, regenerative, angiogenic, and osteoinductive properties of the organo-mineral complex from the inner layer of the shells of the aforementioned mollusks have been demonstrated. These properties are related to the presence of various collagens and growth factors.

If we compare the physico-chemical composition of bone tissue with that of aragonite in the shells of considered mollusks, we observe a strong similarity in the organic components, which represent 32% in bone tissue and 3 to 5% in aragonite. The mineral phases, 43% in bone, essentially calcium phosphate, account for 95 to 97% in aragonite in the form of calcium carbonate; the proportions of the other minerals being very close.

Considering the role of the biopolymers contained in the organic fraction of the natural marine biomaterial, the inventors found it appropriate to modify their composition by increasing the proportion of these biopolymers in the formulation of a new semi-synthetic hybrid biomaterial.

It is known that the organic fraction of the internal aragonitic and external calcitic layers of the shells of the relevant mollusks contains soluble, diffusible molecules with osteogenic properties involved in the mineralization and growth of calcified tissues. Insoluble structural proteins have also been identified in the peri-crystalline and inter-lamellar envelopes of the aragonite.

Moreover, the molecules contained in the organic fraction of the outer calcitic layer of the shell are similar to those contained in the inner aragonitic layer of the shell of the mollusks concerned by the invention.

This is why it seemed appropriate to extract and concentrate not only the organic molecules closely associated with the biocrystals and intercrystalline lamellae that make up the aragonite of nacreous shell tests, but also those contained in the external calcitic layer of the shells of the relevant mollusks.

The extraction of biopolymers from the organic fraction of the biomaterial aims to provide both soluble and insoluble molecules. The objective is to increase, by supplementing with extracted insoluble and soluble biopolymers, the organic-to-inorganic structural ratio, in order to optimize the cellular and tissue regeneration properties, healing, osteoinduction, and angiogenesis of the resulting biomaterial.

This is how the present inventors have found that it is possible, starting from the shell of a selected mollusk among Tridacnae maxima, Tridacnae gigas, Tridacnae derasa, Tridacnae tevaroa, Tridacnae squamosa, Tridacnae crocea, Hippopus hippopus, Hippopus porcelanus, Pinctada maxima, Pinctada margaritifera, and other Pinctadines, to obtain a material meeting these requirements by simultaneously adding both soluble and insoluble biopolymers and calcium carbonate transformed by carbonation.

Thus modified, the new semi-synthetic, powdered, bioresorbable material according to the invention is intended for example for the manufacture of bone substitutes, injectable cements or cement for prosthetic fixation, or for the production of osteosynthesis devices and molded bioresorbable implants.

Thus, the invention relates, according to a first aspect, to a semi-synthetic, powdery material derived from a natural marine biomaterial, compounded with insoluble and soluble biopolymers and calcium carbonate transformed by carbonation.

The invention also relates to a process for preparing this semi-synthetic material.

It also concerns a composition including soluble and insoluble biopolymers or calcium carbonate transformed by carbonation, which is implemented in the semi-synthetic material.

Finally, it concerns the use of semisynthetic materials or compositions for the manufacture, for example, of bone substitutes, injectable cements, or cementing cements for endoprostheses, or also for the development of osteosynthesis devices and molded, biodegradable implants.

Detailed description of the invention:

According to a first aspect, the invention relates to a semi-synthetic, powdery material derived from a natural marine biomaterial, supplemented with insoluble and soluble biopolymers and calcium carbonate transformed by carbonation.

The material according to the invention is derived from a natural marine biomaterial, which is the internal aragonitic layer of the shell of selected bivalve mollusks belonging to the group including Pinctadines, notably Pinctada maxima, margaritifera, and Tridacnes, notably Tridacna gigas, maxima, derasa, tevaroa, squamosa, crocea, Hippopus hippopus, Hippopus porcelanus, said aragonitic layer being in a powdered form.

The pulverulent semi-synthetic material according to the invention is biodegradable.

According to one embodiment, the particle size is from 5 nm to 100 µm, preferably from 20 nm to 50 µm, and more preferably from 50 nm to 20 µm.

Soluble and insoluble biopolymers are extracted from the internal aragonitic layer and/or the external calcitic layer of the shell of selected bivalve mollusks belonging to the group including Pinctadines, such as Pinctada maxima, margaritifera, and Tridacnes, such as Tridacna gigas, maxima, derasa, tevaroa, squamosa, crocea, Hippopus hippopus, and Hippopus porcelanus.

A method for extracting these polymers is described below.

According to a particular method of realization, the addition of soluble and insoluble extracted biopolymers is done according to a ratio of soluble biopolymers to insoluble biopolymers corresponding to that existing in the original biomaterial.

The calcium carbonate treated by carbonation, used in the semi-synthetic material of the invention, comes from a natural terrestrial, marine or precipitated calcium carbonate, or from the inorganic fraction of the aragonitic layer after extraction of the insoluble and soluble biopolymers, which has been treated by carbonation. It is known that calcium carbonate, crystallized in the orthorhombic or rhombohedral system, when subjected to a thermal treatment between 800 and 1100°C, acquires new properties through pyrolysis and oxidation, which are manifested by significant adhesive power and plasticity allowing easy shaping. This phenomenon is called carbonation, according to the following reaction: CaCO3 + thermal treatment → Ca(OH)2 + CO2 → CaCO3 + H2O.

During this reaction, during which the temperature rises and is maintained for a duration of 20 to 40 minutes, calcium carbonate chemically transforms into lime, and then, under the action of CO2 and ambient humidity, becomes amorphous calcium carbonate. This chemical transformation takes place over several days depending on the ambient humidity.

Thus, the pulverulent semi-synthetic material according to the invention comprises a powder derived from a natural marine material, whose organic fraction is supplemented with extracted insoluble and soluble biopolymers, and whose mineral fraction consists of calcium carbonate of marine, sedimentary or coral origin, or of terrestrial sedimentary or precipitated origin, which has been transformed by a carbonation process.

According to a particular embodiment, the pulverulent semi-synthetic material according to the invention comprises aragonite in a powdered form having a particle size of 5 nm to 100 µm, preferably 20 nm to 50 µm, more preferably 50 nm to 20 µm, insoluble and soluble extracted biopolymers, and calcium carbonate transformed by carbonation.

By adding insoluble and soluble extracted biopolymers, the proportion of the organic fraction of the original material is increased within a range of 1% to 10%, preferably maintaining the existing ratio between insoluble and soluble biopolymers in the original material. By adding calcium carbonate produced through carbonation, the proportion of the mineral fraction of the original material is increased within a range of 1% to 10%, depending on the desired physicochemical characteristics.

According to a particular embodiment, the semi-synthetic material according to the invention comprises: for 100 g of finely powdered aragonite with a particle size ranging from 5 nm to 100 µm, preferably from 20 nm to 50 µm, more preferably from 50 nm to 20 µm; from 1 g to 50 g, preferably from 5 g to 25 g, more preferably from 10 g to 15 g of extracted insoluble and soluble biopolymers; and from 0.5 g to 50 g, preferably from 1 g to 25 g, more preferably from 2 g to 10 g of calcium carbonate transformed by carbonation.

During the extraction of biopolymers, the inventors highlighted that in the internal aragonitic layer and the external calcitic layer of the mollusks used in the implementation of the invention, the proportion of insoluble biopolymers ranges from 2.6% to 4.3%, and that of soluble biopolymers ranges from 0.4% to 0.7% of the total weight. The addition of biopolymers into the material according to the invention is carried out such that the ratio of soluble biopolymers to insoluble biopolymers is similar to the ratio found in the natural original product.

The invention also relates to a process for preparing a pulverulent semi-synthetic material, as described above.

According to the method of the invention, the constituent elements are prepared separately and then mixed in order to obtain the material according to the invention. Thus, the powder material derived from a natural marine biomaterial is prepared, along with the insoluble and soluble biopolymers extracted from a natural marine biomaterial, and the calcium carbonate transformed by carbonation.

More specifically, the preparation process comprises mixing a crushed natural biomaterial, insoluble and soluble polymers extracted from the internal aragonitic layer and/or the external calcitic layer of the shells of selected bivalve mollusks belonging to the group including Pinctadines, such as Pinctada maxima, margaritifera, and Tridacnes, such as Tridacna gigas, maxima, derasa, tevaroa, squamosa, crocea, Hippopus hippopus, Hippopus porcelanus, and calcium carbonate transformed by carbonation.

In a particular method of realization, the crushed natural biomaterial is the internal aragonite layer of the shell of mollusks. The grinding is performed in order to obtain an average particle size ranging from 20 nm to 50 µm. The resulting particles can be spheroidized to improve the flowability and compressibility of the powder.

In the process according to the invention, the insoluble and soluble biopolymers are extracted respectively by super-centrifugation, and by tangential ultrafiltration combined with reverse osmosis after hydrolysis. Before extraction, the internal aragonitic layer and/or the external calcitic layer of the mollusk shell may be cross-linked. To facilitate extraction, the internal aragonitic layer and/or the external calcitic layer of the mollusk shell are ground and sieved to a particle size ranging between 250 µm and 50 µm.

These different steps are described successively below.

The naturally derived marine biomaterial used as a raw material is selected from the group including Pinctadines, such as Pinctada maxima, margaritifera, and Tridacnes, such as Tridacna gigas, maxima, derasa, tevaroa, squamosa, crocea, Hippopus hippopus, and Hippopus porcelanus.

Each component can come from the same marine biomaterial or from different marine biomaterials.

The selected shells are cleaned, decontaminated, and optionally reticulated; the calcitic layer is separated from the inner layer. The inner layer is crushed. A portion of the crushed inner layer constitutes the base component of the material according to the invention. Soluble and insoluble biopolymers are extracted from the calcitic layer and/or the inner layer. Calcium carbonate, which may come from the mineral part recovered after extraction of the biopolymers, is transformed by carbonation. The extracted biopolymers and the calcium carbonate transformed by carbonation are added to the previously obtained base component.

A specific method of implementing the process according to the invention is described in detail below. Of course, those skilled in the art will know how to adapt the conditions of this process to the specific starting biomaterials and the desired final uses.

I. PREPARATION OF THE COMPONENTS:

After removing the epibionts by scraping, the shells from the selected marine biomaterial undergo the following treatments:

I.1) Decontamination of the shells:

The shells are decontaminated by immersion in a tap water bath supplemented with a 2% active chlorine hypochlorite solution.

I.2) Treatment of shells with ultrasound:

The shells are then rinsed and treated in an ultrasonic bath filled with microbiologically controlled tap water, for example at a temperature of 55°C, to which a cleaning and disinfecting solution is added at a dilution of 1 part of solution to 127 parts of water. The treatment duration is about 30 minutes at a frequency of approximately 40 kHz.

I.3) Rinsing and drying of the shells:

The shells are then rinsed, for example, for 20 minutes in a demineralized water bath at a temperature of 90°C, supplemented with Calbénium® at a 2% dilution, for 30 minutes. They are then dried.

I.4) Shell Reticle:

According to another embodiment, in order to impart enhanced biological properties to the natural biomaterial, particularly for optimizing cellular metabolism and strengthening antioxidant properties, the shells can be cross-linked as follows: In a translucent glass or plastic container of variable capacity, a mixture of tap water supplemented with 10% riboflavin is prepared; the whole is kept at a temperature above 20°C, with the agitation of the mixture generating a flow perpendicular to UVA radiation.

The shells are placed vertically and exposed on both sides to UVA lamps with a wavelength of 365 nanometers, at an intensity of 2300 microjoules per square centimeter for 180 minutes. The entire setup is kept under vacuum throughout the entire treatment.

The shells are then rinsed and dried with a stream of hot air at 40°C.

One can also use the method described in the French patent application FR 14 50204 filed on January 10, 2014.

I.5) Removal of the outer calcitic layer:

The outer calcite layer of the shells is removed by grinding with a fine-grit wheel.

The product is reserved and constitutes the "Extraction Batch of Bio-Polymers from the Outer Calcitic Layer."

I.6) Freezing of etched tests after grinding:

According to the invention, the nacre tests are frozen at a temperature of -18°C for 120 minutes.

I.7) Crushing of nacre tests and recovery of batches:

Next, the nacreous tests are crushed, for example in a tungsten carbide jaw crusher under suction, in order to recover the suspended particles, which also contain nano-grains.

The crushing operation is repeated at least three times, and after sieving, two batches are reserved: The first batch, with a particle size ranging from 20 microns to 50 nanometers, will constitute the mixed aragonite portion of the product according to the invention hereinafter referred to as "mixed aragonite batch." By "mixed aragonite batch," we mean the powdered form obtained after grinding, containing both organic and inorganic components. The second batch, with a particle size ranging from 250 to 50 microns, is reserved for the extraction of insoluble and soluble biopolymers. It will be called "batch for extraction of biopolymers from the internal aragonite layer."

The use of a laser particle analyzer will determine the size and range of the powder particles obtained.

I.8) Spheroidization of the mixed aragonite batch:

The mixed aragonite batch is subjected to a mechanical treatment aimed at homogenizing the particles through spheroidization, the purpose being to round the edges and corners of the grains by friction.

This treatment has the effect of improving the flowability and compressibility of the resulting powder, thus promoting densification and interparticle bonding during the material's application according to the invention, particularly as bone substitutes, cements, injectable cements, osteosynthesis devices, and bioresorbable molded implants.

For this step of spherical formation, the following procedure can be used: place in a cylindrical glass or zirconium container, with a horizontal axis of rotation, equipped with glass blades of varying width, a mixture in equal parts of powdered material from the mixed aragonite batch and wood chips of a few mm², for example oak, sterilized in an autoclave.

The container is rotated for a variable duration and speed, depending on the size of the container and the amount of product to be processed.

After the spherification process, the entire mixture, consisting of a mixed aragonite and chip material, is recovered in an inert container filled with an adequate amount of water, which is continuously stirred for about 15 minutes. After settling, the floating wood chips on the surface are removed by suction.

The solution is then filtered through a nylon filter with mesh openings of 20 microns in diameter, and the residue is dried using a Rotavapor® at 40°C and packaged.

According to another embodiment, the mixed aragonite batch can also be equally added with sodium chloride in the form of grains with random diameters ranging from 1 to 3 mm. After treatment, the sodium chloride is removed by dissolving it in hot water at 90°C and filtering through a nylon filter, followed by washing with hot water at 90°C and drying with warm air at 40°C.

II. EXTRACTION OF BIOPOLYMERS II.1 Extraction of insoluble biopolymers:

According to the invention, an appropriate amount of powder from the batch of extracted biopolymers from the internal aragonite layer obtained at step I.5) is mixed with a sufficient amount of demineralized water and then injected into a hydrolysis reactor, where a determined amount of 25% citric acid is added; the whole mixture is cooled to a temperature fluctuating between 4 and 5°C under constant stirring. The inventors favored the use of citric acid due to its pH-lowering and surface tension-reducing properties.

The pH, controlled by a pH meter, is maintained above 4.5 by adding 2.5 N sodium hydroxide to prevent alteration of the biopolymers; it is then adjusted back to 7 at the end of the step by adding 0.1 liter of 5N sodium hydroxide per 100 liters of hydrolysate.

Once the powder is completely dissolved, the hydrolysate is transferred to a storage tank, still under constant agitation, and then transferred to a centrifugal separator where it is subjected to a force of 18 to 20,000G in the cyclone.

The operation is repeated if necessary after checking the solution with a turbidimeter and adjusting with citric acid if needed, the temperature being kept between 4 and 5°C.

According to the results provided by the turbidimeter, the hydrolysate can undergo super centrifugation again. At each cycle of super centrifugation, the sediment of insoluble biopolymers collected is washed and reserved. The wash water from the sediments is treated with oxalic acid to check for the presence or absence of calcium.

After the last ultra-centrifugation, we therefore obtain a pellet containing all the insoluble biopolymers, in the form of a moist brown cake, which is dried by lyophilization or zedration, ending with gray spheres of 2 to 3 mm in diameter, resulting from the coiling of proteins under the action of centrifugal force.

The insoluble extracted biopolymers are crushed, for example, in a planetary mill until a powder with a random particle size of 5 micrometers to 100 nanometers is obtained, which is recovered after sieving.

II.2 Extraction of soluble biopolymers:

The permeate and wash water are conveyed to be desalted in a tangential ultrafiltration system, for example with cassettes having a cut-off threshold of 1 kD.

An appropriate amount of 2.0 mol/L sulfuric acid is added to the permeate in order to cause the precipitation of calcium sulfate salts.

The solution is filtered, and the permeate is concentrated under vacuum in a Rotavapor® at a boiling temperature of 33°C to remove the citric acid in the form of crystals.

The distillate containing low-molecular-weight proteins as well as monovalent and polyvalent ions is extended.

The cutoff threshold of the membranes does not retain all the proteins, especially those with very low molecular weight, so the distillate is subjected to reverse osmosis.

The distillate is transferred to undergo liquid-phase separation by membrane permeation, for example with pore diameters of 0.0001 microns, under a pressure gradient of 40 to 80 bars.

The distillate was passed in order to retain all monovalent and polyvalent ions such as iron, magnesium, zinc, etc.

The recovered retentate from the reverse osmosis membranes is collected and diluted with pyrogen-free water, then concentrated, for example, by means of a Rotavapor® under vacuum at a temperature of 40°C, and then lyophilized by zedratisation or cryodesiccation.

A very fine grayish-white powder is obtained, which is then ground, for example in a planetary mill, to obtain after sieving a powder with a random particle size distribution ranging from 5 microns to 100 nanometers.

We check the presence or absence of proteins in the permeate by taking an aliquot of the solution, which is then treated using the Bradford colorimetric method.

II.3 Extraction of biopolymers from the biopolymer extraction batch of the outer calcitic layer

According to another embodiment, the extraction of biopolymers from the external calcitic layer is performed in the same way as that of biopolymers from the internal aragonitic layer.

III. CARBONATION OF CALCIUM CARBONATE:

It is known that calcium carbonate, crystallized in the orthorhombic or rhombohedral system, when subjected to thermal treatment between 800 and 1100°C, acquires new properties through pyrolysis and oxidation, which result in significant adhesive power and plasticity allowing easy shaping. This phenomenon is called carbonation, according to the following reaction: CaCO3 + thermal treatment → Ca(OH)2 + CO2 → CaCO3 + H2O

During this reaction, which causes an increase in temperature and maintains it for a duration of 20 to 40 minutes, calcium carbonate chemically transforms into lime. Then, under the action of CO2 and ambient humidity, it becomes amorphous calcium carbonate. This chemical transformation takes place over several days depending on the ambient humidity.

According to other methods of realization, all calcium salts other than calcium carbonate can, through chemical precipitation reactions, give rise to calcium carbonate that can then be converted by carbonation. Thus, for example, carbonated calcium carbonate can be obtained from calcium hydroxide, calcium acetate, calcium oxalate, calcium sulfate, and calcium citrate; it will be up to the skilled person to implement the known chemical processes suitable for these precipitations.

Calcium carbonate can also come from the aragonite internal shells of bivalve mollusks such as Pinctadines in general, and particularly Pinctada: maxima, margaritifera, and Tridacna: gigas, maxima, derasa, tevaroa, squamosa, crocea, Hippopus hippopus, Hippopus porcelanus, after extraction of biopolymers. It can also be of madrepore origin.

IV. FORMULATION OF A MIXTURE FROM THE ARAGONITE MIXED BATCH, THE EXTRACTED INSOLUBLE AND SOLUBLE BIOPOLYMERS, AND THE CALCITE CARBONATE OBTAINED BY CARBONATION

A certain amount of insoluble and soluble biopolymers, extracted from the two internal aragonitic and external calcitic fractions, determined according to the desired organic fraction content, and a defined amount of calcium carbonate transformed by carbonation, are mixed with a defined amount of the mixed aragonitic batch to form a product formulation according to the invention.

The mixing is performed, for example, in a knife mixer until a homogeneous powder is obtained, which is then packaged.

According to another aspect, the invention relates to the use of the material according to the invention as a bone substitute in a custom formulation, for the healing and regeneration of substance loss, for the treatment of burns, pressure ulcers, ulcers, erythematous skin lesions, or in the manufacture of molded devices or implants.

The pulverulent semi-synthetic material according to the invention can also be used in the manufacture of molded devices or implants with controlled biodegradation, including sutures with time-delayed biodegradation.

It can also be used for the formulation of bone substitute preparations for on-demand use, porous collagen-based bone substitutes, mineral scaffold bone substitutes of animal or human origin, osteosynthesis devices and molded bioresorbable implants, devices with controlled bioresorption, cement for fixation of endoprostheses, injectable cements for minimally invasive surgery in vertebroplasty, kyphoplasty, and bone tumor surgery.

According to another embodiment, the product according to the invention can be associated with a porous collagenous support such as Spongia officinalis that has undergone mechanical and thermochemical treatment intended for bacterial and viral decontamination, removal of any possible pigments, and neutralization of an immunogenic response. It is known that Spongia officinalis is composed of spongin, which itself consists of fibers of a carbonate scleroprotein related to collagen. This protein is poorly soluble and plays a protective and supportive role for all tissues: connective tissue, tendons, bone tissues, muscle fibers, skin, hair, and nails. Spongin is a collagen-like structural and storage protein; it is inert, insoluble in water, hydrophobic, and does not denature easily. It forms a porous support suitable for osteoconduction. Therefore, it can be used in combination with the material according to the invention for the production of bone substitutes.

The material according to the invention can be associated with calcium salts such as anhydrous or hemi-hydrated calcium sulfate, calcite, anhydrous calcium hydroxyphosphate, β-TCP, and calcium hydroxide. The material according to the invention can be associated with mineral matrices of bone tissues of animal or human origin.

It can also be associated with biodegradable polymers such as collagen, hyaluronic acid, chitosan, starch, alginate, or with synthetic resorbable polymers such as polyglycolide, poly(DL-lactide-co-glycolide), poly(L-lactide), or with acrylic polymers such as poly(hydroxyethyl methacrylate), methyl methacrylate, polymethyl methacrylate, as well as with powdered medicaments such as non-steroidal anti-inflammatory drugs, antibiotics, antimitotics, or any other therapeutic substance.

Considering the disadvantages related to the use of methyl methacrylate-based bone cements, the inventors propose bone cements manufactured with the product according to the invention, which is naturally radiopaque and provides primary mechanical retention of the endoprosthesis due to its adhesive properties, eventually leading to tissue integration due to its osteomimetic, osteoinductive, osteoconductive, and bioactive properties, induced by the presence of signaling molecules that initiate biomineralization. These signaling molecules stimulate local endogenous factors of biomineralization in situ, resulting in the formation of metaplastic bone.

According to another aspect, the invention relates to the use of calcium carbonate that has undergone carbonation as implemented in the material according to the invention or as prepared according to step III of the process described above, in compositions comprising calcium salts, natural or synthetic polymers, collagen, mineral matrices of animal or human bone tissue. It may also be associated with biodegradable polymers such as collagen, hyaluronic acid, chitosan, starch, or alginate, or with synthetic resorbable polymers such as polyglycolide, poly(DL-lactide-co-glycolide), poly(L-lactide), or acrylic polymers such as poly(hydroxyethyl), methyl methacrylate, poly(methyl methacrylate), as well as with powdered medicinal substances such as non-steroidal anti-inflammatory drugs, antibiotics, antimitotics, or any other therapeutic substance.

It is known that both insoluble and soluble biopolymers contained in the organic fraction of aragonitic and calcitic layers have healing and regenerative properties, not only for hard tissues such as bone and cartilage, but also for soft tissues such as skin, muscles, and mucous membranes. Some of these non-collagenous biopolymers, particularly low-molecular-weight glycoproteins, can be compared to growth factors such as BMP, TNFβ, EGF, TGFβ, IGF, FGF, etc., as well as to cytokines, which are mediators of inflammation.

The invention also relates to the use of soluble and insoluble biopolymers implemented in the material according to the invention or such as they are extracted by step II of the process described above as adjuvants of powder compositions comprising calcium salts, natural or synthetic polymers, collagen, mineral matrices of bone tissues from animal or human origin. They can also be associated with bioresorbable polymers such as collagen, hyaluronic acid, chitosan, starch, alginate, or with synthetic resorbable polymers such as polyglycolide, poly(DL-lactide-co-glycolide), poly(L-lactide), or with acrylic polymers such as poly(hydroxyethyl), methyl methacrylate, poly(methyl methacrylate), as well as with medicamentous substances in powder form, such as non-steroidal anti-inflammatory drugs, antibiotics, antimitotics, or any other therapeutic substance. They can also be associated with calcium carbonate transformed by carbonation.

The invention will be described in more detail with the aid of the following examples, given only for illustration, and the attached drawings, on which: Figure 1 and Figure 2 are photographs of mixtures: of mother-of-pearl powder and calcium carbonate with whole blood (No. 1) and of mother-of-pearl powder and calcium carbonate that have undergone carbonation with whole blood (No. 2), taken respectively 2 minutes and 15 minutes after addition of whole blood.

EXAMPLES:

In order to verify the pharmacological properties of the product according to the invention, the inventors prepared therapeutic formulations and used them in clinical observation studies.

EXAMPLE 1:

The pulverulent semi-synthetic material according to the invention was prepared as follows:

I. PREPARATION OF THE COMPONENTS:

After removing the epibiont by scraping, the shells undergo the following treatments:

I.1) Decontamination of the shells:

The shells are decontaminated by immersion in a tap water bath supplemented with a 2% active chlorine hypochlorite solution.

I.2) Treatment of shells with ultrasound:

The shells are then rinsed and treated in an ultrasonic tank filled with microbiologically controlled tap water at a temperature of 55°C, to which a cleaning and disinfecting solution is added at a dilution of 1 part of solution to 127 parts of water. The treatment duration is 30 minutes at a frequency of 40 kHz.

I.3) Rinsing and drying of the shells:

The shells are then rinsed for 20 minutes in a demineralized water bath at a temperature of 90°C, supplemented with Calbénium® at a 2% dilution for 30 minutes. They are then rinsed and dried.

I.4) Removal of the outer calcitic layer:

The external calcite layer of the shells is removed by grinding with a fine-grit wheel. The product is set aside and constitutes the "Extraction Batch of Bio-Polymers from the External Calcite Layer."

I.5) Freezing of glazed tests exposed after grinding:

The nacre tests obtained in step I.4) are frozen at a temperature of -18°C for 120 minutes.

I.6) Crushing of nacre tests and recovery of batches:

Next, the nacreous tests are crushed in a tungsten carbide jaw crusher, brand ESSA®, under suction, in order to recover the suspended particles, which also contain nano-grains.

The crushing operation is renewed at least three times, and after sieving, two batches are reserved: The first one, with a particle size ranging from 20 microns to 50 nanometers, will constitute the mixed aragonite portion of the product according to the invention hereinafter referred to as "mixed aragonite batch." By "mixed aragonite batch," we mean the powdered form obtained after grinding, containing both organic and inorganic components. The second batch, with a particle size ranging from 250 to 50 microns, is reserved for the extraction of insoluble and soluble biopolymers. It will be called "batch for extraction of biopolymers from the internal aragonite layer." The size and range of the powder particles are determined using a laser particle size analyzer.

I.7) Spherification of the mixed aragonite batch:

The mixed aragonite batch is subjected to a mechanical treatment aimed at homogenizing the grains through spheroidization, the purpose being to round the edges and corners of the grains by friction.

A mixture of equal parts of the pulverulent material from the mixed aragonite batch and 5 mm² chips of hard wood, such as oak, sterilized in an autoclave, is placed into a cylindrical zirconium vessel with a horizontal axis of rotation, equipped with glass paddles of variable width.

The container's rotations are carried out for a variable duration and speed, depending on the size of the container and the amount of product to be processed.

After the spherification process, the entire mixture, consisting of a mixed aragonite and chip mixture, is recovered in an inert container filled with an adequate amount of water, which is continuously stirred for a duration of 15 minutes. After a resting period of 30 minutes, the floating wood chips on the surface are removed by suction.

The solution is then filtered through a nylon filter with mesh openings of 20 microns in diameter, and the residue is dried using a Rotavapor® at 40°C and packaged.

II. EXTRACTION OF BIOPOLYMERS II.1 Extraction of insoluble biopolymers:

An adequate amount of powder from the extraction batch of the inner aragonite layer is mixed, by suction into the feed tank of Zone I, with a sufficient amount of demineralized water, and then injected into Zone II of the hydrolysis reactor, where a defined amount of 25% citric acid is added; everything is cooled to a temperature ranging between 4 and 5°C under constant stirring. The pH, controlled by a pH meter, is kept above 4.5 by adding 2.5 N sodium hydroxide solution in order to prevent degradation of the biopolymers; it is then adjusted to 7 at the end of the step by adding 0.1 liter of 5N sodium hydroxide per 100 liters of hydrolysate.

Once the powder is completely dissolved, the hydrolysate is transferred to the storage tank, still under constant stirring, and then transferred to the centrifugal separator where it is subjected to a force of 18 to 20,000G in the cyclone.

The operation is repeated if necessary after checking the solution with a turbidimeter and adjusting with citric acid if needed, the temperature being kept between 4 and 5°C.

According to the results provided by the turbidimeter, the hydrolysate undergoes another super centrifugation.

At each cycle of super-centrifugation, the sediment of insoluble biopolymers collected is washed and reserved. The washing water from the sediments is treated with oxalic acid to check for the presence or absence of calcium.

After the last ultra-centrifugation, a pellet containing all the insoluble biopolymers is obtained, in the form of a moist brown cake, which is then freeze-dried, resulting finally in grayish spheres of 2 to 3 mm in diameter, formed by the coiling of proteins under the action of centrifugal force.

The insoluble extracted biopolymers are crushed in a planetary mill until a powder with a random particle size ranging from 5 microns to 100 nanometers is obtained, which is recovered after sieving.

II.2 Extraction of soluble biopolymers:

The permeate and washing water are conveyed to be desalted in the tangential ultrafiltration cassette assembly unit, Millipore® units each with a 1 kDa molecular weight cut-off, connected in series for a total surface area of 15 m², under a pressure of 5 bars, with a flow rate of 10 to 15 liters per hour, at a temperature of 40°C.

An adequate amount of 2.0 mol/L sulfuric acid is added to the permeate in order to cause the precipitation of calcium sulfate salts. The solution is filtered, and the permeate is concentrated under vacuum using a Rotavapor® at a boiling temperature of 33°C to remove the citric acid in the form of crystals.

The distillate containing low-molecular-weight proteins as well as monovalent and multivalent ions is extended.

Since the cutoff threshold of the membranes does not retain all the proteins, especially those of very low molecular weight, the distillate is subjected to reverse osmosis.

The distillate is then transferred to undergo liquid-phase separation by membrane permeation, where the membrane pore diameter is 0.0001 micron, under a pressure gradient of 40 to 80 bars and.

The distillate is passed to retain all monovalent and polyvalent ions such as iron, magnesium, zinc, etc. The retentate recovered from the reverse osmosis membranes is collected and diluted with pyrogen-free water, then concentrated by Rotavapor® under vacuum at a temperature of 40°C, and finally lyophilized by zedration.

A very fine grayish-white powder is obtained, which is then ground in a planetary mill to obtain, after sieving, a powder with a random particle size ranging from 5 microns to 100 nanometers.

We check for the presence or absence of proteins in the permeate by taking an aliquot of the solution, which is then treated using the Bradford colorimetric method.

III. CARBONATION OF CALCIUM CARBONATE:

The recovered calcium carbonate after the above extraction of biopolymers is subjected to thermal treatment between 800 and 1100°C for 20 to 40 minutes, then slowly cooled to room temperature. This process is called carbonation, according to the following reaction:        CaCO3 + thermal treatment → Ca(OH)2 + CO2 → CaCO3 + H2O

During this reaction, calcium carbonate chemically transforms into lime, and then, under the action of CO2 and moisture, reverts back to amorphous calcium carbonate. This chemical transformation takes place over several days depending on the ambient humidity.

IV. FORMULATION OF A MIXTURE FROM THE MIXED ARAGONITE BATCH, IN SOLUBLE AND SOLUBLE BIOPOLYMERS EXTRACTS, AND THE CALCITE CARBONATE OBTAINED BY CARBONATION

During the extraction of biopolymers, it was found that in the internal aragonitic layer and the external calcitic layer of the shells used, the proportion of insoluble biopolymers ranged from 2.6% to 4.3%, while that of soluble biopolymers ranged from 0.4% to 0.7%.

The material was prepared according to the invention by mixing the aragonite mixed batch, the insoluble polymers obtained in step II.1, the soluble polymers obtained in step II.2, and the calcium carbonate that underwent carbonation obtained in the above step III. The specific amounts of the different components are specified in each of the application examples given below.

The mixing is carried out in a knife mixer until a homogeneous powder is obtained, which is then packaged.

EXAMPLE 2:

It is done as in Example 1 above, except that an additional crosslinking step as described below is added at the end of Step I.3.

In a transparent glass or plastic container, a mixture of tap water enriched with 10% riboflavin is prepared; the entire mixture is kept at a temperature above 20°C, and the agitation of the mixture generates a flow perpendicular to the UVA radiation. The shells are placed vertically and exposed on both sides to UVA lamps with a wavelength of 365 nanometers, at an intensity of 2300 microjoules per square centimeter for 180 minutes. The entire setup is maintained under vacuum throughout the treatment.

The shells are then rinsed and dried with a current of hot air at 40°C.

EXAMPLE 3:

In order to check the adhesion and cohesion properties of the calcified calcium carbonate, the following procedure is carried out: In two Dappen cups, named respectively Dappen No. 1 and No. 2, each containing 1 g of the mother-of-pearl powder obtained at the end of step I.7 of the process from Example 1, the following are added: 0.1 g of natural calcium carbonate (Dappen No. 1), and 0.1 g of naturally calcified calcium carbonate, resulting from step III of the process of Example 1 (Dappen No. 2).

After mixing, the contents of each Dappen well are mixed with 2 cc of whole blood.

A photograph of each Dappen cup is taken 2 minutes (Figure 1) and 15 minutes (Figure 2) after mixing with whole blood.

As shown in Figure 1(1), the mixture of Dappen No. 1 remains in the form of a red powder, no coagulum is formed. After 15 minutes, no coagulum has formed (Figure 2(1)).

As shown in Figure 1 (2), the mixture of Dappen No. 2 quickly forms a clot and gradually changes from red to brown, gains mass, can be shaped, becomes sticky, and hardens after 15 minutes (Figure 2(2)).

EXAMPLE 4: Formulation for an improvised bone substitute

A case of critical clinical situation is presented as a chip fracture of the cannon bone of a one-year-old filly, treated by osteosynthesis. After failure of osteosynthesis, resulting in fracture of four screws, a pseudoarthrosis with sepsis followed by a secondary comminuted fracture with small fragments, leaving euthanasia as the only alternative, the decision was made to use the material according to the invention, formulated as follows: 40 g of a mixed aragonite powder with a particle size ranging from 50 nanometers to 20 microns, obtained from step I.8 of Example 1; 0.070 g of insoluble biopolymers extracted at step II.2 of Example 1; 0.010 g of soluble biopolymers extracted at step II.1 of Example 1; 2 g of calcified calcium carbonate obtained from step III of the process of Example 1; 10 ml of autologous venous blood to form a clot, shaped into a cylinder 10 cm long and 2 cm in diameter, placed in the bone defect after removal of the bone sequestra.

The member protected by bandages was casted. Post-operative X-rays showed the presence and integration of the bone substitute according to the invention, followed by consolidation at 4 months, after which the filly could run and jump over obstacles. Subsequent control X-rays showed complete restoration of the bone shaft with reconstruction of the medullary canal.

The same formulation was also used by preparing a clot spontaneously with 2.5 ml of water for injection (PPI) at room temperature.

EXAMPLE 5: Formulation of a skin healing cream

A preparation of the product according to the invention is made according to the following centesimal formulation: 10 g of a mixed aragonite material with a particle size of 50 nanometers to 20 micrometers obtained according to Example 2; 0.035 g of insoluble biopolymers extracted obtained at step II.2 of Example 1; 0.005 g of soluble biopolymers extracted obtained at step II.1 of Example 1; 0.5 g of calcined calcium carbonate; 15 drops of an essential oil complex containing per 100 ml: Lavandula spica: 1 ml; Salvia officinalis: 2 ml; Rosa rubiginosa: 10 ml; Helichrysum italicum: 1.5 ml; Wheat germ vegetable oil: 50 ml; Evening primrose oil: 10 ml; Sweet almond oil: 20 ml; H.E. emulsion qsp 100 g.

This preparation was applied on a cutaneous necrosis of the sternum in a horse, extending from the base of the neck to the axillae, covering a height of 32 cm and a width of 18 cm. Clinical observation showed exceptional healing of 1 cm per day in both height and width, with reconstruction of the different aponeurotic, subcutaneous, and skin layers, and simultaneous regrowth of the hair without depigmentation, resulting in complete healing of the skin within 28 days.

EXAMPLE 6: Formulation for a dermatological preparation for the treatment of psoriasis

It is known that psoriasis is an inflammatory skin condition characterized by accelerated cell renewal without apoptosis, leading to the formation of thick, scaly plaques. Apart from corticotherapy and local treatments based on coal tar and phototherapy, which have inconsistent and disappointing results, there are more aggressive treatments with dangerous side effects for the patient.

A centesimal preparation of the product according to the invention is prepared according to the following formulation: 3 g of insoluble biopolymers extracted obtained in step II.2 of Example 1; 0.45 g of soluble biopolymers extracted obtained in step II.1 of Example 1; 0.5 g of calcium carbonate obtained in step III of Example 1; 10 drops of an essential oil complex containing per 100 ml: Lavandula spica: 1 ml; Salvia officinalis: 2 ml; Rosa rubiginosa: 10 ml; Helichrysum italicum: 1.5 ml; Wheat germ vegetable oil: 50 ml; Evening primrose oil: 10 ml; Sweet almond oil: 20 ml; H.E. emulsion qsp 100 g.

This emulsion is applied daily on severe psoriasis lesions located on the chest, back, arms, and legs. After the third application, the redness disappears, indicating the calming of the inflammatory process, the scales, and the itching, as well as the reduction of secondary infections with a significant aesthetic improvement. The improvement of clinical signs reflects the trophic, anti-inflammatory, and regenerative properties of the insoluble and soluble biopolymers.

EXAMPLE 7: Formulation of a wound dressing for burns

The exceptional tissue regeneration properties of the insoluble and soluble bio-polymers extracted according to step II of Example 1 were highlighted in a case of deep second-degree and third-degree burns after failure of keratinocyte grafting, using the following formulation: For 100 g: 50 g of a mixed aragonite granulometry ranging from 50 nanometers to 20 microns obtained according to Example 2; 0.174 g of insoluble bio-polymers extracted at step II.2 of Example 1; 0.026 g of soluble bio-polymers extracted at step II.1 of Example 1; Cera de Gallien in cherry laurel water, q.s.p. 100 g.

The preparation is applied to all burned surfaces under an occlusive dressing and renewed every 72 hours.

Repeated clinical exams showed a sedation of the exudative phenomenon, significant angiogenesis, pain reduction, re-epithelialization of the affected areas, and a notable decrease in fibroplastic tension.

EXAMPLE 8: Formulation for a bioresorbable molded bone substitute

The material according to the invention can be used for the manufacture of osteosynthesis devices and molded biodegradable implants.

According to the invention, for 100 g: 80 g of a mixed aragonite powder with a particle size ranging from 50 nanometers to 20 micrometers obtained in step I.8 of Example 1; 0.139 g of insoluble biopolymers extracted in step II.2 of Example 1; 0.021 g of soluble biopolymers extracted in step II.1 of Example 1; 20 g of polyethylene glycol 400; and 4 g of calcium carbonate obtained in step III of Example 1.

The mixture is kneaded in a mixer for 10 minutes at room temperature until a homogeneous, pliable, extrudable, and moldable paste is obtained.

We take impressions of adapted shapes, based on the digital modeling of the anatomy of the potential insertion areas for osteosynthesis devices and/or implants.

A sufficient amount of the previously obtained paste is injected into the compression chamber of a mold having one or more cavities.

The assembly is then compressed under a gradually increasing pressure ranging from 100 to 220 N; the pressure is maintained for a variable period and gradually decreases until it reaches the value 0.

Once demolded and dried at 40°C, the device is packaged in double packaging and sterilized by ionizing radiation at 25 kGy.

EXAMPLE 9: Preparation for a controlled biodegradable bone substitute

It has been observed that the bio-resorption of a bone substitute or a bioresorbable device is directly related to the diameters of the interconnected pores, which must range from 5 to 100 microns to allow colonization by new blood vessels and cells involved in bone remodeling.

This is the reason why inventors propose the development of bone substitutes or molded implants, whose interconnected porosity would be controlled. To do this, prepare for 100 g: 80 g of a mixed aragonite granular material with a particle size ranging from 50 nanometers to 20 micrometers, obtained as in Example 2; 0.139 g of insoluble biopolymers extracted obtained at step II.2 of Example 1; 0.021 g of soluble biopolymers extracted obtained at step II.1 of Example 1; 20 ml of a 50% hydroxypropylmethylcellulose (HPMC) solution; 20 mm³ of monofilament synthetic absorbable sutures, 5 mm long, ranging from diameter 5/0 to 12/0.

These absorbable threads are polymers such as glycolic acid, glycolide copolymer, polyglactin (Vicryl Rapid or irradiated), and chitosan. These threads have a staggered absorption period ranging from 12 to 90 days.

As in the previous example, the paste is injected into the molds, then compressed. The devices or implants are then demolded, dried, packaged in double packaging, and sterilized as previously done at 25 KGy.

EXAMPLE 10: Preparation for Injectable Bone Substitute and Prosthesis Cement

A cement is prepared whose composition is as follows per 100 g: 80 g of the material according to the invention, composed of: 73 g of a mixed aragonite material with a particle size ranging from 50 nanometers to 20 microns obtained at the end of step I.8; 2.702 g of insoluble biopolymers extracted at step II.2 of Example 1; 0.405 g of soluble biopolymers extracted at step II.1 of Example 1; 3.699 g of calcium carbonate obtained from step III of Example 1; 20 g of high viscosity aqueous solution of HPMC at 50%.

The resulting product is packaged under vacuum or in a controlled atmosphere in syringes with variable capacities, for example from 0.5 cm³ to 1 cm³, with straight or curved nozzles, stored at a temperature of about 4° C.

This preparation can also be used as a cementing agent, and it helps prevent the cementing material from entering the circulatory system, for example, when cementing the prosthesis stem into the medullary cavity.

Moreover, considering its composition, it does not cause the release of volatile substances that could affect the respiratory system.

Such a composition is also proposed for vertebroplasty and kyphoplasty in minimally invasive surgery.

EXAMPLE 11: Preparation for a collagen-based bone substitute

Bone substitutes are prepared with the following composition: For 100 g, it is prepared as follows: 50 g of a mixed aragonite powder with a particle size ranging from 50 nanometers to 20 microns obtained in step I.8 of Example 1; 0.087 g of insoluble biopolymers extracted in step II.2 of Example 1; 0.013 g of soluble biopolymers extracted in step II.1 of Example 1; 2.5 g of calcium carbonate obtained in step III of Example 1; 50 g of polyethylene glycol (PEG) with a molecular weight gradient of 400.

The mixture is kneaded until a gel with a viscosity of about 10 Pa·s is obtained.

To this gel, 30 g of commercially available Spongia officinalis is added, reduced to fragments of 2 mm in size.

The mixture is kneaded until a homogeneous paste with a viscosity of approximately 108 Pa·s is obtained. The mixture is then injected into a mold containing impressions of osteosynthesis devices or implants. After demolding, the devices or implants are dried in a hot air current at 40°C, packaged in double packaging, and sterilized according to the current protocol.

EXAMPLE 12: Preparation for bone substitute

According to another embodiment, the bio-polymers extracted solely from the aragonitic fraction and/or the calcitic fraction can be added to any other synthetic or natural biomaterial in order to optimize or induce certain properties, particularly osteoinductive or osteomimetic properties, which they lack.

This is how osteoconductive substitutes, such as certain calcium salts, were supplemented with the biopolymers extracted from the aragonite layer, according to the following formulation for 100 g: 95 g of βTCP granules with a particle size ranging from 50 to 250 microns; 4.4 g of insoluble biopolymers extracted at step II.2 of Example 1; and 0.6 g of soluble biopolymers extracted at step II.1 of Example 1.

This mixture combined with autologous blood is inserted into a bone defect created by the excision of a cyst at the apex of the upper central incisor.

At the same time, βTCP is compacted in a substance loss created by the excision of a periapical granuloma from the upper canine.

A radiological examination performed at two weeks showed a more significant and rapid bone density increase in the cystic cavity treated with the βTCP + insoluble and soluble extracted biopolymers mixture, compared to the second cavity, where βTCP particles were clearly visible and only osteoconduction was expressed. In contrast, in the cystic cavity, osteoinduction occurred simultaneously with osteoconduction, indicating that βTCP has acquired a new property.

Claims (17)

1. Pulverulent semisynthetic material, derived from a natural marine biomaterial, with addition of insoluble and soluble biopolymers and calcium carbonate transformed by carbonation, said semisynthetic material being bioabsorbable and the natural marine biomaterial is the aragonitic inner layer of the shell of bivalve molluscs.
2. Semisynthetic material according to Claim 1, characterized in that the bivalve molluscs selected from the group comprising Pinctadines, notably Pinctada maxima, margaritifera, and Tridacnes, notably Tridacna gigas, maxima, derasa, tevaroa, squamosa, crocea, Hippopus hippopus, Hippopus porcelanus, said aragonitic layer being in pulverulent form.
3. Semisynthetic material according to Claim 1 or 2, characterized in that the natural biomaterial in pulverulent form has a granulometry from 5 nm to 100 µm, preferably from 20 nm to 50 µm, even more preferably from 50 nm to 20 µm.
4. Semisynthetic material according to any one of Claims 1 to 3, characterized in that the insoluble and soluble biopolymers are extracted from the aragonitic inner layer and/or from the calcitic outer layer of the shell of the bivalve molluscs selected from the group comprising Pinctadines, notably Pinctada maxima, margaritifera, and Tridacnes, notably Tridacna gigas, maxima, derasa, tevaroa, squamosa, crocea, Hippopus hippopus, Hippopus porcelanus.
5. Semisynthetic material according to any one of Claims 1 to 4, characterized in that the calcium carbonate transformed by carbonation is derived from a natural terrestrial, natural marine or precipitated calcium carbonate, or from the inorganic fraction of the aragonitic layer after extraction of the insoluble and soluble biopolymers.
6. Method for preparing a material according to any one of Claims 1 to 5, comprising mixing a ground natural biomaterial, insoluble and soluble polymers extracted from the aragonitic inner layer and/or from the calcitic outer layer of the shell of the bivalve molluscs selected from the group comprising Pinctadines, notably Pinctada maxima, margaritifera, and Tridacnes, notably Tridacna gigas, maxima, derasa, tevaroa, squamosa, crocea, Hippopus hippopus, Hippopus porcelanus, and calcium carbonate transformed by carbonation.
7. Method for preparing according to Claim 6, characterized in that the ground natural biomaterial is obtained by grinding the aragonitic inner layer of the shell of the bivalve molluscs selected from the group comprising Pinctadines, notably Pinctada maxima, margaritifera, and Tridacnes, notably Tridacna gigas, maxima, derasa, tevaroa, squamosa, crocea, Hippopus hippopus, Hippopus porcelanus.
8. Method for preparing according to Claim 6 or 7, characterized in that it comprises a spherification step after grinding.
9. Method according to any one of Claims 6 to 8, characterized in that the insoluble and soluble biopolymers are extracted respectively by supercentrifugation, and by tangential ultrafiltration coupled to reverse osmosis after hydrolysis.
10. Method according to Claim 9, characterized in that the aragonitic inner layer and/or the calcitic outer layer of the shell of the molluscs is(are) crosslinked before extraction.
11. Method according to Claim 9 or 10, characterized in that the aragonitic inner layer and/or the calcitic outer layer of the shell of the molluscs is(are) ground and sieved to a granulometry between 250 µm and 50 µm before extraction.
12. Pulverulent semisynthetic material according to any one of Claims 1 to 5, or obtained according to the method of one of Claims 6 to 11 for use as bone substitute for extemporaneous formulation, for healing and regeneration of losses of substance, for treating burns, sores, ulcers, or erythematous skin lesions.
13. Use of the pulverulent semisynthetic material according to any one of Claims 1 to 5, or obtained according to the method of one of Claims 6 to 11 in the manufacture of devices or moulded implants.
14. Use of the pulverulent semisynthetic material according to any one of Claims 1 to 5, or obtained according to the method of one of Claims 6 to 11, in the manufacture of devices or moulded implants with controlled bioabsorption comprising suture threads with bioabsorption staggered over time.
15. Use of the pulverulent semisynthetic material according to any one of Claims 1 to 5, or obtained according to the method of one of Claims 6 to 11, for formulation of preparations for bone substitutes for extemporaneous use, for extrudable bone substitutes, notably packaged in a syringe under vacuum, bone substitutes with a porous collagen support, bone substitutes with a mineral structure of animal or human origin, bioabsorbable osteosynthesis devices and moulded implants, devices with controlled bioabsorption, cements for sealing endoprostheses, and injectable cements for minimally invasive surgery in vertebroplasty and kyphoplasty.
16. Use of the calcium carbonate that has undergone carbonation employed in the material according to any one of Claims 1 to 5, as additive that is plastic, modellable and adhesive, in compositions comprising calcium salts, natural or synthetic polymers, collagen, and mineral structures of bone tissues of animal or human origin.
17. Use of the insoluble and soluble extracted biopolymers employed in the material according to any one of Claims 1 to 5, as additives in pulverulent compositions comprising calcium salts, natural or synthetic polymers, collagen, and mineral structures of bone tissues of animal or human origin.