CA2338021A1 - Odontostomatologic use of apatite-based nanostructured materials - Google Patents

Abstract

Biocompatible nanocrystalline materials based on apatite with high specific surface properties, having average size of the crystallites comprised betwee n 0.5 and 200 nm and possible lattice deformations and/or defects, are propose d for use in the fields of dentistry and dental hygiene, in particular for the production of preparations, solutions, toothpastes and materials for the remineralisation of enamel and dentine and for the treatment of dentine hypersensitivity. The apatite-based nanostructured materials appear to posse ss specific physico-chemical properties connected to their nanocrystalline and defective nature, such as the remarkable surface chemical reactivity and the hygroscopicity, which allow the material to recrystallise as a result of the metastable character of the nanocrystalline system, to be used for the remineralisation of enamel and dentine. In addition, they possess the most suitable grain sizes to easily and deeply penetrate mechanically within the dentinal tubules, and also cause it to cement the internal surface thereof, both by swelling and by readily reacting with the natural tissues.

Description

I~'.
~cv.v. rWV:I:YA-ML~E.IVC.'H~~) t~ :.~w.~_i0= 0 : 7:39 : OGOOUOUOOU-~ -~-4J 89 2~994465:~k 8 ' ~ ; : r~. t~:.. : 0aa~caa 0i ~~,. 2am ~~: ~7 ~a Qt3~11'~TC~ST~3iV#ihTt~LOC~tC USE fli= APATITE~.BASiED
NA~IQSTRt~G"tt.iREO ~iTER', SPEC#F1CAT#tJhl The present invention concerns sdm~P odontostorrsato#agic uses of apatite-based nanc~structured material. Il~iare spe~cifirall~r, the invention con-cerns the application c~f biocornpatible crystalline products based on apatite with high specific surface properties, #~aving sing#e oanstituent crystals ~afi ~a nartometric size, in the treatment of dentine hypersensitivity and in the remin.-eraiisatian of denta# tissue_ As it is known, dentine sensitivity ar hypersensitivity is a complex s~mptomato(ogy, ci ~aract~ised by pain and s~,nsitiv"ety to therma#, mechanica#
(i.e. taco#ea, cherrrica# and osmotic $timuii caused by exposure of the dentin ~
~5 the oral environrnentr Several data on the ~p#derniology of dentine hypersen-sitivity have been pubiisheGl in the #atest y~ar:~, r$vealing, inter alto, that such ~c3inica! condition afiFects more than 9 5°~b of the population in the age range between 20 and 4~ years, with a particular preFerence for females.
C3entine hypersensitivity may be primary, i.e: not resulting Pram any 24 therapeutic intervention on the ~dents# components, or it may be secnndary to t previous periodontal interventions andlor to injuries resulting it dentine expo-sure, or to restorations {inc#uding dental pro~~theses) vvitft projecting margins or having an i~acorrect shape. After an exfens#ve periodontal intervention, espeoiaiiy of resa~ctive surgery, Patients quite; frequently report a hy~ersensi-a~ tivity naf previ~s#y present !n such cases the affection is also referred to as radicular tar cervica# hypersensitivity.
Accord#ng to the therrry advanced by ~r~nnstrorn {i,e., the hydrody-namic theory of pain induction; see l~I. 8ra~nnstrs~m, A. AstrQm: The hydrody-r~mic~ of the der~ian: its possib#e retationshi~p to dentina# pain. tni. Dent.
.1., 3c 22:29-227 {19T2)) and. cater, by P sh#ey (~.H. Pashley, N#echanisms of deritin sensitivity. fler~t C:in. ~#orfh Am:; 34= 443-473 (1990)y, the presence of ~'..'~#~D ~i _2-exposed dentine and of open dentinal tubules ;allows the dentine fluid to move within the said tubules. Such displacement is induced by mechanical stimuli (such as, for instance, by passing the specill~um over the tooth surface), by osmotic and chemical stimuli (such as by the. contact with food, beverages, etc.) and by thermal stimuli (bath hot and, specially, cold). However, it has not been ascertained, so far, whether the displacement of the dentine fluid causes a nervous stimulation (for instance, a stimulation of intratubular nerve fibres) or whether it directly stimulates the odontoblasts projections (which occupy about 2/3 of the whole tubule length).
In .all cases, the greater is the expo;>ed dentine surface (with non-obstructed tubules), the greater is the likelihood that the patient be sensitive to any stimuli. In other terms, 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 ~s 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, Transmis-sion electron microscopic character9sation of hypersensitive human radicufar dentiri, J. Dent. Res.; 69: 1293-1297 {1990)).
As a direct clinical consequence of the foregoing, any dental treatment 2o involving the removal of enamel and dentine, and thus the opening of the dental tubules, unavoidably results in an increased sensitivity. Accordingly, 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 andlor of gum cov-eying) with open tubular orifices, and no kind of surface debris or smear layer 25 (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 tubulEa.
Food may play a primary role in causing the onset or, more properly, the becoming acute again, of dentine hypersensitivity. As a matter of fact, so several food items or beverages have acid properties, as they contain citric, phosphoric or malefic acid, and they may easily effect the chemical removal of the surface smear layer, thus opening the df:ntine tubules. Also other food items or beverages, such as red wine, citrus fruits and unr#pe fruits in general, have been reported to have effects on the denitine hypersensitivity. In addition, as pointed out before, 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. Also other periodontal proce-dures may be related to the occurrence of dentine hypersensitivity: for in-stance, during the operative procedures of dental calculus removal, root scalling, root planning, root polishing and surFace cleaning, any residual ce-ment (which is often already absent) and any infected dentine are removed, ~o thus resulting in the removal of large root surfiaces and in the exposure of an extremely high number of open dentine tubules. In the event that such expo-sure allows 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 tile exterior of the pulp cavity to ~s the interior thereof, a temporary pulpitis occurs, accompanied by a painful symptomatology. The latter is normafly felt aftf;r some hours or days from the intervention. Very often, the symptoms spontaneously decrease owing to the spontaneous obstruction of the tubules, resulting from the deposition of bacte-rial debris, the precipitation of proteins and fibrinogen and the production of a 20 smear layer by the patient himself during the normal dental hygiene prose=
dures with the toothbrush (Pashley, 1990, ioc. ~;it.).
Since the presence of open dentine tubules represents the critical stage of dentine hypersensitivity, the most reliable therapy aims, basically, at occluding the said tubules. A different therape~~utic approach, aiming at reduc-25 ing the receptor sensitivity, consists in increa;>ing the intratubular concentra-tion of potassium and strontium ions (by means of the application of dentifrice or other agents), in order to make the dentinal nerve fibres less excitable.
However, the hypothesis of an activity of the IK* and Sr2* ions in influencing the nerve stimulus conduction has found limited clinical confirmation (D.G.
3o Gillam, J.S. Bulman, R.J. Jackson, H.H. Newman, Efficacy of a potassium nitrate mouthwash in alleviating cervical dentine sensitivity (CDS), J. Clin.
Pertodontol. 23: 993-99? ( 1996)).

As far as home-therapy is concerned, several dentifrices and gels specific for sensitive teeth are commercially available, containing, for instance, strontium chloride hexahydrate, potassium nitrate or citrate or stannous fluo-ride, and also some mouthwashes and periodontal gels have been proposed.
s Data reported in the literature show that several toothpaste products possess some desensitising action, which is often independent of the kind of active ingredient employed (D.G. Gillam, J.S. Buiman, R.J. Jackson, H.N. Newman, Comparison of 2 desensitizing dentifrices with a commercially available fluo-ride dentifrice in alleviating cervical dentine sensitivity, J. Periodontol., 67:
~0 737-742, {1996)). Very often, also the other components of the toothpaste play an important role, in that they are able themselves to seal the dentine tubules. In addition, the placebo effect plays a role considered by someone to be a primary one.
The most studied and well-known products for home therapy include 15 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 den~tinal nerve fibres stimulation. It is believed that the Sr2+ ion reacts with the ap<~tite matrix of the dentine, thus forming with said matrix an insoluble strontium-apatite. The latter settles as a 20 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. fluoroapatite or calcium--fluoride), which com-plexes are able to obstruct the tubular lumen. As tar as potassium citrate is concerned, it has been shown that the citrate ion chelates the calcium con-es tained in the tooth hydroxyapatite, thus resulting in the formation of a soluble calcium citrate {Misra D.N., Interaction of citrate Acid with Hydroxyapatite:
Surface exchange of ions and precipitate of calcium citrate, J. Dent. Res., 75(6), 1418-1425, June 1996).
In this connection, other dentifrices and desensitising preparations 3o 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. For instance, the US patent No. 4634589 (Wurttember-gische Parfumerie-Fabrik} discloses the use oaf a crystalline apatite having a particle size of less than 8 pm as an abrasive; and polishing agent, active in the treatment of dentine hypersensitivity and also having a remineralising s action on the dental tissue. The dentifrice formulation proposed contains at least 15% by weight of such ingredient. Similarly, the European patent appli-cation No. 0346957 (Unilever) discloses the use of hydroxyapatite as a de-sensitising abrasive agent for dental products. In this case, the hydroxyapatite particle size is 1-15 Nm, and the formulation proposed also comprises a source of potassium andlor strontium {as a further desensitising agent).
The treatment of 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. fn addition, the said results are often only partial, as a great amount of co~-operation is required from the 15 patient. In view of that, 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 20 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 insolu-ble precipitates; 3} impregnation and obstruction of the dentine tubules by means of adhesives; 4} application of dental restoration materials, optionally 2s together with intraoral devices for sustained release of fluorine. In the most serious cases, as well as in those cases where any other kind of therapy is ineffective, the dental restoration and the endodontal treatment are often in-eluded in order to finally solve the problem.
As regards the first one of the treatnnents mentioned above, since 3o dentine is not permeable when a suitable smear layer is present, it is often possible; by simply applying an abrasive paste (such as the Nupro paste} and by working the dentine surface by means of silicone or rubber cups, to obtain a thin and uniform smear layer and, consequ~:ntly, the obstruction of the tu-bules. Although simple, such treatment does not give long-lasting results, as the smear layer is soluble (specially in an acidl environment), and therefore it may be easily removed by the patient. An incorrect brushing of the teeth or the ingestion of acid liquids (such as fruit juices, beverages, etc.) are often suffi-cient to cause the smear layer removal. Therefore, the adoption ofi the above treatment is limited to non serious cases, e.g. to treat patients after a perio-dontal intervention with exposure of limited deintine surfaces and without any previous history of dentine hypersensitivity.
~o The second kind of medical treatment mentioned in the foregoing consists, for instance, in applying oxalates (such as potassium, iron or alu-minium oxalate, and oxalic acid) on the affected dentine surface. Such agents may react with the dentinal apatite constituents, thus forming insoluble cal-cium oxalate microcrystals on the surface and in the interior of the dentine ~s 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 prob-lems due to the toxicity of oxalates. Also silver nitrate, another agent em-2o 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 tubutes. _ ..
The same kind of therapeutic approach also includes the direct appli-cation of microcrystalline apatite, as mentioned in the foregoing in connection 25 with products for home therapy. More specifically, the medical treatment in-cludes the use of an ultramicronised apatite, ouch as that disclosed, for in-stance, in the Italian patent No. 1271874, where: the particle size of about 70%
of the apatite microcrystals is less than '! pm (0.2-1 Irm}. Such product is con-sidered to be more active than the micronisedl apatite products disclosed in 3o the foregoing, on the basis of the simple consideration that the dentine tubules have an average diameter of about 1.3-3.5 um. Therefore, crystals having slightly submicronic size may, theoretically, pE;netrate within the dentine tu-WO 00/03747 PCTIIT99l00224 bules, thus performing a better mechanical occlusion. On the contrary, the micron-sized crystals disclosed in the two previously cited patent documents may only create an occlusive layer on the dentine surface, without penetrating into the tubules.
s Considering the medical treatments, however, the procedures be-longing to the third group referred to above, consisting in dentine impregnation by means of adhesive resins, are considered to be more effective. The resin impregnation technique, proposed by Nordenvall and Brannstrom (K.J. Nor-denvall, M. Brannstrom, In vivo resin imprecanation of dentinal tubules, J.
~o Prosthet. Dent.; 44:630-637 (1980)), has become extremely reliable only after the introduction of dentinal adhesive systems consisting of highly hydrophilic resins, which are able to infiltrate through the dentine and to penetrate within the tubules for some tens of microns. tt has been shown that the dentine sur-face, treated with etching acid (for instance, with phosphoric or malic acid) ~s becomes demineralised up to 4-10 Nm of depth, and rich in partially collapsed collagen fibres only. This treatment enhances the penetration of the resin (usually, a primer) within the dentine tubules and within the collapsed colla-gen. The resin, by penetrating into the tubule;>, forms long plugs (referred to as resin tags) which occlude the tubular lumen and reduce the dentine per-2o meability: In addition, the resin impregnates .and occludes the small lateral channels as well (as it has recently been confirmed by some studies by scan-ping electron microscopy); thus contributing to the formation of a thick resin network effective in stably blocking any movernent of the dentine fluid. A by brid layer made of resin and collagen is thus formed. From the point of view of 2s the mechanism of action, it is not clear whetlher other factors co-operate in reducing the sensitivity, in addition to the tubules occlusion and the conse-quent permeability reduction (which is, very Ilikely, the main mechanism of action). It has been hypothesised that some of the components of the dentinal primers also have a pharmacological action such as to reduce the symptoms.
3o At the present state of knowledge, they impregnation treatment of the exposed dentinal surface with resin is to be considered specific for patients wherein few dental elements show a medium or serious dentine hypersensi-_g-tiVity.
Finally, in the occurrence of widespread and serious dentine hyper-sensitivity, a distinction has to be made between lesions with deficiency of periodontal tissue, wherein surgery represents the treatment of choice, and lesions with extensive enamel loss, wherein a conservative dental restoration is indicated. For the latter purpose, the same dentinal adhesive systems of the previously disclosed method may be used, followed, obviously, by the appli-cation of a composite resin-restorative material; in the alternative, photocuring glass-ionomer cements may be used. This kind of intervention, as pointed out before, is reserved to cases showing a marked deficiency of tissues, or when the hypersensitivity is extremely serious. The further therapeutic step is, obvi-ously, the endodontic treatment of the affected tooth.
In addition to the well established treatments referred to in the fore-going, further literature, mainly patent publications, proposes (besides the ~5 micronised and ultramicronised apatite materials mentioned above) the use of amorphous calcium phosphates, both in order to remineralise (and, optionally, to fluoridate) the dental tissue, and in order to reduce the dentine hypersensi-tivity. The US patent No. 5268167 {American Dental Association Health Foun-dation) proposes, to such aim, the use of amorphous calcium phosphates (i.e., 2o ACP or amorphous calcium phosphate; ACPIF or amorphous calcium fluoro-phosphate; ACCP or amorphous calcium carbonate phosphate). According to the disclosure; 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 25 to crystalline apatite, thus performing a remineralising action and reducing dentine sensitivity. In the same frame, the PCT patent application No. WO
94/04460 (American Dental Association Healtlh Foundation) also presents the ACCPF phosphate (i.e., amorphous calcium carbonate fluorophosphate), as a new compound particularly interesting for the same purpose.
3o In the light of the prior art referred to above, it is an object of the pres-ent invention to provide a biocompatible product substantially based on apa-tite, able to satisfactorily comply with various therapeutic requirements in the ,:.....~n~-rrn-nn:~vLt~:v u9- :Za-10--0 : 7::39 : OOU0000000> -t-5~;~ 8:3 :2:3f~:# 9 I~. TEL : ~0~00IO~F30 fli Gen. 20~i 67: 48 r 9 field of dental cage and, frstiy, to effectively and Lastingly counteract dQrr#ine hypersensitivity, by deeply occhding and cemE:nting the derctina tubules, and by exerting a desensitising action that withstands the attack of any chemically aggressive agent presertt in the bras environment, such as acid t~evareges and food.
tn a4cordance with the invention, it has been found that the apatite materials characterised by a nat~a~ysta111ne, preferably defective structure {as opposed to the lcncv~m amorphous and sr~ierocrystaitine Structures, including the uitrarnlcronised forms) lend themselves m~c~ch better to the objects men-30 tinned above, as they allow to achieve remarikable therapeutic results, up to the complete suppression of det~ine hypersensitivity and, in addition, with a considerable ren~ineralisa~on of the denfai (issue. Such results are made possible by the unir~ue features of the cryst;~lline nanostructures, that sub-stantiate the great potential of these advanced materials in many applicative ~5 fields.
In general, ate naraocrystaaiine materials ere artificially synthesised rnaterials, cha~ractertsecD by a constituent phase, of by granular structures, modulated bn a Length scale normally smaller' than 10Q rim. According to the number of dirnensions in which they show a nanametric s~uc~ure, the nano ~c crystalline materials are considered to be with dimensionality eguai to zero t' (clusters of atoms - for instance, dispersed inn a non-nanocrystaliine matrix , fiiarrtents ar tubules), ~Nith dimensicnaiity on~i (multilayers, i.e., layers which are nanornetric in t1-~e only direction of tt~e tt~tiacitnessj, with dimensionallty two tgranular s~uperposltions, ultrafine, or buried layers), or with dimensionafity three ~nanophasic materials, wherein all of the ronstituerrt phases are of nanometric proportions on three dirnensions',t (R.111~, Siegel, in Ma~teriais Sci-enc~ and Technology, Voi. 'I5: Processing oil Metals and Alloys, R_li'V_ Calm, 583 (199~i ~). '1~he particular facnperties c~f the nana~crys#atline materials in com-parison with the conven~i~nai materials res~rEt from the cambirration of their 3o three basic and distinctive features, namely: ij atomic domains (i.e., clusters, grains, I$yers or phases) limited in space to less than ~i00 rim; ii) s#gn~cant atornic fractir~ns asscxiated writh irrter~ace erw~iranmer~ts (that is, grain bounda-'~~g ~~T

ries, interfaces and heterophase; and free surfaces); and iii) interactions among their constituent domains.
At present, it is possible to produce inexpensively, by means of sev-eral physical and chemical processes, clusters of atoms in the range of the nanometric dimensions, containing from -hundreds to tens of thousands of atoms, in such a number as to be assemblablle into materials which advanta-geously incorporate into one single material a multiplicity of effects due to the dimension. The said effects range from the electronic effects of quantum di-mension caused by the spatial confinement of the valence electrons, to the suppression of mechanisms of lattice defects, such as the dislocation genera-tion and the migration towards limited granular dimensions. The basis of the particular pertormances 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. There-fore, for instance, a metal that is conventionally ductile owing to the usual ease in creating and displacing dislocations through the crystal lattice thereof wil! become remarkably harder when the grain size is reduced down to a criti-2o cal point wherein the dislocation sources are r~o more able to work at the low levels of the applied stress.
in addition to being characterised by t:he dimensions- of-their-ultrafine domain (i.e. grains or layers), the nanocrystalline materials are also charac-terised 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 t;he 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%.

...... .w~a.a n-mc.~W .tiClY V~ :l:L-lll- V : 'I~~1) : VVV~1VVVVVV-~ ~~ tS~
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h1. Tt'~'-.~ : 000~000000 (31 G~t't: r~~01. 07: d8 P26 In view of the general features of they nanocrystaliine materials re-ferred to in .the faregain~, for the use prop~osec~ according to the invention the apatite-based nanostructured rr~ateriais appe~~r to possess, firstly, the most suitable gt.anufa~r dimensions to easily and deeply penetrate within the dentine ~ tubules which, as pointed out before, have disameters in the range of 1.3-3.~
Nm). In addition, as pointed out before, the concerned materials show specific physiemiaal properfies connected with #h~eir nanocrystatiine nature, such as the re~arkatxle sur~ac~ lira! reactivity and the hygrnscapiaty, which not t~niy allow the material to penetrate mechanically within the tubules, but to also cause it to cement the internal surfaces thereof, both bar swelling and by readily reacting with the natural tissue, and recrystaliising as a result of the rnetastabie character of the nanocrystalline system. Actually, the tatter may have, in this spec~~fic case, a high defective content Apafite-based materials characterisecl by a nanoaystaHine structure appear to have somehow been descrJbed in the PCT patent application No.
9IV0 9711725 ~Etex Gor~r. ), concerning the to~v temperature synthesis of a law crystaJtinity apattte for use in bone tissue gram, Actually, the definition of claw crystailinEty material" given in such text gene~ricatly includes bath amorphous materials and nanocrystatiine materials (witi~i nanometre-siaed Qr Angstram-zo sized aystaDtine domairtsj. such document, ho~revrer, excie~siveD~r concerns the production of resvrbat~le synthetic bane ma#Exiais, which, in view of their in-tended use, are formed in moulds to give s~~iid elements. The only relevant requirement .For such use is the ability of closely repraducing~ the natural bone,.
tissues, so that the synthetic bone graft is integrated and resorted in the saint 2~ tissues. The applications of the canaerne:d material in the arthQpaedic field differ from the present denfat application in that in the fast case the rf,ateriat must comply with stability requirE;rnents, it must be able to eas-ily integrate within the bone and, in soma oases, i't must be able to in-duce bone growth ~direGt relationship between material and celtsj, While ap in the second case the material must interact with mineaai tissue,having chemical and structurat properties different #rom bone, and having a different Qrganic component. Conset~uentlfy, in the,iatter case character-A~,~"~D

I I' ,w,~. mv~Grw:nu~twnuv trt ~s:. -1V~ V ~ t~'3~V . t:~VVVVVVVVV~~ 'tg~ t3~
:~i3~~~3~be7~3f11 ~ ~ N. TEL : 0A0000a~ 01 Gen. 2'301 67: 49 Pi1 istics of the material such as surface reactivity and metastabiiity will have to prevail. Furthermore, the ortt~apaedic applications that irnroive intenrentir~rts on the bone tissue are concerned with a systs:m interaEcting with the blood stream and unaffected by variations induced by the cx~ntaot with the external envi-ronment (such as pH changes nor cnrpositional rrariations afi various finds).
This is right the opposite c~f what happens in the oral environment, and on the tooth surtaae.
Accordingly, the present invention speciftcaily provides the use of an apatite-based nanostructured material of the general fr~rrnuia:
Ca1 o,.xll~Ix~l~C?4)~gs~43H~ z wherein t~! is a nation r3ifferent from Ca~~', B is pan anion di~'erent from R0~3-, A
is chosen from the group cansis~ng of Ow, CO~', F' and Cl-, x is a number from 0 to 9, y is a number from 0 to 5, z is a number frs~m t~ to 2, wherein the said numbers may also be firactionai, with the proviso that the sum of the ~ s charges of the Ca and ~ catlons is equal to tf~a sum of the charges of the F~OQ3', B, A, anti t3H~ anions, wherein the average size of the crystallites of said nanostructured material is comprised between '1 and 50 nm, for the praduGtion of preparations useful for th$ restoration and the protection of dental tissue and, specificaity, fs~r the the~~py of denfine hypersensitivity and for the remineralisatiorz of enamel and dE3ntine. Specifically, the said na-nostructured rr~aterials may show lattice defarrnations and deflects.
in the above definition, the reference to the terrxt "remineralisation~ is intended to include bflth a proper remineralisatidn of enarnei arrd dentine and a reduction of derr~ineraiisatian, or a prevention of demineralisation. P~ an alternative, the present inv~ntian specificadiy provides the use of an apatite-based nanostructured material of the above cieneral farm~rla wherein the vari-ors syrnbois have the meanings specified above, wherein the average size of the crystallites oaf said n~ostructured material is 3o c~ortiprised betweetz 4.5 and 20B nm and said nanostn:ctured materials show lattice deformations and defects, for the production of preparations useful far the therapy of dentine hypersertsi-A~f '~~t~ ~T

i m...r. rvm~crwnvcr~n.rac« v-r .a-iv- v : i~~t ~ vvvvvvvvvv-~ rr~ c~a t.~a~rrot~~xt:
. I~l : H. TEL : 000~6~60 fl1 Gin. ~~: 0'~: 49 P1? .
-'i''c~-tivity and for the rernineralisa~on aF enamel and dentine.
_ fps it is known, apatites represent the rr~ait~ inorganic process of c~ici-fication c~f normal tissue (i.e. enartoel, dentine, ce~°ne~t, bone) and are found associated ~rrith oifier phosphatEC arid nan-pho,~phatic minerals in path~rlogi~ai calcifications. The main one of these compo~nnds, i.e. hydroxy~apatita or hy_ droxyiapatite ~HA~, ha ink the st~aichiam~tric formula Ca~Q(PCt~)s(dN)2 for Ca~~Pt7~ai?H), and being - in its synthe#ic ~bic~compatible) form - the apa#ite rrraterial most Snrideiy exploited at a commerciail Level for several indica#ions in d~eniistry, orthopaedy and maxilio-tacial surgery, is neYer found in a pure state ~ o in the hiola~ical tissues. This is due to the possible Esomorphaus replacements :.
~~e ~a~~, pp~,~- and C~H- ions. Tl~e calcium ion may be totally or partially replaced by a number of cations generally (but not exclusively) having oxida-tion number +2; the phosphate ion (site B} rnay be replaced by carbonate, acid phosphate, pyrophosphate, sulphate, alurninate and silicate ions, and the hydroxyl ion (site A} may be replaced by halogenide, carbonate and oxide ions.
In comparison with the value 1.67 of the stoichiometric hydroxyapatite, biological apatites have a Ca/P molar ratio co3mprised between 1.53 and 1.74 (in particular, comprised between 1.53 and 1.64 for the dental enamel, be-tween 1.62 and 1.68 for the dentine and between 1.72 and 1.80 for the bone).
In general, the oscillations in the CaIP ratio may be caused by: 1 ) lattice va-cancies; 2) ions isomorphically replaced or adsorbed on the lattice surface (for instance, the substitution of the P04s- anion with acid phosphate, which is divalent, bungs about a reduction in the calcium contents); 3) coexistence or presence of possible precursors consisting of phosphates with different CaIP
~s ratio. Among the latter, in particular, there rnay be mentioned (i-tricalcium phosphate {~i-TCP, i.e. Ca3{P04)z, an orthophosphate also known as tribasic calcium phosphate), amorphous calcium phosphate (ACP), octocalcium phos-phate (OCP, Ca$Hz(P04}6~5H20), dicalcium phosphate dehydrate (DCPD, CaHP04~2H20, also known as calcium acid phosphate, dibasic calcium phos-2o phate or dicalciurn orthophosphate). As it m~iy be deduced from the corre-sponding formulas; the Ca/P molar ratio valuEa for some of the phosphates considered are as follows:
HA Ca5{P04)3OH 1.67 (i-TCP Ca3{PO4)z 1:50 25 OCP Ca8Hz(P04)6~5H20 1.33 DCPD CaHP04~2H20 1.00 More correctly, the biological apatites may be described as non-stoichio-metric carbonatoapatites containing, in general as impurities, different ions.
For this reason, biological apatites have variable rnorphology, crystalline charac-3o teristics, chemical and phisico-chemical properiries.
Among the possible phosphates and a~patite materials, either naturally occurring or obtainable by synthesis, suitable for the production of materials for use in the odontostomatologic and biomedic fields, hydroxyapatite [Ca5(P04)30H~ has already been presented as the most widespread material.
In its structure, the phosphate and calcium ions are placed approximately according to a hexagonal prism; in the direction of elongation (crystallographic s axis c) the prism is crossed by a channel having a diameter of 3-3.5 A, hous ing any OH- groups or other possible replacing ions (e.g., fluorine and chlo rine). HA may crystallise in two forms: a monocl'ine form {spatial group P2~/b) and a hexagonal form (spatial group P63/m): In the monocline form, a binary symmetry axis is present along the axis c, vvhile in the hexagonal form the ~o symmetry axis becomes hexagonal.
Some Authors have hypothesised for the biological samples a simul-taneous presence of amorphous calcium phosphate (ACP) and of scarcely crystalline hydroxyapatite. These two phosphates would differ from each other in the intensity of the diffracted X-rays, while having the same broadening of ~5 the peaks. The same Authors hypothesised that the bone tissue contains both phases, the first one to be deposited being ACP; with time, the latter phase would undergo a transformation into microcrystalline HA according to a partial solubilisation process, with subsequent renucleation. The transformation pro-cess of ACP is apparently controlled by environmental factors (ATP, pyro-2o phosphates, Mg2+, etc.), which stabilise with their presence one of the two phases.
Also octocalcium phosphate [Ca$H2(;P04)6-5H20] {OCP) has been found in biological samples. Its crystalline structure appears to be very similar to the structure of hydroxyapatite. Actually, Ot~P may be transformed into HA
25 through a simple chemical mechanism such as the in situ hydrolysis of the already formed crystals. This kind of transformation, considered to be irre-versible, allows foreign ions to be incorporated in the crystal lattice. Such process might also be controlled by a "layer by layer" transformation, meaning that as soon as OCP is deposited in a layer of the thickness of a unit cell, this so is hydrolysed and transformed into two layers consisting of two unit cells of HA. OCP may be considered to be a mediator between the aqueous phase and the apatitic phase. The kind of product obtained (i.e. OCP or HA) depends on the relative rates of the OCP precipitation process and of the transforma-Lion thereof into HA.
As concerns the possible cation replacement in the apatite formula, although this problem has been studied in thE: literature, the only information s of some interest is that the solid solutions are structurally more ordered when the size of the replacing cation is large. It is not possible to theoretically fore-see the ability of calcium of being replaced by chemically and crystallographi-tally similar ions; in addition, also when this is the case, the extent of the iso-morphaus replacement may be partial. However, it is ascertained that the ~o preparation method has a substantial influence on the extent of the replace-ment.
In an aqueous environment and under conditions similar to the physiological ones, the Mg2* ions inhibit the hydroxyapatite precipitation and promote the formation of (i-tricalcium phosphate. Actually, when the magne-~s slum concentration exceeds 10% the simultaneous formation of ~HA and ~i-TCP occurs, the latter being the only product that precipitates when the con-centration exceeds 25%. The fact that magnesium replaces calcium in (i-TCP
is made evident by the displacement of the X-ray diffraction peaks in the pow-der diffraction spectra of the same samples.
2o However, the magnesium contents in biological apatites is very low {about 1 %), and is limited to a surface deposition. This is evidenced by the fact that during the initial dissolution stage oi' biological apatites (both bone apatite and tooth enamel) a prevailing relea;>e of magnesium ions may be observed. In the apatites with magnesium replacement, the lattice parameters 2s show a slight contraction, and the infra-red {LR.) spectrum bands are shifted towards lower frequencies, in accordance with the lower atomic mass of mag-nesium and with the different energy of the Mg-O interaction.
Strontium is present in biological apa~tites only at the impurity level, and may replace calcium, thus causing an expansion of the a and c axes. The 3o presence of this element in apatites for odontostomatological use is consid-ered 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.
As concerns barium, this cation does not isomorphicaily replace cal-cium in the whole concentration interval. As a matter of fact, the variation of the lattice parameters results in an expansion of the cell parameters.
As pointed out before, the basic structure of hydroxyapatite may also be substituted with anionic groups, in particular, in carbonatoapatite the car-bonate ion may replace the hydroxyl ion (site .A) or the phosphate ion (site B) or both. Although the replacement of the carbonate ion in the site B is prefer-~o ential in the biological samples, it is also possible to obtain carbonatoapatites of the type A synthetically, by means of high temperature reactions. On the other hand, ca~bonatoapatites of the type B o~r mixed type A + B are mainly obtained by precipitation from solutions.
The presence of the carbonate ion in the different sites, which is ~5 hardly detectable by X-ray diffractometry, may be evidenced through LR.
spectroscopy, since the position of the absorption bands of the carbonate ion directly depends on the occupied site. 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 2o greater size of the C032- ion with respect to the OH- ion; if, on the contrary, the carbonate ion occupies the B site the same parameter undergoes a con-traction, due to the smaller O-O distance in the C032--ion with respect-to the P043- ion. In addition, it has been observed that the inclusion of the carbonate ion in apatite results in a reduction and in a diifferent morphology of the crys-25 tats, which appear to change their shape from a needle-like one to one of comparable length in the various crystal dimensions. The solubility increases as well, while the thermal stability decreases.
In fluoro- and chioroapatites the F- and CI- ions, respectively, replace the hydroxy ion; the said replacement, theoretically, may be total. Fluoroapa-3o 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-s roapatite with respect to fluoroapatite derives from the remarkable difference in ionic radius between the two halogens: in l7uoroapatites 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 slrability decreases: It is possible to observe the presence of solid solutions t~etween fluoroapatites and hy droxyapatites and also between chloroapatites and hydroxyapatites in the whole concentration range; solid solutions may also occur befinreen fluoroapa ~5 tites and chloroapatites.
The non-stoichiometric character of biological apatites may also be caused by the presence of the acid phosphate ion HP042-; this ion is con-tained specifically in the tooth enamel, in amounts comprised between 5% and 15%, and is effective in increasing the hydroxyapatite solubility. The presence 20 of acid phosphate in biological and synthetic apatites is not easily detectable, since the carbonate ion, which is almost aiways present, overlaps the LR.
absorption bands and causes a similar variation of the cell parameters: The original presence of HP042- may be evidenced by the pyrophosphate forma-tion, obtained by heating the apatite samples between 400 a 500°C.
25 According to some specific embodiments of the invention, therefore, the M cation is chosen among the following ones: H+, Na+, Mg2+, K+, gr2+, Ba2+ and Fe2+, and preferably the x value is comprised between 0 and 2. The B anion is chosen, preferably among C032-, HP042-, HC03-, and P2074-, while the y value may be comprised, for instance, between 0 and 2. As far as so the A anion is concerned, according to a specific choice the latter is absent, the z value being equal to 0 (hydroxyapatites), while according to another specific choice the A anion is F-, with z = 2 (fluoroapatites).
The nanostructured apatites according to the invention may be pro-duced by any one of the several known methods, already in use for the pro-duction of nanocrystalline materials, such ass 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 plhase separation), and the meth-odg borrowed from nature (i.e., biologically mirnicked systems).
The conventional deposition of layers of material by electrolytic proc-esses or by vapour condensation has been exploited in recent times to de-posit materials of nanometric dimensions with remarkable control and accu-racy. The new or improved methods inctude~, in particular, increasingly ad-vanced mono- or multi-bath systems for elecl:rodeposition and new chemical 15 or physical vapour depcssition methods, such as molecular beam epitaxy (MBE), metal-organic chemical vapour deposition (MOCVD) and chemical vapour synthesis (CVS). Such methods not only afford an accurate control on the chemistry and the thickness of the layer deposited on a nanometric scale, but they also afford, in some cases, to control the nature of the interfaces 2o within the same layers (t_.E. McCandlish, D.E:. Polk, R.W. Siegel, and B.
H.
Kear, Multicomponent Ultraflne Microstructure:;: Mater. Res. Soc. Symp. Proc.
132 (1989); G. Gumbs; S. Luryi, B. Weiss, and G.W. Wicks: -Growth, Proc-essing and Characterisation of Semiconductor Heterostructures: Mater. Res.
Soc. Symp. Proc., 326 (1994}).
25 Several new opportunities also exist in the meld of the production of nanophasic materials assembled from atomic clusters synthesised through physical and chemical methods. For instance, chemical precipitation repre-sents one of the conventional methods for synthesising ultrafine powder or colloidal suspensions that has been successifully applied in the synthesis of so nanometre-sized clusters with narrow dimensional distribution, for instance by applying the sol-gel technique or the inverse nnicelle method: Also many high-temperature gas reaction methods are presently available for the synthesis of nanometric clusters, or of nanostructured powders of bigger size (R.W. Siegel, 1991, loc: cit. ).
As far as the latter group of techniques is concerned, the synthesis of nanocrystalline materials by in sifu consolidation, under vacuum, of ultrafine particles condensed in the form of nanometre-sized gas starts when a precur-sor material, be it an element or a compound; is evaporated into a gas main-tained under low pressure, generally well below 1 atmosphere. The evapo-rated atoms loose energy as a result of the collisions with the atoms or mole-to 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. In order to keep the clusters dimen:>ions small while reducing to a minimum the growth with further atoms or molecules or the coalescence among the clusters, it is necessary to rapidly remove the previously nucleated ~5 clusters from the highly supersaturated zone. Since the clusters are already suspended in the condensation gases, this may be readily obtained by pro-viding for the conditions to displace the said gias, for instance by natural con-vection or by forced circulation. The gas-suspended clusters are then con-veyed towards a collecting surface (the collection occurring by thermophore-2o sis} and are then placed in a piston-anvil device for consolidation. It is also possible to cant' out, for instance, a direct deposition of clusters as thin ~tms or filaments.
Only three basic parameters, working in connection with each other, control the atom clusters formation in the gas condensation process described 25 in the foregoing (McClandish et al., 1989, loc, cif.), namely: the rate of atoms supply to the supersaturation area wherein thE; condensation occurs, the rate of energy removal from hot atoms through the condensation medium, i.e. the gas, and (Siegel, 1991, loc. cit.) the removal rate of the previously nucleated clusters from the supersaturation area.
3o The mechanical attrition method (i.e. (lattice destabilisation) allows to produce nanostructures, rather than by cluster coalescence, through the de-composition of coarse-grained structures, caused by a severe mechanical ~CA 02338021 2001-O1-17 deformation. The nanometre-sized grains fornn 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 s surface wear phenomena (E: Hellstern, H.J. F'echt, 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 deforma-tion densities (R. Valiev, in Mechanical Properties and Deformation Behaviour of Materials Having Ultra-Fine Microstructures, M. Nastasi, D.M. Parkin, ,and ~o 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.
This quite direct and relatively easy method offers a ready access to the ultrafine grain dimensions useful for the purposes of the invention, and ~5 allows to produce commercial amounts of material. 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 accoirding to the invention was pro-duced starting from apatite with microcrystalliine structure through lattice de-_ 2o 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 im-pacts from hard balls contained within the same chamber. The kinetic energy of the balls is generated by a rotation rnovemE;nt of the chamber with respect z5 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 me-chanical 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-~ torr, or with liquids 30 (alcohol, ethers, oils and other organic molecules).
The above method reduces the crystallites dimension through subse-quent introduction of lattice defects (which may be evaluated by means of X-ray diffraction techniques). In particular, the n~anoapatite 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.
s 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 totallx impermeable. The dimensional features, the properties of surface activity and the defective contents of the nanoapatites, preferably obtained according to the previously disclosed method, allow to easily obtain both the mechanical filling of the tubules and the fixing reaction with the tubule surface {i.e. remineralisation). Therefore, the clentine sensitivity resulting from the hydraulic conductivity through the tubule structure of dentine is practically removed. As noted in the foregoing, 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 stimulii are present, the movement of dentinal fluid, perceived at the neural structure level as a pain stimulus.
According a preferred embodiment of the invention, the nanocrystal-20 line apatite materials are protected from acid attack by subjecting them to a treatment with protective aqueous solutions containing tartaric acid andlor its - salts, such as; e.g., aqueous solutions contaiining potassium sodium tartrate {or Seignette's salt, or Rochelle's salt, NaC>OC(CHOH)2COOK~4H20) and hydrous calcium acetate {Ca{CH3C00)2~H20;~, or, in the alternative, with a 2s solution containing tartaric acid (HOOC(CHOH~ COOH). Preferably, a 0.1-1.0 M solutioh 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 ac-cording to the invention, preferably at 37°C, for periods of time comprised between 5 and 30 minutes. Specifically, the rjanoapatite is immersed in the 3o 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 ii K1,1.. 1~U:~ ~ ~.Yt1-hll~tWCaIt:IV U4 ::?'?- lU- U : 'T : ij.. j ..
UIIUUUt)Ui3UU-> +4a t3J '.,~.~~~bti:~: # 1~
DR : t~l. TEL t SflflC~00i~3~ 01 Gen. 2~0t 07: 50 Flew agents: rncno-, bi-, tri-, tetra-, polycarba~cyiated caiciurn gluconate in aqueous solution at a co~aer~tration of 0.0~ _5°~ by v~eiyhtt or in aE:cohoiic or hydroatco-hoiic solution at a r"ancentratlon 'sn the range from O.t3l °~ and 15~k by weight;
or with art acetic andfor rrialic a~nd~or hyalt~ronic solution of ohitosac~ at a ccsn-s centration in the rarsge from 5°~ to 2(3°~ by vrei~~ht; or with a solution of human, bovir~, swine, rat or turi~ey tendorx collagen, either isotonic or not, at a con-centration in the range from ~.5a/a to 5°~o by weight. The said treatments result in a protection of the r~anostructure~i materi~a from acid environments. The said mater;at, however, rnay also be used vtrith~out any further treatment.
~ o the invention Rather concerns compo:~tiar~ for the #herapy of dentine hypersensitivity and for the r~e~roinsr~eiisafion of enamel and dertti~
(~mprisirtg the apatite-Eased nanostruclured materials desait5e~d in the far~egoing, toge~er-w~h further possible ingredients and exclpients of the ;same kind as those used in p~aa-rations frx dental h3rc~ier~ and for the therapy of the oral cavity. The oampositions, ~ s that are pre~r ably in the fame of ~too~te, haste, get, suspension or solution, preferably an from ~.5°!o to 50°k by weight of nanocrystaliine ratite rrtateriai, either treated with protect~re agents or not Fur~~ preferred flees of the cornpo- .
sitions according to the inventjon, with particular reference to oral fonnutations in the get form, are ' Ced in the fuhher dependent dairr~s.
2o Some specific embodiments of the irwention are described below for merely itluatrative purposes, together with the results of tine experimental studies carried out on the proposed rranvstr«cfured tarQducics, ir~ciuding com-parati~re tests with other non-nanocrystalline <~patite materials.
EXAMi'L~ 'E
A rnicracrystaliine carbonate-apatite materiat, nor.-stoiahte~rt7etric as regards the hydroxy ion, was subjected to a lattice destabilisation treatment in a controlled environment, under high energyi, by using the r~eact~on charrrber r~escribed in the foregoing. The resulting pn~duct ~nanospatite) is character iced by average crystallite sues of 15-2C3 nrn anct by an average rniorostrain so content comprised between '! ~~' and 1 Ct's.
A portion of the nanocrystatline ap~~tite thus obtained which will be referred to in the fooltowing as nanoapatite 't or r~ano 't ) has also been sub-~~~ 'r~D ..: . ~

jected to treatment with tartrate in order to increase its resistance to acid at-tack in the conditions of use in the oral cavii;y. To that aim, 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, Miiano) at 37°C for 20 min-utes.

An apatite material of the same nature and composition as the mate-rial 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 compri:>ed between 5~10' and 10'x.
Also in this case, a portion of the nano~crystalline apatite thus obtained (i.e. nanoapatite 2 or nano 2) was treated with potassium sodium tartrate and ~5 hydrous calcium acetate, according to the same procedure of Example 1.
~ Dentine permeability test The effectiveness of the nanocrystailine apatite products according to the invention in the treatment of dentine hypersensitivity was evaluated in 2o terms of reduction of the hydraulic conductance within the dentine tubules, according to a well established protocol (Pas~hley, 1990, loc. cft.), in agree-ment with the international literature. In ordear to carry out such test, sound human molars have been used, extracted for orthodontic reasons from young patients, in order to avoid the problem of sclE;rotic dentine. Each crown was 25 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 sE;gment, projecting into the pulp 3o 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 tuibular 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-s ~ tem.
The tests reported below where carried out; by means of the de-scribed equipment, by applying on the exposed dentine surface a car-boxymethyl cellulose-based gel, to which the single active ingredients under test were added in tum. Each batch of gehwas obtained by dissolving fi g of ~.o carboxymethyl cellulose at 50°C in an aqueous solution containing 0.2% by weight of paraben, and-stirring the solution until a transparent gel, of the right consistency, was obtained. The various products under test were mixed to 3 ml aliquots of the gel thus produced, according to the following scheme:
1. control, consisting of the only gel vehicle;
15 2. commercial microcrystalline hydroxyapatite (Merck, Darmstadt, Germany, Art. 2196);
3. commercial microcrystalline hydroxyapatite treated with potassium sodium tartrate and hydrous calcium acetate as described in Example 1;
4. nanoapatite 1, produced according to Example 1;
20 5. nanoapatite 1 treated with potassium sodium tartrate and hydrous calcium acetate according to Example 1;

- 6. nanoapatite 2, produced according to Example 2;

7. nanoapatite 2 treated with potassium sodiuim tartrate and hydrous calcium acetate as described in Example 1.
25 According to the well established procedure referred to in the forego-ing, for each test the following passages were carried out:
~ Formation of a smear layer by rubbing with abrasive paper under manual pressure, followed by washing in deionised water.
~ Treatment with 0.5 M EDTA (ethylene diammine tetraacetic acid, Sigma, 3o St. Louis, USA) at pH 7.4 for 5 minutes. The permeability was measured after leaving to stand for 2 minutes and subEsequently washing the sample.
The value so obtained was taken as a reference for the following tests, by associating it to a permeability of 100%.
Treatment with the control gel or the gel containing the apatite under test (either treated or non treated} by brushing the tooth for 3 minutes. The said time has been chosen with reference to the correct brushing time in the s normal toothbrushing operation. The tooth was painted rather than brushed, in order to avoid forming a smear layer, which would interfere with the evaluation of the product activity.
~ Etching with 37 wt. % ortophosphoric acid (PJlerck, Darmstadt} for 1 minute, and washing. The permeability was evaluated after leaving to stand for 1 a minute.
The permeability tests were pertormed seven, times for each of the set times {30, 60, 120 seconds) after treatment wiith each single gel, and repeat-ing each stage of the above procedure.
The results thus obtained are presented in the following Table in re ~s sped of all of the procedural steps. In order to f;vidence the behaviour of each gel in the presence of acid attack, the data corresponding to the evaluation at 60 minutes after the gel application and after treatment with ortophosphoric acid are also presented in the histogram of the <~ttached Figure. ~' WO 00/03747 _2~_ PCT/IT99/00224 0 ~ ~ N

N ~ O N 00 .
O h M

d' M M M

r> ~

O M M O ~ ~ r C i' D

I ~ M O M
~

N
r' t d' ' ~t' .~,, tB

L M ~ M O ~ ~ ~ , ' C C7) e ' D -M ~ j o ~ C ~ M

J

_ N N ~ ~ ~ ~

O O t O

.
~ N M

C
l~d d M C~ M
' "

, O

UJ

a U

~ O ~ ~ O

C M N
O

("" ~ ~ ' M M M M w-~' 00II d .

~

O Cs c ~

L
M N O ~ ~ ~ 0 ~. ~ 0 N

a ~ ~ N , LJ! CD ~ ' d ~ M
d' ' O
' 't o o o o o a o o 0 0 z mu ~ o o o o a~
-- - --~ , ~ ~ r.. ~

.~

3 a~

ina ~ r' c v ~ o M
~ c ~-oo~ ~ rf Sri ~ ai cDcD cD co cD so cfl J !

O JJ

... ~ N ~

~ ~ a M ~ ~ ~ ~

~ M A ~ N

C C C C
D D D D

L
m a C

M O ~ N

l d: M l~ r o ~ o ~, ~ ~ ~ ~- N N .C

~ _ N ~ ~ ~ ~ cUC
~

V

O
~ 0 0 U

I= Z Z Z Z Z Z

From the observation of the foregoing table the following conclusions may be drawn, which are even more evident from the enclosed histogram: no positive result was obtained with the control (c;arboxymethyl cellulose gel), as the dentine was extremely permeable, and particularly susceptible to acid attack, so much so that upon treatment with H3P04 the permeability values are above 100% (see Figure).
The use of commercial microcrystallline hydroxyapatite may bring about a partial occlusion of the dentine tubulE;s, with reduced permeability in comparison with the smear layer formation step. However, after the action of H3P04 no detectable resistance is noted. Some resistance is present, on the other hand, if 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 histo-gram.
~5 With the use of -nanoapatites 1 and 2 the dentine permeability is greatly reduced, to the point that the permeability falls from 100% after treat-ment with EDTA to about 40% after reatmE;nt 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 20 interesting data.
The effectiveness of the treatment with nanocrystalline apatite ac-cording to the invention is even more-evident when nanoapatites treated with Seignette's salt and calcium acetate are used. These not only produce a re-markable reduction in the dentine permeability in the absence of an acid at-25 tack, but, most remarkably, afford an even more considerable reduction after the final acid attack by H3P04, with permeability values not exceeding about 35% of the value after treatment with EDTA.
Although the statistic analysis did not evidence any significant differ-ence between nanoapatites 1 and 2, nanoap~atite 2 gave a lower standard 30 deviation in the measurements, and therefore a greater reproducibility in the results.
From the experimental results presented in the foregoing it will be iI.
twV.Vt)tV:~t'A-A'lU~.~!C'.H~.'~i 04 ~'?~-10- 0 : ~:~I ~ UUUUUUUt?UV-~ +ga ts~ -:a~~~tr~a:W ~r ht. Ta. : fl00~006Q~ 01 G ~. e''.~i 07: 50 P14 .- 28 -appreciated that tl~e r~anastr~tctur2d apatite materials according tc~ the inven-tion can be vaiiclly praposed as biot~mpatibie matariais having a marked and prolonged actian in reducing the hydraulic ccrrduGtance within the dentine tui'uies, ~d suitable to become stably arid deeply integrated Era the dentine $ tissue. Accordingly, tt~e said rnatetials may advarttageousiy be ert~plo~red, either as such or included in powder, taot~apaste, paste, gel, solul~orrs, mruth-wash, suspensions, tabltrt, capsule, resin or c:e:rnent c~rrEpositions, in prepara-tions for the protection of dentine; son tt~e therapy of dentine hypersensitivity, for the remineratisation of enamel, dentine arnd denial tissues, for the dosure ~a oaf dentine tubules or the reduction of their functional diameter, fior ttie protec-lion t~f dental pulp ark as sealants for enamel .and dentine.
'~"r~e present invantian has been disclosed with particular reference to some specific era~t~dirnents thereof, hut it should be understood that rnodi~-cations and ,~,hanges may be unade by the persons skilled in the art without ~ 5 departing from the scope t~F tl~e invention as defined in the appended dairns.

Claims (27)

1. Use of an apatite-based nanostructure material of the general formula:
Ca10-x M x(PO4)6-y B y A z(OH)2-z wherein M is a cation different from Ca2+, B is an anion different from PO4 3-, A
is chosen from the group consisting of O2-, CO3 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 PO4 3-, B, A, and OH- anions, wherein the average size of the crystallites of said nanostructured material is comprises between 1 and 50 nm, for the production of preparations for the therapy of dentine hypersensitivity and for the remineralisation of enamel and dentine.

2. Use according to claim 1, wherein the said nanostructured material shows lattice deformations and/or defects.

3. Use of an apatite-based nanostructured material of the general formula:
Ca10-x M x(PO4)6-y B y A z(OH)2-z wherein M is a cation different from Ca2+, B is an anion different from PO4 3-, A
is chosen from the group consisting of O2-, CO3 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 PO4 3-, B, A, and OH- anions, wherein the average size of the crystallites of said nanostructured material is comprises between 0.5 and 200 nm and said nanostructured materials show lattice deformations and defects, for the production of preparations useful for the therapy of dentine hypersensi-tivity and for the remineralisation of enamel and dentine.

4. Use according to claims 1 or 3 wherein the M cation is chosen from the group consisting of: H+, Na+, Mg2+, K+, Sr2+, Ba2+ and Fe2+.

5. Use according to any one of claims 1-4 wherein x is comprises between 0 and 2.

6. Use according to any one of claims 1-5 wherein the B anion is cho-sen from the group consisting of: Co3 2-, HPO4 2-, HCO3-, and P2O~ 4-.

7. Use according to any one of claims 1-6, wherein y is comprised between 0 and 2.

8. Use according to claim 1, wherien said material is hydroxyapatite, x, y and z being are equal to 0.

9. Use according to claim 1, wherein said material is a fluoroapatites, x and y being equal to 0, and the A anion being F-, with z = 2.

10. Use according to any one of claims 1-9, wherien the said prepara-tions for odontostomaatologic applications are powder, paste, gel, solutions, moutwash, suspenstions, tablet, capsule, resin or cement compositions for the protection fo the hard dental tissues, the therapy of dentine hypersensitiv-ity, the remineralisation of enamel, dentine and dental tissues, the closure of dentine tubules or the reduction of their functional diameter and/or for the protection of dental pulp and as sealants for enamel and dentine.

11. Use according to any one of claims 1-10, wherein the said nanos-stuctured apatite materials are treated, in the said production, with protective aqueous solutions containing tartaric acid and/or its salts.

12. Use according to claim 11, wherein a first solution of 0.1-1.0 M
potassium sodium tartrate and a second solution of 0.1-1.0 M hydrous calcium acetate are used in a sequence.

13. Use according to claim 12, wherein the said nanostructured apa-tite material is immersed in the first solution, at 37°C, and kept therein for 5 minutes, then it is withdrawn from the said first solution and immediately im-mersed in the second solution, at 37°C, and kept therein for 20-30 minutes.

14. Use according to any one of claims 1-10, wherein the said nanos-tructured apatite materials are treated, in the said production, with protective aqueous solutions containing mono-, bi, tri, tetra- or poycarboxylated cal-cium gluconate at a concentration of 0.01-5% by weight, or in alcoholic or hydroalcoholic solution at a concentration in the range fresh 0.01 to to 15%
by weight.

15. Use according to any one of claims 1-10, wherein the said nanos-tructured apatite materials are treated, in the said production, with a protective acetic and/or malic and/or hyaluronic solution of chitosan at a concentration in the range from 5% to 20% by weight.

16. Use according to any one of claims 1-10, wherein the said nanos-tructured apatite materials are treated, in the said production, with a protective aqueous 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.

17. A composition for the therapy of dentine hypersensitivity and for the remineralisation of enamel and dentine comprising an apatite-based na-nostructured material as defined in claim 1.

18. A composition for the therapy of dentine hypersensitivity and for the remineralisation of enamel and dentine comprising an apatite-based na-nostructured material as defined in claim 3.

19. The composition according to claims 17 or 18, wherein the said apatite-based nanostructured material is hydroxyapatite.

20. A composition according to any one of claims 17-19 in the form of a powder, toothpaste, paste, gel, solution, mouthwash, suspension, tablet, capsule, resin or cement.

21. The composition according to claim 20 in the form of a paste, a gel, or a suspension, containing from 0.5% to 50% by weight of the said apatite-based nanostructured material.

22. A composition according to claim 21 in the form of a gel, wherein the said gel contains a mixture of polyacrylic acid and/or derivatives thereof and polyethylene glycol, or a mixture of polymethacrylic acid and/or deriva-tives thereof and polyethylene glycol, or a mixture of different polyethylene glycols.

23. The composition according to claim 22, wherein the said gel also contains glycerol.

24. A gel composition according to claims 22 or 23, wherein the said apatite-based nanostructured material is contained into polyethylene glycol microspheres.

25. A composition according to claim 21 in the form of a gel, wherein the said gel contains one or more of the following gel-forming ingredients:
carboxymethyl cellulose, hydroxymethyl cellulose, optionally substituted chito-san.

26. A composition according to claim 21 in the form of a gel, wherein the said gel is a hydrogel obtained from hydrophylic polymers of the polyvinyl or polysaccharide type, mixed with glycerol and/or polyethylene glycol.

27. A composition according to any one of claims 17-26, wherein the said nanostructured apatite material is protected by treatment with aqueous solutions containing tartaric acid and/or its salts, or with aqueous, alcoholic or hydroalcoholic rotations containing mono-, bi-, tri-, tetra- ,or polycarboxylated calcium gluconate, or with acetic and/or malic and/or hyaluronic solutions of chitosan, or with aqueous solutions of human, bovine, swine, rat or turkey tendon collagen.