Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/19—Cosmetics or similar toiletry preparations characterised by the composition containing inorganic ingredients
  • A61K8/24—Phosphorous; Compounds thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K6/00—Preparations for dentistry
  • A61K6/15—Compositions characterised by their physical properties
  • A61K6/17—Particle size
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K6/00—Preparations for dentistry
  • A61K6/20—Protective coatings for natural or artificial teeth, e.g. sealings, dye coatings or varnish
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K6/00—Preparations for dentistry
  • A61K6/50—Preparations specially adapted for dental root treatment
  • A61K6/52—Cleaning; Disinfecting
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K6/00—Preparations for dentistry
  • A61K6/50—Preparations specially adapted for dental root treatment
  • A61K6/54—Filling; Sealing
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K6/00—Preparations for dentistry
  • A61K6/70—Preparations for dentistry comprising inorganic additives
  • A61K6/71—Fillers
  • A61K6/74—Fillers comprising phosphorus-containing compounds
  • A61K6/75—Apatite
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K6/00—Preparations for dentistry
  • A61K6/80—Preparations for artificial teeth, for filling teeth or for capping teeth
  • A61K6/849—Preparations for artificial teeth, for filling teeth or for capping teeth comprising inorganic cements
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K6/00—Preparations for dentistry
  • A61K6/80—Preparations for artificial teeth, for filling teeth or for capping teeth
  • A61K6/884—Preparations for artificial teeth, for filling teeth or for capping teeth comprising natural or synthetic resins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q11/00—Preparations for care of the teeth, of the oral cavity or of dentures; Dentifrices, e.g. toothpastes; Mouth rinses
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
  • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
  • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
  • A61K2800/41—Particular ingredients further characterized by their size
  • A61K2800/413—Nanosized, i.e. having sizes below 100 nm

Definitions

• the present invention concerns the use in odontostomatology of apatite-based nanostructured materials. More specifically, the invention concerns the application in the fields of dentistry and dental hygiene of biocompatible crystalline products based on apatite with high specific surface properties, having single constituent crystals of nanometric size. Such products are particularly useful in the treatment of dentine hypersensitivity and in the remineralisation of dental tissue.
• dentine sensitivity or hypersensitivity is a complex symptomatology, characterised by pain and sensitivity to thermal, mechanical (i.e. tactile), chemical and osmotic stimuli caused by exposure of the dentin to the oral environment.
• Several data on the epidemiology of dentine hypersensitivity have been published in the latest years, revealing, inter alia, that such clinical condition affects more than 15% of the population in the age range between 20 and 40 years, with a particular preference for females.
• Dentine hypersensitivity may be primary, i.e. not resulting from any therapeutic intervention on the dental components, or it may be secondary to previous periodontal interventions and/or to injuries resulting in dentine exposure, or to restorations (including dental prostheses) with projecting margins or having an incorrect shape. After an extensive periodontal intervention, especially of resective surgery, patients quite frequently report a hypersensitivity not previously present. In such cases the affection is also referred to as radicular or cervical hypersensitivity.
• Such displacement is induced by mechanical stimuli (such as, for instance, by passing the speculum over the tooth surface), by osmotic and chemical stimuli (such as by the contact with food, beverages, etc.) and by thermal stimuli (both hot and, specially, cold).
• mechanical stimuli such as, for instance, by passing the speculum over the tooth surface
• osmotic and chemical stimuli such as by the contact with food, beverages, etc.
• thermal stimuli both hot and, specially, cold
• the exposed dentine behaves as a semi-permeable membrane, wherein the dentine tubules represent the membrane pores. Therefore, the diameter and number of the exposed tubules largely affect the degree of sensitivity, as it has been ascertained in vivo by Yoshiyama et al. (M. Yoshiyama, Y. Noiri, K. Oaki, A. Uchida, Y. Ishikawa, H. Ishida, Transmission electron microscopic characterisation of hypersensitive human radicular dentin, J. Dent. Res.; 69: 1293-1297 (1990)).
• any dental treatment involving the removal of enamel and dentine, and thus the opening of the dental tubules unavoidably results in an increased sensitivity.
• the patient with dentine hypersensitivity is found to have, at the same time, an area of exposed dentine (i.e., an area deprived of enamel and/or of gum covering) with open tubular orifices, and no kind of surface debris or smear layer (the latter term actually meaning the layer of debris that normally covers the dentine and closes the tubules with plugs, i.e. the so-called smear plugs) obstructing, at least partially, the dentine tubules.
• Food may play a primary role in causing the onset or, more properly, the becoming acute again, of dentine hypersensitivity.
• several food items or beverages have acid properties, as they contain citric, phosphoric or maleic acid, and they may easily effect the chemical removal of the surface smear layer, thus opening the dentine tubules.
• other food items or beverages such as red wine, citrus fruits and unripe fruits in general, have been reported to have effects on the dentine hypersensitivity.
• a particular situation may occur in patients who have undergone a resective periodontal intervention, about 80% of whom reports dentine sensitivity after the periodontal therapy.
• Periodontal procedures may be related to the occurrence of dentine hypersensitivity: for instance, during the operative procedures of dental calculus removal, root scalling, root planning, root polishing and surface cleaning, any residual cement (which is often already absent) and any infected dentine are removed, thus resulting in the removal of large root surfaces and in the exposure of an extremely high number of open dentine tubules.
• any residual cement (which is often already absent) and any infected dentine are removed, thus resulting in the removal of large root surfaces and in the exposure of an extremely high number of open dentine tubules.
• the passage of intratubular fluid from the interior of the pulp cavity to the exterior thereof with loss of plasma proteins and electrolytes, and, vice- versa
• the passage of liquid and toxins from the exterior of the pulp cavity to the interior thereof a temporary pulpitis occurs, accompanied by a painful symptomatology. The latter is normally felt after some hours or days from the intervention.
• the symptoms spontaneously decrease owing to the spontaneous obstruction of the tubules, resulting from the deposition of bacterial debris, the precipitation of proteins and fibrinogen and the production of a smear layer by the patient himself during the normal dental hygiene procedures with the toothbrush (Pashley, 1990, loc. cit).
• dentifrices containing strontium chloride hexahydrate The most studied and well-known products for home therapy include dentifrices containing strontium chloride hexahydrate.
• the latter as pointed out in the foregoing, is used in view of its ability of closing the dentine tubules, rather than for its activity in depressing the dentinal nerve fibres stimulation. It is believed that the Sr 2+ ion reacts with the apatite matrix of the dentine, thus forming with said matrix an insoluble strontium-apatite. The latter settles as a layer of microcrystals, that are able to reduce the functional diameter of the dentine tubules. Also stannous fluoride is believed to act by forming insoluble complexes with dentine (i.e.
• dentifrices and desensitising preparations have been proposed, which, rather than being based on products that form insoluble precipitates by reacting in the oral environment, are based on the direct use of apatite, i.e., the basic material making up the dentine, in the microcrystalline state.
• apatite i.e., the basic material making up the dentine
• the US patent No. 4634589 discloses the use of a crystalline apatite having a particle size of less than 8 ⁇ m as an abrasive and polishing agent, active in the treatment of dentine hypersensitivity and also having a remineralising action on the dental tissue.
• the dentifrice formulation proposed contains at least 15% by weight of such ingredient.
• the European patent application No. 0346957 discloses the use of hydroxyapatite as a desensitising abrasive agent for dental products.
• the hydroxyapatite particle size is 1-15 ⁇ m
• the formulation proposed also comprises a source of potassium and/or strontium (as a further desensitising agent).
• dentine hypersensitivity by home therapy, however, has the drawback of requiring long periods of time (often more than four weeks) to reach any appreciable clinical results. In addition, the said results are often only partial, as a great amount of co-operation is required from the patient.
• the treatment of dentine hypersensitivity is preferably carried out at the dentist's consulting room, by directly treating the affected tooth (or teeth). Also in this case, the main aim is to occlude the open dentine tubules. Several techniques and materials have been proposed to that aim, according to the degree of seriousness of the symptoms.
• Such techniques can be schematically divided into four main groups: 1 ) mechanical treatment of the sensitive surface in order to obtain a new smear layer; 2) application of agents effective in occluding the dentinal tubules, either directly or by forming insoluble precipitates; 3) impregnation and obstruction of the dentine tubules by means of adhesives; 4) application of dental restoration materials, optionally together with intraoral devices for sustained release of fluorine.
• the dental restoration and the endodontal treatment are often included in order to finally solve the problem.
• the adoption of the above treatment is limited to non serious cases, e.g. to treat patients after a periodontal intervention with exposure of limited dentine surfaces and without any previous history of dentine hypersensitivity.
• the second kind of medical treatment mentioned in the foregoing consists, for instance, in applying oxalates (such as potassium, iron or aluminium oxalate, and oxalic acid) on the affected dentine surface.
• oxalates such as potassium, iron or aluminium oxalate, and oxalic acid
• Such agents may react with the dentinal apatite constituents, thus forming insoluble calcium oxalate microcrystals on the surface and in the interior of the dentine tubules.
• the solutions containing the concerned agent may easily be applied on the dentine surface by means of little brushes, and they may be left in situ for 2-3 minutes, this period of time being enough for the said microcrystals to form. It is to be noted, however, that this technique may involve some problems due to the toxicity of oxalates. Also silver nitrate, another agent em- ployed for a long time for the same purpose, has a mechanism of action based on the formation of precipitates (Ag chloride) which occlude the dentine tubules.
• precipitates Ag chloride
• the same kind of therapeutic approach also includes the direct application of microcrystalline apatite, as mentioned in the foregoing in connection with products for home therapy.
• the medical treatment includes the use of an ultramicronised apatite, such as that disclosed, for instance, in the Italian patent No. 1271874, where the particle size of about 70% of the apatite microcrystals is less than 1 ⁇ m (0.2-1 ⁇ m).
• Such product is considered to be more active than the micronised apatite products disclosed in the foregoing, on the basis of the simple consideration that the dentine tubules have an average diameter of about 1.3-3.5 ⁇ m.
• the resin by penetrating into the tubules, forms long plugs (referred to as resin tags) which occlude the tubular lumen and reduce the dentine per- meability.
• the resin impregnates and occludes the small lateral channels as well (as it has recently been confirmed by some studies by scanning electron microscopy), thus contributing to the formation of a thick resin network effective in stably blocking any movement of the dentine fluid.
• a hybrid layer made of resin and collagen is thus formed. From the point of view of the mechanism of action, it is not clear whether other factors co-operate in reducing the sensitivity, in addition to the tubules occlusion and the consequent permeability reduction (which is, very likely, the main mechanism of action).
• the said amorphous salts, or solutions that may originate such salts by precipitation are applied on the dental tissue surface, and settle thereon and within the same tissue.
• the amorphous salts are then converted to crystalline apatite, thus performing a remineralising action and reducing dentine sensitivity.
• the PCT patent application No. WO 94/04460 (American Dental Association Health Foundation) also presents the ACCPF phosphate (i.e., amorphous calcium carbonate fluorophosphate), as a new compound particularly interesting for the same purpose.
• the apatite materials characterised by a nanocrystalline and/or defective structure lend themselves much better to the objects men- tioned above, as they allow to achieve remarkable therapeutic results, up to the complete suppression of dentine hypersensitivity and, in addition, with a considerable remineralisation of the dental tissue.
• Such results are made possible by the unique features of the crystalline nanostructures, that substantiate the great potential of these advanced materials in many applicative fields.
• the nanocrystalline materials are artificially synthesised materials, characterised by a constituent phase, or by granular structures, modulated on a length scale normally smaller than 100 nm.
• the nano- crystalline materials are considered to be with dimensionality equal to zero (clusters of atoms - for instance, dispersed in a non-nanocrystalline matrix , filaments or tubules), with dimensionality one (multilayers, i.e., layers which are nanometric in the only direction of the thickness), with dimensionality two (granular superpositions, ultrafine, or buried layers), or with dimensionality three (nanophasic materials, wherein all of the constituent phases are of nanometric proportions on three dimensions) (R.W.
• the basis of the particular performances of the nanostructured materials is to be found in the fact that a physical property of matter becomes altered when the entity or the mechanism responsible for such property (or the combination thereof) are confined within a space (defined by the dimension of the atoms set) smaller than a given critical length associated with such entity or mechanism. Therefore, for instance, a metal that is conventionally ductile owing to the usual ease in creating and displacing dislocations through the crystal lattice thereof will become remarkably harder when the grain size is reduced down to a criti- cal point wherein the dislocation sources are no more able to work at the low levels of the applied stress.
• the nanocrystalline materials are also characterised by the high number of interfaces they contain. Since the number of interfaces present in the nanocrystalline materials is much higher than in the conventional materials, a suitable control, in the course of the synthesis, on the nature of the interfaces created between the constituent phases leads to a control on the nature of the interactions through the said interfaces. In order to have an idea of the importance of the interface environment in a nanocrystal- line material it is sufficient to consider, for instance, that in a material with an average grain size of 5-10 nm the percentage of atoms comprised in the grain boundaries is in the range from 15 to 50%.
• the apatite-based nanostructured materials appear to possess, firstly, the most suitable granular dimensions to easily and deeply penetrate within the dentine tubules (which, as pointed out before, have diameters in the range of 1.3-3.5 ⁇ m).
• the concerned materials show specific physico-chemical properties connected with their nanocrystalline nature, such as the remarkable surface chemical reactivity and the hygroscopicity, which not only allow the material to penetrate mechanically within the tubules, but also cause it to cement the internal surfaces thereof, both by swelling and by readily reacting with the natural tissue, and recrystallising as a result of the metastable character of the nanocrystalline system.
• the latter may have, in this specific case, a high defective content.
• Apatite-based materials characterised by a nanocrystalline structure appear to have somehow been described, so far, only in the PCT patent application No. WO 97/17285 (Etex Corp.), concerning the low temperature synthesis of a low crystallinity apatite for use in bone tissue graft.
• the definition of "low crystallinity material” given in such text generically includes both amorphous materials and nanocrystalline materials (with nanometre- sized or Angstrom-sized crystalline domains).
• Such document exclusively concerns the production of resorbable synthetic bone materials, which, in view of their intended use, are formed in moulds to give solid elements.
• the only relevant requirement for such use is the ability of closely reproducing the natural bone tissues, so that the synthetic bone graft is inte- grated and resorbed in the said tissues.
• the applications of the concerned material in the orthopaedic field differ from the odontostomatological application in that in the first case the material must comply with stability requirements, it must be able to easily integrate within the bone and, in some cases, it must be able to induce bone growth (direct relationship between material and cells), while in the second case the material must interact with mineral tissue having chemical and structural properties different from bone, and having a different organic component. Consequently, in the latter case character- istics of the material such as surface reactivity and metastability will have to prevail.
• the orthopaedic applications that involve interventions on the bone tissue are concerned with a system interacting with the blood stream and unaffected by variations induced by the contact with the external envi- ronment (such as pH changes or compositional variations of various kinds). This is right the opposite of what happens in the oral environment, and on the tooth surface.
• the present invention specifically provides the use of apatite-based nanostructured materials of the general formula: Ca 10- ⁇ M x (P ⁇ 4)6-yByA 2 (OH) 2-z wherein M is a cation different from Ca 2+ , B is an anion different from P0 4 3 ⁇ , A is chosen from the group consisting of O 2 ", CO 3 2 ", F” and Cl", x is a number from 0 to 9, y is a number from 0 to 5, z is a number from 0 to 2, wherein the said numbers may also be fractional, with the proviso that the sum of the charges of the Ca and M cations is equal to the sum of the charges of the P0 4 3_ , B, A, and OH" anions, having average size of the crystallites comprised between 0.5 and 200 nm, for the production of preparations for odontostomatologic applications, useful for the restoration and the protection of dental tissue and, specifically, for the therapy of dentine hypersensitivity and for the
• apatites represent the main inorganic process of calcification of normal tissue (i.e. enamel, dentine, cement, bone) and are found associated with other phosphatic and non-phosphatic minerals in pathological calcifications.
• normal tissue i.e. enamel, dentine, cement, bone
• phosphatic and non-phosphatic minerals in pathological calcifications.
• hydroxyapatite or hy- droxylapatite having the stoichiometric formula Ca- ⁇ 0 (P ⁇ 4 ) 6 (OH) 2 (or Ca 5 (P0 2 ) 3 ⁇ H), and being - in its synthetic (biocompatible) form - the apatite material most widely exploited at a commercial level for several indications in dentistry, orthopaedy and maxillo-facial surgery, is never found in a pure state in the biological tissues. This is due to the possible isomorphous replacements of the Ca 2+ , PO 4 3 - and OH " ions.
• the calcium ion may be totally or partially replaced by a number of cations generally (but not exclusively) having oxidation number +2;
• the phosphate ion (site B) may be replaced by carbonate, acid phosphate, pyrophosphate, sulphate, aluminate and silicate ions, and the hydroxyl ion (site A) may be replaced by halogenide, carbonate and oxide ions.
• biological apatites have a Ca/P molar ratio comprised between 1.53 and 1.74 (in particular, comprised between 1.53 and 1.64 for the dental enamel, between 1.62 and 1.68 for the dentine and between 1.72 and 1.80 for the bone).
• the oscillations in the Ca/P ratio may be caused by: 1 ) lattice vacancies; 2) ions isomorphically replaced or adsorbed on the lattice surface (for instance, the substitution of the P0 4 3_ anion with acid phosphate, which is divalent, brings about a reduction in the calcium contents); 3) coexistence or presence of possible precursors consisting of phosphates with different Ca/P ratio.
• ⁇ -tricalcium phosphate i.e. Ca 3 (P0 4 ) 2
• an orthophosphate also known as tribasic calcium phosphate
• ACP amorphous calcium phosphate
• OCP octocalcium phosphate
• DCPD dicalcium phosphate dihydrate
• DCPD CaHP0 4 -2H2 ⁇ , also known as calcium acid phosphate, dibasic calcium phos- phate or dicalcium orthophosphate.
• the Ca/P molar ratio values for some of the phosphates considered are as follows: HA Ca 5 (P0 4 ) 3 OH 1.67 OCP Ca 8 H 2 (P04) 6 -5H 2 0 1.33
• biological apatites may be described as non-stoichio- metric carbonatoapatites containing, in general as impurities, different ions. For this reason, biological apatites have variable morphology, crystalline charac- teristics, chemical and phisico-chemical properties.
• hydroxyapatite [Ca 5 (P ⁇ ) 3 ⁇ H] has already been presented as the most widespread material.
• the phosphate and calcium ions are placed approximately according to a hexagonal prism; in the direction of elongation (crystallographic axis c) the prism is crossed by a channel having a diameter of 3-3.5 A, housing any OH" groups or other possible replacing ions (e.g., fluorine and chlorine).
• HA may crystallise in two forms: a monocline form (spatial group P2 1 /b) and a hexagonal form (spatial group P6 3 /m).
• a monocline form a binary symmetry axis is present along the axis c, while in the hexagonal form the symmetry axis becomes hexagonal.
• OCP octocalcium phosphate
• HA octocalcium phosphate
• the Mg 2+ ions inhibit the hydroxyapatite precipitation and promote the formation of ⁇ -tricalcium phosphate.
• the magne- sium concentration exceeds 10% the simultaneous formation of HA and ⁇ - TCP occurs, the latter being the only product that precipitates when the concentration exceeds 25%.
• the fact that magnesium replaces calcium in ⁇ -TCP is made evident by the displacement of the X-ray diffraction peaks in the powder diffraction spectra of the same samples.
• the magnesium contents in biological apatites is very low
• Strontium is present in biological apatites only at the impurity level, and may replace calcium, thus causing an expansion of the a and c axes.
• the presence of this element in apatites for odontostomatological use is considered to be important in connection with a possible cariostatic effect thereof (in addition to the hypothesised effect of reduction of the dentine sensitivity), and confers on the apatite a lower solubility and a higher resistance to thermal treatments.
• this cation does not isomorphically replace calcium in the whole concentration interval.
• the variation of the lattice parameters results in an expansion of the cell parameters.
• hydroxyapatite may also be substituted with anionic groups.
• the carbonate ion may replace the hydroxyl ion (site A) or the phosphate ion (site B) or both.
• site A hydroxyl ion
• site B phosphate ion
• the replacement of the carbonate ion in the site B is prefer- ential in the biological samples, it is also possible to obtain carbonatoapatites of the type A synthetically, by means of high temperature reactions.
• carbonatoapatites of the type B or mixed type A + B are mainly obtained by precipitation from solutions.
• the presence of the carbonate ion in hydroxyapatite modifies the lattice dimensions: if the said ion occupies the A site an increase in the a parameter is obtained, as a consequence of the greater size of the C0 3 2_ ion with respect to the OH" ion; if, on the contrary, the carbonate ion occupies the B site the same parameter undergoes a contraction, due to the smaller 0-0 distance in the CO 3 2 - ion with respect to the PO 4 3 - ion.
• Fluoroapa- tite is characterised by an increase in the crystal dimensions, by a decrease in the a parameter of the unit cell, by a lower solubility and by an enhanced thermal stability.
• the lower solubility, and therefore the higher lattice stability, of the fluoroapatites substantiates the present use of the fluoride ion in the therapy of bone affections and of dental caries.
• Chloroapatite is characterised by an expansion of the a side and a contraction of the c side of the unit cell.
• the different lattice behaviour of chlo- roapatite with respect to fluoroapatite derives from the remarkable difference in ionic radius between the two halogens: in fluoroapatites the fluoride ion is placed on the senary axis located on the plane defined by the three calcium ions, while in chloroapatites the chloride ion is slightly displaced from the plane of the metal ions. This phenomenon results in the above-mentioned parameter variations of chloroapatite, while the crystallinity does not seem to be significantly affected, even if the thermal stability decreases.
• the non-stoichiometric character of biological apatites may also be caused by the presence of the acid phosphate ion HPO 4 2 -; this ion is contained specifically in the tooth enamel, in amounts comprised between 5% and 15%, and is effective in increasing the hydroxyapatite solubility.
• the presence of acid phosphate in biological and synthetic apatites is not easily detectable, since the carbonate ion, which is almost always present, overlaps the I.R. absorption bands and causes a similar variation of the cell parameters.
• the original presence of HP0 4 2 " may be evidenced by the pyrophosphate formation, obtained by heating the apatite samples between 400 e 500°C.
• the M cation is chosen among the following ones: H + , Na + , Mg 2+ , K + , Sr 2+ , Ba 2+ and Fe 2+ , and preferably the x value is comprised between 0 and 2.
• the B anion is chosen, preferably, among C ⁇ 3 2 ⁇ , HP0 2 ⁇ , HC0 3 " , and P2O7 4" , while the y value may be comprised, for instance, between 0 and 2.
• the nanostructured apatites according to the invention may be produced by any one of the several known methods, already in use for the production of nanocrystalline materials, such as the synthesis methods from atomic or molecular precursors (e.g., chemical or physical vapour deposition, condensation in gas, chemical precipitation, reactions from aerosol), the methods of production from mass precursors (e.g. by mechanical attrition, by crystallisation from the amorphous state, by phase separation), and the methods borrowed from nature (i.e., biologically mimicked systems).
• atomic or molecular precursors e.g., chemical or physical vapour deposition, condensation in gas, chemical precipitation, reactions from aerosol
• mass precursors e.g. by mechanical attrition, by crystallisation from the amorphous state, by phase separation
• the methods borrowed from nature i.e., biologically mimicked systems.
• the conventional deposition of layers of material by electrolytic processes or by vapour condensation has been exploited in recent times to deposit materials of nanometric dimensions with remarkable control and
• the new or improved methods include, in particular, increasingly advanced mono- or multi-bath systems for electrodeposition and new chemical or physical vapour deposition methods, such as molecular beam epitaxy (MBE), metal-organic chemical vapour deposition (MOCVD) and chemical vapour synthesis (CVS).
• MBE molecular beam epitaxy
• MOCVD metal-organic chemical vapour deposition
• CVS chemical vapour synthesis
• the synthesis of nanocrystalline materials by in situ consolidation, under vacuum, of ultrafine particles condensed in the form of nanometre-sized gas starts when a precursor material, be it an element or a compound, is evaporated into a gas maintained under low pressure, generally well below 1 atmosphere.
• a precursor material be it an element or a compound
• the evaporated atoms loose energy as a result of the collisions with the atoms or mole- cules of the gas, and undergo a homogeneous condensation suitable to form clusters of atoms in the highly supersaturated area close to the precursor source.
• the nanometre-sized grains form a nucleus within slip bands of highly deformed precursors materials, thus transforming a coarse-grained structure into a nanophasic one.
• a great deformation is normally obtained by means of a high energy crushing, but it may also occur as a consequence of surface wear phenomena (E. Hellstern, H.J. Fecht, Z. Fu and W.L. Johnson., J. Appl. Phys., 65 (1989); C.C. Koch, Nanostructured Mater., 2, 109 (1993)), or it may be obtained through other methods of introduction of high deformation densities (R. Valiev, in Mechanical Properties and Deformation Behaviour of Materials Having Ultra-Fine Microstructures, M. Nastasi, D.M. Parkin, and H. Gleiter. 303 (1993)). In the course of the hard mechanical work on the precursors it is also possible to react different materials so as to have them form new phases and compounds.
• the method has been used for the synthesis of the nanostructured apatite materials according to the invention that underwent the comparative experimentation described further on.
• the nanostructured apatite material according to the invention was produced starting from apatite with microcrystalline structure through lattice de- stabilisation in a controlled environment, under high energy, by subjecting the starting material to a high mechanical energy transfer treatment. The latter is carried out into a cylindrical reaction chamber by means of high energy impacts from hard balls contained within the same chamber.
• the kinetic energy of the balls is generated by a rotation movement of the chamber with respect to its main axis and by a revolution movement of the same with respect to an axis parallel to the main one.
• the transformation of kinetic energy into mechanical energy of lattice destabilisation occurs through impacts between the balls and the starting material.
• the chamber may work either in air or in inert gas or under vacuum, down to a pressure value of 10 "6 torr, or with liquids (alcohol, ethers, oils and other organic molecules).
• the above method reduces the crystallites dimension through subsequent introduction of lattice defects (which may be evaluated by means of X- ray diffraction techniques).
• the nanoapatite material obtained by the disclosed synthesis method and employed in the applications reported herein shows an average dimension of the crystallites of about 15 nm, with an average strain ranged between 10 "5 and 10 "2 .
• the apatite-based nanocrystalline products according to the present invention represent a material that simulates the dentine composition and is perfectly compatible, both biologically and structurally, with the dental tissue. The material is able to efficiently become stably integrated with the dentine, thus making it totally impermeable.
• the dimensional features, the properties of surface activity and the defective contents of the nanoapatites allow to easily obtain both the mechanical filling of the tubules and the fixing reaction with the tubule surface (i.e. remineralisation). Therefore, the dentine sensitivity resulting from the hydraulic conductivity through the tubule structure of dentine is practically removed.
• the said permeability is a critical factor in the dentinal pain stimulation theory, as the presence of exposed and open tubules causes, when suitable external stimuli are present, the movement of dentinal fluid, perceived at the neural structure level as a pain stimulus.
• the nanocrystal- line apatite materials are protected from acid attack by subjecting them to a treatment with protective aqueous solutions containing tartaric acid and/or its salts, such as, e.g., aqueous solutions containing potassium sodium tartrate (or Seignette's salt, or Rochelle's salt, NaOOC(CHOH) 2 COOK-4H 2 0) and hydrous calcium acetate (Ca(CH 3 COO) 2 -H 2 0), or, in the alternative, with a solution containing tartaric acid (HOOC(CHOH) 2 COOH).
• protective aqueous solutions containing tartaric acid and/or its salts such as, e.g., aqueous solutions containing potassium sodium tartrate (or Seignette's salt, or Rochelle's salt, NaOOC(CHOH) 2 COOK-4H 2 0) and hydrous calcium acetate (Ca(CH 3 COO) 2 -H 2 0), or, in the alternative, with
• a 0.1-1.0 M solution of potassium sodium tartrate and a 0.1-1.0 M solution of hydrous calcium acetate are used in a sequence, by dipping therein the material according to the invention, preferably at 37°C, for periods of time comprised between 5 and 30 minutes. Specifically, the nanoapatite is immersed in the first solution and kept therein for 5 minutes, then it is withdrawn from the first solution and immediately immersed in the second solution for 20-30 minutes.
• the material can also be individually treated with one of the following agents: mono-, bi-, tri-, tetra-, polycarboxylated calcium gluconate in aqueous solution at a concentration of 0.01-5% by weight, or in alcoholic or hydroalco- holic solution at a concentration in the range from 0.01% and 15% by weight; or with an acetic and/or malic and/or hyaluronic solution of chitosan at a con- centration in the range from 5% to 20% by weight; or with a solution of human, bovine, swine, rat or turkey tendon collagen, either isotonic or not, at a concentration in the range from 0.5% to 5% by weight.
• agents mono-, bi-, tri-, tetra-, polycarboxylated calcium gluconate in aqueous solution at a concentration of 0.01-5% by weight, or in alcoholic or hydroalco- holic solution at a concentration in the range from 0.01% and 15% by weight;
• the said treatments result in a protection of the nanostructured material from acid environments.
• the said material may also be used without any further treatment.
• the invention further concerns compositions for odontostomatologic use comprising the apatite-based nanostructured materials described in the foregoing, together with further possible ingredients and excipients of the same kind as those used in preparations for dental hygiene and for the therapy of the oral cavity.
• the compositions that are preferably in the form of toothpaste, paste, gel, suspension or solution, preferably contain from 0.5% to 50% by weight of nanocrystalline apatite material, either treated with protective agents or not.
• Further preferred features of the compositions according to the invention, with particular reference to oral formulations in the gel form are specified in the further dependent claims.
• EXAMPLE 1 A microcrystalline carbonate-apatite material, non-stoichiometric as regards the hydroxy ion, was subjected to a lattice destabilisation treatment in a controlled environment, under high energy, by using the reaction chamber described in the foregoing.
• the resulting product (nanoapatite) is characterised by average crystallite sizes of 15-20 nm and by an average microstrain content comprised between 10 "4 and 10 "5 .
• nanoapatite 1 or nano 1 A portion of the nanocrystalline apatite thus obtained (which will be referred to in the following as nanoapatite 1 or nano 1 ) has also been sub- jected to treatment with tartrate in order to increase its resistance to acid attack in the conditions of use in the oral cavity.
• the nanoapatite was treated with a 0.1 M aqueous solution of potassium sodium tartrate (Sei- gnette's salt, Carlo Erba, Milano) at 37°C for 5 minutes, and then with a 0.1 M solution of hydrous calcium acetate (Carlo Erba, Milano) at 37°C for 20 minutes.
• EXAMPLE 2 An apatite material of the same nature and composition as the material of Example 1 was subjected to a similar lattice destabilisation treatment, with a different value of the energetic content transferred. The resulting nanoapatite was characterised by average crystallite dimensions of 10-14 nm and by an average microstrain content comprised between 5-10 "4 and 10 "4 .
• nanocrystalline apatite thus obtained (i.e. nanoapatite 2 or nano 2) was treated with potassium sodium tartrate and hydrous calcium acetate, according to the same procedure of Example 1.
• the effectiveness of the nanocrystalline apatite products according to the invention in the treatment of dentine hypersensitivity was evaluated in terms of reduction of the hydraulic conductance within the dentine tubules, according to a well established protocol (Pashley, 1990, loc. cit), in agreement with the international literature.
• sound human molars have been used, extracted for orthodontic reasons from young patients, in order to avoid the problem of sclerotic dentine.
• Each crown was properly separated from the root and cut in order to obtain a crown segment deprived of the occlusal enamel. After removing the pulp tissue, the crown segment was fixed by means of adhesive onto a Plexiglas base, with the flat surface of occlusal dentine facing upwards.
• the base was crossed through its entire thickness by a tubular stainless steel segment, projecting into the pulp cavity in order to provide the hydraulic connection, below the Plexiglas base, with the hydraulic conductance measuring device.
• the latter was made of a simple hydrodynamic device consisting of a set of capillary tubes filled with deionised water and connected through the tubular stainless steel segment to the pulp cavity. The passage of water from the pulp cavity to the occlusal surface through the dentine was evidenced by the displacement of an air bubble in a graduated microcapillary tube located in the hydrodynamic sys- tern.
• nanoapatite 1 produced according to Example 1 ; 5. nanoapatite 1 treated with potassium sodium tartrate and hydrous calcium acetate according to Example 1 ;
• the permeability tests were performed seven times for each of the set times (30, 60, 120 seconds) after treatment with each single gel, and repeating each stage of the above procedure.
• Nanoapatite 1 62.28 64.47 64.11 100.00 44.40 35.92 32.85 47.67 41.63 38.40
• Nanoapatite 1 treated 63.31 67.13 65.19 100.00 42.92 37.09 33.02 37.91 33.93 34.02
• Nanoapatite 2 treated 64.11 62.96 63.37 100.00 39.89 34.45 30.67 38.18 32.18 30.81
• microcrystalline hydroxyapatite may bring about a partial occlusion of the dentine tubules, with reduced permeability in comparison with the smear layer formation step.
• H3PO4 no detectable resistance is noted.
• microcrystalline hydroxyapatite is treated with the Seignette's salt and calcium acetate solutions.
• the behaviour before and after acid attack, with or without the protective treatment, is evident from the enclosed histogram.
• nanoapatites 1 and 2 the dentine permeability is greatly reduced, to the point that the permeability falls from 100% after treatment with EDTA to about 40% after treatment with the nanoapatites, and further improves up to 120 seconds. In this case, even the acid attack does not result in any appreciable negative effect, this being certainly the most interesting data.
• nanocrystalline apatite according to the invention is even more evident when nanoapatites treated with Seignette's salt and calcium acetate are used. These not only produce a remarkable reduction in the dentine permeability in the absence of an acid at- tack, but, most remarkably, afford an even more considerable reduction after the final acid attack by H 3 PO 4 , with permeability values not exceeding about 35% of the value after treatment with EDTA.
• nanoapatite 2 gave a lower standard deviation in the measurements, and therefore a greater reproducibiiity in the results.
• the nanostructured apatite materials according to the invention can be validly proposed as biocompatible materials having a marked and prolonged action in reducing the hydraulic conductance within the dentine tubules, and suitable to become stably and deeply integrated in the dentine tissue.
• the said materials may advantageously be employed, either as such or included in powder, toothpaste, paste, gel, solutions, mouth- wash, suspensions, tablet, capsule, resin or cement compositions, in preparations for the protection of dentine, for the therapy of dentine hypersensitivity, for the remineralisation of enamel, dentine, dental tissues and supporting tissues, for the closure of dentine tubules or the reduction of their functional diameter, for the protection of dental pulp and of prosthetic abutments, as well as in preparations for use as bases and liners for dental restorations with amalgam, as cements for endodontic fillings and for dental prostheses, and as orthodontic cements and sealants for enamel and dentine.
• the present invention has been disclosed with particular reference to some specific embodiments thereof, but it should be understood that modifications and changes may be made by the persons skilled in the art without departing from the scope of the invention as defined in the appended claims.

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• 1998
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