Composition for use as a dentin substitute - Patents.com

Abstract

The present disclosure relates to compositions comprising collagen microparticles containing greater than 90% by weight collagen, biomimetic hydroxyapatite or a biomimetic hydroxyapatite precursor, and a physiologically compatible aqueous solvent for use in dentin repair.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to the field of dentin repair and regeneration. [Background technology]

[0002] 2. Background of the Invention The longevity of the world's population, together with the desire of patients to keep their teeth for life, is influencing the way dental professionals deal with the caries process. The traditional surgical model approach for dental treatment consisting of "cavity restoration - new carious restoration, replacement tooth, extraction of weakened tooth" is gradually being replaced by a much more conservative medical model in which minimally invasive dentistry is widely practiced. In this context, dentists are increasingly interested in preserving as much enamel and dentin as possible. Moreover, remineralization of the tooth matrix can be considered the basis for preventive and non-surgical treatment strategies for caries.

[0003] Dentin is mainly composed of hydroxyapatite (HAp) crystals and type I collagen. Caries disease is due to bacteria that produce acids that dissolve HAp crystals and destroy collagen fibers. Unless the demineralization process is reversed towards mineralization by increasing the local pH, which favors mineral deposition in the dentin, the caries process will progress until a cavity in the tooth is formed. This cavity needs to be filled by different types of materials, and this is the basis of the dentist's daily clinical work to fill cavities that are only late symptoms of a disease that initially began with simple demineralization from the tooth surface. Sometimes the extent and depth of the cavity will compromise the mechanical integrity of the tooth and the vitality of the dental pulp (nerve).

[0004] WO 2007 / 009477 discloses a bone repair composition comprising a matrix-building polymer (i.e. collagen) by including hydroxyapatite particles (50% of said particles have a size of 5 nm or less) as a biomaterial for medical applications (such as bone implant material or dental cement). The hydroxyapatite used in WO 2007 / 009477 is different from biomimetic hydroxyapatite at least in terms of chemical composition, size and shape. Also, the composition is not sufficiently concentrated in collagen to obtain a liquid crystal structure. In addition, WO 2007 / 009477 teaches that collagen may be replaced by gelatin or that the composition may be prepared at temperatures up to 45° C., thus allowing working above 40° C., i.e. above the temperature at which collagen irreversibly denatures in vitro and turns into a gelatinized material. Finally, all of the compositions exemplified in WO 2007 / 009477 are dried prior to use to form a powder, which results in the collagen being a brittle sponge-like material rather than a hydrogel containing banded fibrils.

[0005] In this context, tooth-like materials that are able to "repair" carious dentin are desirable, in particular tooth-like materials that are able to repair demineralized dentin at later stages of caries disease, i.e. when cavities have already formed. Summary of the Invention

[0006] The present invention relates to a composition for use in dentin repair and regeneration, comprising: Non-crosslinked and non-denatured collagen microparticles containing more than 90% by weight of collagen; biomimetic hydroxyapatite or a biomimetic hydroxyapatite precursor; and Physiologically compatible aqueous solvent The present invention relates to a composition comprising:

[0007] The present invention also relates to preformed implantable matrices comprising the compositions as disclosed herein.

[0008] Further aspects of the present invention are as disclosed herein and in the claims. [Brief description of the drawings]

[0009] [Figure 1] TGA thermograms of hybrid collagen materials showing good agreement between the initial weight (collagen microparticles contain approximately 10% water by weight) and the measured organic and inorganic content (initial collagen / hydroxyapatite ratio of 1:1). [Diagram 2] DSC analysis of different collagen materials prepared with saline solution showing similar endothermic peaks typical of collagen denaturation. [Diagram 3] PLM observation of hybrid collagen solutions: bright birefringent structures evidence anisotropic organization. [Figure 4] SEM micrographs of mineralized collagen material before fibrillogenesis (left) showing partially dissolved collagen microparticles. After fibrillogenesis (right), the material exhibits better defined collagen fibrils. [Diagram 5] SEM micrograph of a mineralized collagen gel (1:1 collagen / hydroxyapatite ratio) showing aligned domains of fibrils in a dense matrix. [Figure 6] TEM micrograph of an unstained ultrathin section of a collagen / HA (50:50) matrix with high dry matter content showing the co-alignment of collagen fibrils and hydroxyapatite nanoplatelets. [Figure 7] Energy dispersive X-ray spectroscopy (EDS) chemical profile at the dentin / mixture interface (right bar: mean C; middle bar: mean P; left bar: mean Ca in each case). [Figure 8]Cross-sectional scanning electron microscope (SEM) micromorphology of the composition of Example 2 in contact with demineralized dentin. The presence of a biomodified layer is shown between the two white lines. [Figure 9] Cross-sectional SEM micromorphology of the composition of Example 3 in contact with demineralized dentin. The presence of a biomodified layer is shown between the two black lines. [Figure 10] SEM micrograph of vacuum dried pourable hybrid material of Example 4 showing dense organic-inorganic integration of Mixture 3. [Figure 11] SEM micrograph of vacuum dried pourable hybrid material of Example 4 showing dense organic-inorganic integration of Mixture 3. [Figure 12] Cross-sectional SEM micromorphology of the vacuum-dried injectable hybrid material of Example 4 applied to demineralized dentin. The presence of a biomodified layer is indicated between the two white arrows. [Figure 13] Cross-sectional SEM micromorphology of vacuum dried injectable hybrid material of Example 4 showing the biomimetic properties of Mix 3 when applied over dentin. [Figure 14] Cross-sectional SEM micromorphology of the vacuum-dried injectable hybrid material of Example 4 applied to demineralized dentin. The biomimetic boundary is indicated by the white arrow. It can be seen that the biomimetic mixture is very well integrated into the underlying dentin. Description of the Invention

[0010] Detailed Description of the Invention The inventors have discovered that a composition comprising biomimetic hydroxyapatite or amorphous calcium phosphate and dense collagen microparticles can repair demineralized dentin. After injection, the composition is biomimetic in microstructure and can serve as a scaffold to promote dentin repair. The main advantage of such a composition is that due to its similar composition to dentin, it serves as a neo-substrate for dentin bonding.

[0011] Accordingly, the present invention relates to compositions as disclosed hereinafter for use in the repair and regeneration of dentin, particularly for use in repairing damage to dental dentin.

[0012] composition The composition for use according to the invention comprises · High density collagen microparticles (i.e. microparticles containing more than 90% collagen by weight); biomimetic hydroxyapatite or biomimetic hydroxyapatite precursor or amorphous calcium phosphate; and Physiologically compatible aqueous solvent The present invention relates to a method for producing a semiconductor device comprising the steps of:

[0013] The composition is suitable for injection and / or implantation. The composition may then be defined as being injectable and / or implantable.

[0014] The components of the composition are as described in detail herein below.

[0015] High-density collagen particles The term "high density collagen microparticles" as used herein refers to collagen microparticles comprising more than 90% collagen by weight, in particular more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98% collagen by weight, the remainder being water.

[0016] High density collagen microparticles are as disclosed in WO 2016 / 146954.

[0017] High density collagen microparticles are in the form of solid spherical or spheroidal particles formed from undenatured and uncrosslinked collagen. The diameter of the particles is typically in the range of 0.05-20 μm, particularly in the range of 0.25-10 μm, and more particularly in the range of 0.4 μm-3 μm. It should be understood that the particle diameter range refers to the diameter distribution. The particles typically have a diameter ranging from a minimum diameter of 0.05 μm to a maximum diameter of 20 μm.

[0018] The term "spheroidal" as used herein refers to a solid object whose shape resembles that of a sphere.

[0019] The term "diameter" refers to the diameter of a sphere or the maximum diameter of a spheroid. Diameter can be measured, for example, by electron microscopy or by dynamic light scattering.

[0020] The term "non-denatured" as used herein refers to collagen in which the secondary structure of the α-triple helix is ​​preserved. The non-denatured or denatured nature of collagen can be observed, for example, by colorimetry. Denatured collagen has a colorimetric profile characteristic of denatured proteins (gelatin) without any sign of organized macromolecular domains. Dried collagen (which results in a sponge-like material without any banded fibrils) and gelatinized collagen are considered "denatured" collagen. The use of non-denatured collagen is advantageous in that it will improve the ability of the composition to behave as a biomimetic scaffold and allow the recruitment and activation of hard tissue forming cells to stimulate dentin formation and thus regeneration.

[0021] The term "non-crosslinked," as used herein, refers to collagen in which no crosslinks are present, whether these are the result of chemical modification (such as treatment with glutaraldehyde) or enzymatic or physical modification. The absence of crosslinks can be determined, for example, by electrophoresis.

[0022] High density collagen microparticles may be prepared from various collagens. The source of collagen is therefore not problematic. Collagen can be obtained according to the following protocol: A solution of type I collagen is prepared from the tail tendons of Wistar rats. After dissection in a laminar flow cabinet, the tendons are washed in a sterile saline phosphate buffer. They are then immersed in a solution of 4M NaCl in order to remove the remaining intact cells and to precipitate part of the high molecular weight proteins. After washing with saline phosphate buffer, the tendons are solubilized in a sterile 500 mM acetic acid solution. The resulting solution is clarified by centrifugation at 41000g for 2 hours. Proteins other than collagen are selectively precipitated in an aqueous solution of 300mM NaCl and removed by centrifugation at 41000g for 3 hours. Collagen is recovered from the supernatant by precipitation in a solution of 600mM NaCl followed by centrifugation at 3000g for 45 minutes. The resulting pellet is solubilized in an aqueous solution of 500 mM acetic acid and then dialyzed in the same solvent to remove NaCl ions. The solution is kept at 4° C. and centrifuged at 41000 g for 4 hours before use. This detailed protocol can be applied to other types of collagen.

[0023] The collagen of the high density collagen microparticles typically has a molecular weight in the range of 200-450 KDa.

[0024] The collagen of the high density collagen microparticles is typically collagen type I. Nevertheless, the collagen may alternatively be type II, III, V, XI, XXIV, XXVII, and mixtures thereof.

[0025] High density collagen microparticles may be prepared by a spray processing technique as disclosed in WO 2016 / 146954. Briefly, this spray processing technique consists in atomizing an acid-soluble collagen solution (undenatured and uncrosslinked collagen) to form a very thin mist of droplets that are immediately dried by evaporation of the solvent in a controlled atmosphere (due to the large solution / air interfacial area of ​​the droplets). The concentration of collagen in the acidic collagen solution typically ranges from 0.1 to 10 mg / L. The acidic collagen solution has a pH of less than 7. The acid is typically acetic acid. The concentration of acetic acid in the acidic collagen solution typically ranges from 0.1 to 1000 mM. The atomization is typically carried out at a temperature below about 40° C., in particular below about 39° C., below 38° C. or below 37° C., to obtain a powdered composition. The concentration in the collagen droplets is high enough to induce self-assembly of collagen molecules and subsequent liquid crystal ordering (e.g., nematic orientation domains). This strategy allows highly concentrated collagen microparticles to be obtained within seconds, avoiding the large increase in viscosity of type I collagen solutions that normally prevents rapid processing of this protein, and consequently its use at biological concentrations.

[0026] Thus, advantageously, the composition comprises 40 mg / mL of collagen relative to the total weight of the composition.

[0027] Biomimetic Hydroxyapatite The chemical formula of biomimetic hydroxyapatite is Ca 10-x (PO4) 6-x (CO3) x (OH) 2-x (Wherein, 0≦x≦2).

[0028] The term "biomimetic hydroxyapatite" refers to bone-like hydroxyapatite platelets that are typically approximately 10-200 nm in length, 25-100 nm in width, and 1-10 nm in thickness as measured by transmission electron microscopy.

[0029] The biomimetic hydroxyapatite is typically in the form of a powder.

[0030] Biomimetic hydroxyapatite powders may be synthesized according to the procedure described by Nassif et al., Chemistry of Materials, 22(12), pp3653-3663, 2010. Briefly, biomimetic hydroxyapatite is synthesized by vapor diffusion of ammonia (NH3) into an acidic calcium phosphate (CaCl2-NaH2PO4-, or possibly with other salts, especially NaHCO3) solution under thermodynamic conditions to avoid precipitation formation of other calcium phosphate phases. For example, biomimetic hydroxyapatite may be synthesized by vapor diffusion of Ca 10 by precipitation in an acidic solution of CaCl2 / NaH2PO4 (acetic acid, 500 mM) with a calcium to phosphate (Ca / P) molar ratio consistent with the formation of hydroxyapatite, which has the formula (PO4)6(OH2), or by Ca 10-x (PO4) 6-x (CO3) x (OH) 2-x It may be prepared by precipitation of an acidic solution of CaCl2 / NaH2PO4 / NaHCO3 (acetic acid, 500 mM) with a calcium to phosphate plus carbonate (Ca / [P+C]) molar ratio consistent with the formation of hydroxyapatite, which has a formula of 0≦x≦2. Precipitation is induced by the addition of aqueous ammonia (30%, w / w).

[0031] This precipitation method has the advantages of being free of any organic additives, being performed without direct pH control, and being performed within a few hours at room temperature, and does not produce any by-products or undesired (i.e., non-physiological) phases.

[0032] Synthesis of biomimetic hydroxyapatite as disclosed by Nassif et al., 2010, has been shown to result in nanoplatelets that exhibit self-assembly properties in water similar to natural bone apatite (Wang,et al., “Water-mediated structuring of bone apatite”, Nature materials 12.12(2013):1144-1153). The nanoplatelets have been shown to have a crystalline core and an amorphous shell with an X-ray diffraction pattern consistent with that of JCPDS N 9-0432. Nanoplatelets typically measure 200×100×5 nm 3 and carbonate substitution as observed for bone mineral. Such self-assembly properties are not exhibited by non-biomimetic hydroxyapatites, and particularly those having hydroxyapatite particles that do not exhibit an amorphous layer.

[0033] It should be noted that the composition of hydroxyapatite can also be modified, in particular enriched with strontium (up to 10% calcium substitution), in order to combine anti-osteoporotic effects (Tovani et al., 'Formation of stable strontium-rich amorphous calcium phosphate: Possible effects on bone mineral', Acta biomaterialia, 2019). In such cases, strontium-enriched biomimetic hydroxyapatite typically has the formula: Ca 10-x (PO4) 6-x Sr y (CO3) x (OH) 2-x (wherein, 0≦x≦2 and 0≦y≦10−x, where y is, for example, 0.1 * (equal to (10-x)).

[0034] Biomimetic hydroxyapatite precursors The term "biomimetic hydroxyapatite precursor" refers to precursor ions that induce the formation of biomimetic hydroxyapatite under conditions described, for example, in Nassif et al., Chemistry of Materials, 22(12), pp3653-3663, 2010.

[0035] Suitable biomimetic hydroxyapatite precursors include CaCl2.2H2O, NaH2PO4, and NaHCO3, as well as salts that may be found in inorganic bone compositions, including salts of magnesium, zinc, fluorine, and strontium.

[0036] The Ca / P molar ratio is typically in the range of 1.5-2.

[0037] The calcium to phosphate + carbonate (Ca / [P+C]) molar ratio is preferably Ca 10-x (PO4) 6-x (CO3) x (OH) 2-x (where 0≦x≦2) (Von Euw, scientific reports 2019), which is consistent with the formation of hydroxyapatite, which has a formula of (typically 1.67; approximately 1.2-1.5 for bone tissue).

[0038] Amorphous calcium phosphate The term "amorphous calcium phosphate" refers to amorphous calcium phosphate particles.

[0039] The amorphous calcium phosphate is typically in the form of a powder.

[0040] Amorphous calcium phosphate powders may be synthesized by atomization of a biomimetic hydroxyapatite precursor acidic solution using spray processing techniques such as those disclosed in WO 2016 / 146954. Amorphous calcium phosphate powders have an average size, as measured by transmission electron microscopy, that is typically in the range of 3-6 μm.

[0041] Aqueous Solvent The aqueous solvent may include any physiologically compatible aqueous solvent. Non-limiting examples of suitable aqueous solvents include physiological serum, phosphate buffer, sodium bicarbonate, sterile water, normal saline, blood or plasma.

[0042] Advantageously, the weight ratio of aqueous solvent to the mixture of high density collagen microparticles and hydroxyapatite or amorphous calcium phosphate is typically in the range of 1.8-10, preferably in the range of 2-9, more preferably in the range of 3-8.

[0043] When a biomimetic hydroxyapatite precursor is used, the weight ratio corresponds to the weight ratio of aqueous solvent to a mixture of dense collagen microparticles and equivalent hydroxyapatite obtained with said biomimetic precursor.

[0044] This concentration is advantageous in that the composition does not dry out (thus avoiding collagen denaturation) and the composition is sufficiently concentrated to preserve self-assembly of the collagen molecules and subsequent liquid crystal ordering (with nematically oriented domains) while remaining injectable.

[0045] Any Therapeutic or Bioactive Agent The compositions may include one or more therapeutic or bioactive agents, such as, for example, anti-inflammatory agents, saliva, antibiotics, bone morphogenetic proteins, hyaluronic acid, and anti-osteoporotic agents (eg, salts).

[0046] Compositions for use according to the invention typically contain from 20 mg to 100 mg of high density collagen microparticles per mL of composition, preferably from 40 mg to 80 mg of high density collagen microparticles, more preferably from 50 mg to 70 mg of high density collagen microparticles.

[0047] In some embodiments, the weight ratio of high density collagen microparticles to biomimetic hydroxyapatite or amorphous calcium phosphate in a composition (which may be prepared according to Process 1) ranges from 10:90 to 90:10, preferably from 30:70 to 80:20, and more preferably 50:50 or 30:70.

[0048] One of skill in the art will readily adjust the weight ratio of high density collagen microparticles to biomimetic hydroxyapatite or amorphous calcium phosphate to adapt the composition's formulation to its intended use and site of administration.

[0049] The composition of the present invention can be easily embedded or injected or otherwise applied at the site where there is a need for dentin repair.For example, the composition can be suitably injected by syringe directly at the site of the defect to be repaired.The composition has the ability to fill the targeted defect and take the same 3D shape.The composition is sufficiently adhesive / tacky to be fixed and sustained in the defect without the use of external aids or mediators.

[0050] Alternatively, the composition can be poured into a mold to form a preformed matrix. The preformed matrix is ​​implantable. The present invention also relates to a preformed implantable matrix comprising the composition as disclosed herein for use in dentin repair, particularly for use in repairing damage to dental dentin, more particularly for use in dentin repair where a cavity has already been formed.

[0051] Compositions for use according to the present invention may suitably be prepared as disclosed hereinafter.

[0052] When the composition is prepared according to Process 1, the composition more specifically comprises: · high density collagen microparticles (i.e. microparticles comprising more than 90% collagen by weight); Biomimetic hydroxyapatite platelets or amorphous calcium phosphate; and Physiologically compatible aqueous solvent The term "compound" may be defined as comprising:

[0053] When the composition is prepared according to process 2, the composition more specifically comprises: Hybrid dense collagen microparticles (i.e., dense collagen microparticles containing biomimetic hydroxyapatite precursors); and A physiologically compatible aqueous medium optionally containing a biomimetic hydroxyapatite precursor The term "compound" may be defined as comprising:

[0054] When the composition is prepared according to process 3, the composition more specifically comprises: High density collagen microparticles (i.e., microparticles comprising more than 90% collagen by weight); and A physiologically compatible aqueous medium containing a biomimetic hydroxyapatite precursor The term "compound" may be defined as comprising:

[0055] Process for preparing the composition Process 1: Mixing high density collagen microparticles and hydroxyapatite or amorphous calcium phosphate The composition may be prepared by mixing a desired weight of high-density collagen microparticles (typically in the form of a powder) with a desired weight of hydroxyapatite or amorphous calcium phosphate powder. The high-density collagen microparticles, hydroxyapatite powder and amorphous calcium phosphate powder may be prepared as described herein above. The high-density collagen microparticles and hydroxyapatite particles or amorphous calcium phosphate powder are typically mixed in a mortar. The high-density collagen microparticles and hydroxyapatite powder or amorphous calcium phosphate powder are typically mixed in a weight ratio that is suitably selected to reproduce the target tissue and can be adapted to the target application. Non-limiting examples of suitable weight ratios of high-density collagen microparticles to hydroxyapatite powder or amorphous calcium phosphate powder include the following ratios: 10 / 90 to 90 / 10, preferably 30:70 to 80:20, more preferably 50:50 or 30:70.

[0056] After the high density collagen microparticles and the hydroxyapatite powder or amorphous calcium phosphate powder are mixed in a suitable weight ratio, an aqueous solvent is added to the mixture as described above in this specification. The weight ratio of the aqueous solvent to the mixture of the high density collagen microparticles and the hydroxyapatite powder or amorphous calcium phosphate powder is typically in the range of 1.8-10 (i.e., in the range of 0.18 mL-1 mL of solvent per 100 mg of the mixture of the high density collagen microparticles and the hydroxyapatite powder or amorphous calcium phosphate powder), preferably in the range of 2-9, and more preferably in the range of 3-8.

[0057] The mixture may then be supplemented with one or more therapeutic or bioactive agents, such as an anti-inflammatory agent or an anti-osteoporosis agent.

[0058] After mixing, the resulting composition may be loaded into a sterile syringe since it is in a paste or liquid form.

[0059] All steps of the disclosed processes are preferably carried out under sterile conditions.

[0060] The syringe may then be stored in a dry place at a temperature below the denaturation temperature of collagen, preferably at 4° C. in a refrigerator.

[0061] Alternatively, the composition may be prepared by atomizing an acidic solution containing a biomimetic hydroxyapatite precursor and collagen (Process 2), or dense collagen microparticles may be mixed with an aqueous solution containing a biomimetic hydroxyapatite precursor (Process 3).

[0062] Process 2: Atomization of collagen solutions containing biomimetic hydroxyapatite precursors The composition may be prepared by a process that includes the step of atomization of a solution containing a hydroxyapatite precursor and collagen, the solution typically having an acidic pH (i.e., a pH strictly less than 7).

[0063] The spray processing technique is carried out as disclosed in WO 2016 / 146954. Atomization is carried out with an acid-soluble collagen solution (undenatured and uncrosslinked collagen). The concentration of collagen in the acidic collagen solution is typically in the range of 0.1-10 mg / L. The acidic collagen solution has a pH of less than 7. The acid is typically acetic acid. The concentration of acetic acid in the acidic collagen solution is typically in the range of 0.1-1000 mM. The collagen solution is mixed with a desired volume / concentration of a biomimetic hydroxyapatite precursor solution (i.e. the acidic collagen solution is supplemented with an ionic precursor of hydroxyapatite). In a preferred configuration, the biomimetic hydroxyapatite precursor solution is made by dissolving biomimetic hydroxyapatite platelets in an acidic solution.

[0064] Atomization is typically carried out at a temperature below about 40° C., particularly below about 39° C., 38° C., or 37° C., to obtain an undenatured powdered composition.

[0065] The microparticles resulting from the micronization are referred to herein as "hybrid dense collagen microparticles". Hybrid dense collagen microparticles are dense collagen microparticles containing biomimetic ionic precursors (e.g., CaCl2.2H2O, NaH2PO4 and NaHCO3). Hybrid microparticles with different ionic compositions may be obtained. Calcium acetate can be used as an alternative to calcium chloride to avoid NaCl precipitation.

[0066] The hybrid high density collagen microparticles are typically mixed with a physiologically compatible aqueous solvent (with or without a biomimetic hydroxyapatite precursor) in a weight ratio suitably selected to replicate the target tissue and that can be tailored to the target application.

[0067] After mixing, the resulting composition may be loaded into a sterile syringe since it is in a paste or liquid form.

[0068] All steps of the disclosed processes are preferably carried out under sterile conditions.

[0069] The syringe may then be stored in a dry place at a temperature below the denaturation temperature of collagen, preferably at 4° C. in a refrigerator.

[0070] Process 3: Mixing dense collagen microparticles with a biomimetic hydroxyapatite precursor solution The composition may be prepared by mixing a desired weight of high density collagen microparticles (typically in the form of a powder) with a desired volume of a biomimetic hydroxyapatite precursor solution. The high density collagen microparticles and the biomimetic hydroxyapatite precursor solution may be prepared as described herein above. The high density collagen microparticles and the biomimetic hydroxyapatite precursor solution are typically mixed in a mortar. The high density collagen microparticles and the biomimetic hydroxyapatite precursor solution are typically mixed in a weight ratio that is suitably selected to reproduce the target tissue and can be adapted to the target application. The volume of the biomimetic hydroxyapatite precursor solution added to the high density collagen microparticles typically results in a final concentration of collagen of 80 mg / mL.

[0071] After mixing, the resulting composition may be loaded into a sterile syringe since it is in a paste or liquid form.

[0072] All steps of the disclosed processes are preferably carried out under sterile conditions.

[0073] The syringe may then be stored in a dry place at a temperature below the denaturation temperature of collagen, preferably at 4° C. in a refrigerator.

[0074] Hereinafter, the embodiments of the present invention will be described with reference to examples, but these are for illustrative purposes only and are not intended to limit the scope of the present disclosure. EXAMPLES

[0075] Example 1: Injectable hybrid material (collagen / hydroxyapatite ratio, 50:50) in 0.9% saline Synthesis of carbonate-doped hydroxyapatite. The synthesis of carbonate-doped hydroxyapatite was carried out according to the procedure described by Nassif et al., Chemistry of Materials, 22(12), pp.3653-3663, 2010.

[0076] A solution of 110 mM CaCl2.2H2O, 33 mM NaH2PO4 and 33 mM NaHCO3 was prepared in 500 mM vinegar. The pH was adjusted to 2.2 with HCl solution at 37%. Two flasks (35 mL) were filled with 20 mL of this solution and placed in a sealed chamber (i.e., in a 1 L beaker covered with paraffin) in the presence of a third flask containing 8 mL of aqueous NH3 (28-30% by mass). Before closing, these three flasks were covered with parafilm in which six holes were pierced using a needle in order to slow down the gas-phase diffusion of ammonia. The device was then left for 6 days. The precipitate was then collected by centrifugation at room temperature (20 min at 6000 rpm) and washed with ultrapure water until the pH of the supernatant was close to that of the wash water. Finally, the resulting white powder was dried in an oven at 37 °C for 7 days. The dry powder was then pulverized in a mortar with a pestle to obtain a fine powder.

[0077] Synthesis of collagen microparticles by aerosol The synthesis of collagen microparticles was carried out according to the procedures described by Nassif et al, Paris, 2018: Injectable collagen suspensions, the preparation method thereof, and the uses thereof, particularly for forming dense collagen matrices; US Patent Application Publication No. 15 / 558,787 and Lama et al, Self-Assembled Collagen Microparticles by Aerosol as a Versatile Platform for Injectable Anisotropic Materials, Small, p.1902224, 2019.

[0078] A collagen solution concentrated to 1.2 mg / mL was obtained by diluting a stock solution of collagen (usually 1.3-5 mg / mL) with acetic acid (500 mM). 250 mL of the above solution was dried in a spray dryer (Buchi B290). The spray dryer was placed in a fume hood, next to a mobile reversible air conditioner. The temperature in the fume hood should ideally be maintained between 19°C and 21°C (preferably not exceeding 25°C). The injection rate of the collagen solution (at 1.2 mg / mL) was controlled by the peristaltic pump of the atomizer and was equal to 0.6 mL / min. The set temperature of the nozzle is maintained at 30°C. The actual temperature of the nozzle fluctuates between 34°C and 35°C (before starting the peristaltic pump) after 1 hour of stabilization in vacuum. The internal temperature of the system is measured between the drying column and the particle collection cyclone and is between 19°C and 25°C. The air flow responsible for shearing the droplets at the nozzle outlet is 414 L / h. The suction power controlling the drying of the droplets between the nozzle outlet and the collector is set at 50% of the maximum capacity of the drying system, i.e. at 20 m3 / h. The "nozzle" parameter used to prevent agglomeration of the solution at the end of the nozzle is set at 2. Aluminum is installed on both sides of the junction between the column and the cyclone to avoid heat losses as much as possible. The formed particles are collected by a high-performance cyclone connected to a flask. In order to recover all the powder remaining on the cyclone walls and to maximize the yield, the temperature set point is switched off at the end of atomization and the suction is increased from 50% (20 m3 / h) to 100% (40 m3 / h) in 10% steps by waiting 5 minutes per step. The process efficiency is between 50% and 60%. To guarantee sterile conditions, commercial devices of different sizes of filters sold by BEKO technologies can be used. It is also recommended that the entire set-up be sterilized with ethanol above 94° prior to spraying with collagen.

[0079] Preparation of the injectable composition 60 mg of collagen powder obtained as disclosed hereinabove and 60 mg of hydroxyapatite powder obtained as disclosed hereinabove were mixed in a mortar. 1 mL of sterile saline (0.9% NaCl) was added into the mortar. The whole was mixed for about 1 minute to obtain a homogenous paste. The paste was transferred into an empty 1 mL syringe. The plunger was replaced in place. The paste was then ready to be injected into the defect.

[0080] Preparation of preformed matrices The above protocol is repeated. The mixture is injected by syringe into a silicone mold of the desired size and total volume of 1 mL. Fibril formation (gelation) is carried out overnight under ammonia vapor. The gel is then removed from the mold and rinsed with saline until a neutral pH is reached. The material can then be embedded into the cavity that corresponds to the shape of the mold.

[0081] Characterization of injectable and preformed materials method: Thermogravimetric analysis (TGA): Experiments were carried out using a NIETZSCH STA 409PC instrument on a thermal microbalance under oxidizing atmosphere with a heating rate of 5°C / min from room temperature to 850°C.

[0082] Differential Scanning Calorimetry (DSC): Experiments were carried out using a TA Q-20 instrument. The heating rate was set at 5°C / min, and the temperature range was set from 20°C to 80°C. Approximately 20 mg of material pieces were weighed and placed in a sealed aluminum pan. An empty sealed aluminum pan was used as a reference.

[0083] Polarized Light Microscopy (PLM): The material was placed between a slide and a cover slip without any treatment. Observations were performed using a Zeiss AxioImager A2 POL in transmission. The microscope was equipped with standard accessories for examining birefringent samples under polarized light (i.e. crossed polarizers) and an AxioCam CCD camera.

[0084] Scanning electron microscopy (SEM): Samples were fixed in 2.5% glutaraldehyde solution. After washing in cacodylate / saccharose buffer solution, samples were dehydrated through ethanol baths (30% to 100% ethanol). Supercritical CO2 drying was performed by a CPD-300 (Leica). Dried samples were cut into small pieces, placed on carbon tape covering a sample holder, and covered with a 15 nm gold layer. Observations were performed by using a Hitachi S-3400N microscope operating at 3 kV and 30 pA.

[0085] The final composition of the material is consistent with that of the initial mixture, taking into account the presence of water (approximately 10%) in the collagen microparticles (Figure 1).

[0086] The denaturation temperature of collagen is about 48°C. This is close to the denaturation temperature reported for collagen gels (Tiktopulo and Kajava, 1998), indicating that the addition of saline can promote fibril formation. Indeed, when fibril formation is induced by ammonia vapor (mineralized collagen gel), the denaturation temperature remains unchanged. The addition of hydroxyapatite to the mixture of collagen microparticles and saline seems to induce favorable interactions: the denaturation enthalpy is increased and the width at the mid-height of the endotherm is less important (Figure 2). This means that the addition of HA tends to homogenize the collagen fibril (or fibril) population.

[0087] As observed by PLM (Figure 3), the solution exhibits domains of birefringence that attest to the anisotropy of the material and confirms that the addition of hydroxyapatite under these conditions does not interfere with the self-assembly of collagen in the liquid crystalline phase.

[0088] This local anisotropy can be seen by SEM through the observation of aligned mineralized collagen fibrils (Figure 4). Before fibril formation, the material also shows partially dissolved collagen microparticles. The dissolution of the microparticles can be controlled by the mixing time before injection. After fibril formation, more defined fibrils are observed.

[0089] Example 2 : Injectable hybrid material (collagen / hydroxyapatite ratio, 50:50) in 2 mM acetic acid Preparation of injectable hybrid materials 40 mg of collagen powder obtained as disclosed hereinabove and 40 mg of hydroxyapatite powder obtained as disclosed hereinabove are mixed in a mortar. 0.15 mL of 2 mM acetic acid is added to the mortar. The whole is mixed for about 1 minute to obtain a homogenous paste. The paste is transferred to an empty 1 mL syringe. The plunger is returned to its place. The paste is then ready to be injected into the defect.

[0090] Material characterization SEM observations show aligned domains of fibrils (Figure 5). The material appears to be dense.

[0091] SEM reveals areas of co-aligned collagen fibrils and hydroxyapatite nanoplatelets that resemble those seen in compact bone (Figure 6).

[0092] Example 3 : Injectable hybrid material in 2 mM acetic acid (collagen / hydroxyapatite ratio, 30:70) Preparation of injectable hybrid materials 24 mg of collagen powder obtained as disclosed hereinabove and 56 mg of hydroxyapatite powder obtained as disclosed hereinabove are mixed in a mortar. 0.15 mL of 2 mM acetic acid is added to the mortar. The whole is mixed for about 1 minute to obtain a homogenous paste. The paste is transferred to an empty 1 mL syringe. The plunger is returned to its place. The paste is then ready to be injected into the defect.

[0093] Example 4 : Injectable hybrid material (collagen / amorphous calcium phosphate ratio, 30:70) in 2 mM acetic acid Preparation of amorphous calcium phosphate Amorphous calcium phosphate powder is synthesized by atomization of a biomimetic hydroxyapatite precursor acidic solution of 110 mM CaCl2.2H2O, 33 mM NaH2PO4 and 33 mM NaHCO3 in 500 mM acetic acid using spray processing techniques as disclosed in WO2016 / 146954.

[0094] Preparation of preformed hybrid materials 40 mg of collagen powder obtained as disclosed hereinabove and 40 mg of amorphous calcium phosphate powder obtained as disclosed hereinabove are mixed in a mortar. 0.15 mL of 2 mM acetic acid is added to the mortar. The whole is mixed for about 1 minute to obtain a homogeneous paste. The paste can be injected into the mold via a 1 mL syringe or spread into the mold with a spatula. Fibril formation is carried out under ammonia vapor for 3 hours. The gel is then removed from the mold and rinsed with PBS until a neutral pH is reached. The material can then be embedded into the cavities corresponding to the shape of the mold.

[0095] Example 5: Injectable and preformable material (high-density collagen microparticles mixed with a biomimetic hydroxyapatite precursor solution) Preparation of the injectable composition 90 mg of collagen powder obtained as disclosed herein above was mixed with 1 mL of biomimetic hydroxyapatite precursor solution obtained as disclosed herein. The whole was mixed for about 1 minute to obtain a homogenous paste. The paste was transferred to an empty 1 mL syringe. The plunger was returned to its original position. The paste was then ready to be injected into the defect.

[0096] Preparation of preformed matrices The above protocol is repeated. The mixture is injected by syringe into a silicone mold of the desired size and total volume of 1 mL. Fibril formation (gelation) is carried out overnight under ammonia vapor. The gel is then removed from the mold and rinsed with saline until a neutral pH is reached. The material can then be embedded into the cavities that correspond to the shape of the mold.

[0097] Example 6: Synthesis of hybrid collagen microparticles by aerosol The synthesis of collagen microparticles was carried out according to the procedures described by Nassif et al, Paris, 2018: Injectable collagen suspensions, the preparation method thereof, and the uses thereof, particularly for forming dense collagen matrices; US Patent Application No. 15 / 558,787 and Lama et al., Self-Assembled Collagen Microparticles by Aerosol as a Versatile Platform for Injectable Anisotropic Materials, Small, p.1902224, 2019. In addition, salts present in the biomimetic hydroxyapatite precursor were added to the acidic collagen solution at low collagen concentrations before atomization leading to the final composition (2 mg / mL collagen, 500 mM acetic acid, 110 mM CaCl2.2H2O, 33 mM NaH2PO4, and 33 mM NaHCO3). The composition of the ionic precursor was varied to have different mineral / collagen ratios and to contain different therapeutic ions (e.g., Sr 2+ , Mg 2+ , Zn 2+ ) can be modified to form hybrid collagen microparticles loaded with

[0098] For example, SrCl2.6H2O is Ca 2+ 10% Sr in relation to 2+ It may be added to a biomimetic hydroxyapatite precursor solution to obtain (mol / mol).

[0099] Preparation of the injectable composition 90 mg of collagen powder obtained as disclosed hereinabove was mixed with 1 mL of 500 mM acetic acid. The whole was mixed for about 1 minute to obtain a homogenous paste. The paste was transferred to an empty 1 mL syringe. The plunger was put back into place. The paste was then ready to be injected into the defect. Different weights of ionic precursors may be used to obtain different mineral / collagen ratios.

[0100] Preparation of preformed matrices The above protocol is repeated. The mixture is injected by syringe into a silicone mold of the desired size and total volume of 1 mL. Fibril formation (gelation) is carried out overnight under ammonia vapor. The gel is then removed from the mold and rinsed with saline until a neutral pH is reached. The material can then be embedded into the cavities that correspond to the shape of the mold.

[0101] Example 7 Study of Ca and P ion restoration to demineralized deep dentin from Class I cavities by the compositions of Example 2 and Example 3 1. Preparation of Dental Cavity Deep class I cavities were prepared in anonymously collected extracted molars. Intracanal fluid flow with solutions of phosphate buffered saline (PBS) and horse serum was performed to simulate the biological environment of the dentition as disclosed in Bortolotto T, Onisor I, Krejci I, Proximal direct composite restorations and chairside CAD / CAM inlays: Marginal adaptation of a two-step self-etch adhesive with and without selective enamel conditioning, Clin Oral Invest, 2007;11:35-43.

[0102] After cavity preparation, four dentin treatments were examined.

[0103] Positive control for healthy dentin As-prepared cavities in sound dentin were filled with glass ionomer cement (Fuji IX, GC) and stored at 37°C for 15 days under dentin irrigation.

[0104] Negative control for demineralized dentin The as-prepared cavities were etched with 37% H3PO4 aqueous solution for 20 seconds, then filled with glass ionomer cement and stored at 37°C for 15 days under dentin irrigation.

[0105] Preparation of demineralized dentin coated with the composition of Example 2 or Example 3 The as-prepared tooth cavity was etched with 37% H3PO4 aqueous solution for 20 seconds. Afterwards, a layer of the composition of Example 2 or Example 3 was applied. A thin layer of light-cured bonding agent (Optibond FL bond) was placed on top to isolate the mixture layer from the cement and avoid any chemical interaction. After application of the glass ionomer cement, the tooth was stored at 37°C under dentin irrigation for 15 days.

[0106] Material characterization Tooth samples were sectioned and polished to obtain scanning electron microscopy (SEM) micromorphological evaluation and energy dispersive spectroscopy (EDS) chemical profiles at the dentin / composite interface.

[0107] As assessed by EDS, the calcium (Ca), phosphorus (P) and carbon (C) peaks varied depending on the type of dentin surface treatment. The C peak was related to the amount of exposed collagen, since the lowest Ca (8) and P (2) peaks were found in demineralized dentin (DD) reaching a peak of 41 (Figure 7).

[0108] The composition of Example 2 (HAp coll mix 1) and the composition of Example 3 (HAp coll mix 2) gave the peaks closest to sound dentin. This indicates that biological modification occurred at the demineralized dentin surface. SEM micrographs confirmed these assumptions in light of the morphology of the layer well integrated into the dentin surface (Figures 8 and 9).

[0109] It therefore appears that restoration of demineralized dentin was possible, especially with the composition of Example 3 (collagen / hydroxyapatite ratio of 30:70; "HAp coll mix 2"), by integration of a newly formed hydroxyapatite / collagen layer with characteristics similar to those observed in sound dentin.

[0110] Example 8 : Ca and P ion mapping study of demineralized deep dentin from class I cavities using the composition of Example 4 (amorphous calcium phosphate (40 mg) in acetic acid (2 mM) + collagen / amorphous calcium phosphate (44.4 mg) (70 / 30 ratio): Mix 3) The following steps were the same as in Example 7: preparation of dental cavities, positive control for sound dentin, negative control for demineralized dentin, preparation of demineralized dentin coated with the composition of Example 4, and characterization of the material.

[0111] SEM micrographs showed a mineralized collagenous material that represents a very tight integration between both components (Figures 10 and 11). The interface characteristics between dentin and Mix 3 were typical of a biomimetic neolayer with a structure very similar to the underlying dentin (Figures 12-14).

Claims

1. A composition for use in the restoration and regeneration of dentin, - Non-crosslinked and undenatured collagen microparticles containing more than 90% by weight of collagen; • Biomimetic hydroxyapatite or biomimetic hydroxyapatite precursors; and • Physiologically compatible aqueous solvents A composition for use comprising the above.

2. The composition for use according to claim 1, wherein the collagen microparticles have a diameter in the range of 0.05 to 20 μm as measured by electron microscopy.

3. The composition for use according to claim 1, wherein the collagen microparticles are type I collagen microparticles.

4. The composition for use according to claim 1, wherein the physiologically compatible aqueous solvent is physiological serum, phosphate buffer, sodium bicarbonate, or blood.

5. The composition for use according to claim 1, further comprising one or more therapeutic agents or bioactive agents.

6. The composition for use according to claim 1, wherein the weight ratio of collagen microparticles to biomimetic hydroxyapatite or the weight ratio of collagen microparticles to biomimetic hydroxyapatite precursor is in the range of 10:90 to 90:

10.

7. The composition for use according to claim 1, wherein the weight ratio of the aqueous solvent to the mixture of high-density collagen microparticles and hydroxyapatite or amorphous calcium phosphate is in the range of 1.8 to 10.

8. A composition for use according to any one of claims 1 to 7, for use in repairing damage to the dentin of a tooth.

9. A composition for use according to any one of claims 1 to 7, for use in dentin restoration when a cavity has already formed.

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