GB2539490A - Method for the preparation of teeth coatings having morphogenetic activity - Google Patents

Abstract

Solid, biocompatible and biodegradable amorphous calcium polyphosphate microparticles that (i) form a tightly bound polyphosphate layer onto the hydroxyapatite surface, (ii) have a hardness and elastic modulus close to natural enamel, (iii) are able to trigger differentiation of precursor cells into odontoblasts, and (iv) activate the expression of alkaline phosphatase in precursor odontoblasts, for use in sealing dentinal tubules exposed at the tooth surface and filling defects in tooth enamel, cementum and dentin, is provided. Preferably the amorphous calcium polyphosphate microparticles ameliorate dental hypersensitivity or are for the prophylaxis of dental caries. A tooth implant material that stimulates differentiation and activation of odontoblast precursor cells and odontoblasts wherein the material comprises amorphous calcium polyphosphate microparticles is outlined. A method for producing a toothpaste that stimulates differentiation and activation of odontoblast precursor cells and odontoblasts and/or for resealing dentinal tubules to ameliorate hypersensitivity wherein the toothpaste comprises amorphous calcium polyphosphate microparticles is also provided.

Description

METHOD FOR THE PREPARATION OF TEETH COATINGS HAVING

MORPHOGENETIC ACTIVITY

This invention concerns a method for sealing dentinal tubules exposed at the tooth surface as a consequence of enamel defects, based on amorphous calcium polyphosphate microparticles that, in contrast to polyphosphate-calcium salts/complexes, strongly bind both to tooth enamel, cementum and dentin surfaces. The inventive method uses microsized particles, consisting a biocompatible and biodegradable polymer (calcium polyphosphate), can be applied not only for protective teeth coatings but also in the fabrication of morphogeneticallyactive tooth implants that stimulate differentiation of precursor odontoblasts to mature, alkaline phosphatase-expressing cells, with a hardness and elastic modulus close to natural enamel.

Background of Invention

Dental caries and tooth hypersensitivity belong to the most common diseases worldwide. The costs for the public health system caused by them are immense. As result of the loss of enamel or cementum dentinal tubules become exposed to the tooth surface. The consequences are an increased risk of a series of dental diseases/impairments, such as dentin hypersensitivity, caries, and pulp inflammation. Demineralization of enamel and cementum are caused by bacterial biofilm formation, especially by the acid-producing Streptococcus nuttans.

Physiologically, teeth enamel and dentin undergo a permanent remodeling by demineralization and remineralization processes. However, these processes, in particular tooth repair are slow. Calcium and phosphate ions, as well as by fluoride can be administered in order to partially reconstitute the crystal remnants on the subsurface lesions remaining after demineralization. The remineralized crystals are more resistant to acid, but less soluble and more brittle than the original mineral.

The biomineral of tooth enamel, dentin and cementum mainly consists of hydroxyapatite (HA). The formation of HA deposits is controlled, among others, by certain growth factors (e.g., amelogenin and ameloblastin) and enzymes (e.g., ALP and carbonic anhydrase).

Dentin is traversed by a network of tubular structures, termed dentinal tubules. These tubules are shielded by the enamel (crown) and the cementum (root), which form a protective layer of the pulp against external physical and chemical influences, like temperature changes and acids, and prevent affection of the nerve protrusions and dentin hypersensitivity. The diameter of the dentinal tubules which protrude into the dentin layer and are open to the dental surface varies between 1 and 2.5 inn. Patients suffering from tooth hypersensitivity have larger number of open dentinal tubules and/or tubules with a larger in diameter than normal.

Strategies that have been undertaken to reseal and to desensitize dentinal tubules comprise: Occlusion of the dentinal tubules with Na-oxalate (Greenhill JD, Pashley DH (1981) The effects of desensitizing agents on the hydraulic conductance of human dentin in vitro. J Dent Res 60:686-698; Rusin RP, Agee K, Suchko M, Pashley DH (2010) Effect of a new desensitizing material on human dentin permeability. Dent Mater 26:600-607). However: The minerals formed are slowly dissolved in artificial saliva (Suge T, Ishikawa K, Kawasaki A, Yoshiyama NI, Asaoka K, Ebisu S (1995) Duration of dentinal tubule occlusion formed by calcium phosphate precipitation method: In vitro evaluation using synthetic saliva. J Dent Res 74:1709-1714).

Application of Na-fluoride which undergoes transient formation of crystals that remain only relatively loosely attached on the surface (Schlueter N, Hardt M, Lussi A, Engelmann F, Klimek J, Ganss C (2009) Tin-containing fluoride solutions as anti-erosive agents in enamel: An in vitro tin-uptake, tissue-loss, and scanning electron micrograph study. Eur J Oral Sci 117:427-434).

Occlusion of the more surface-exposed sections (up to 10 pm deep) of dentinal tubules by the resealing of the teeth surfaces with roducts present in, e.g. Sensodyne, NovaMin and Colgate Sensitive Pro-Relief (Petrou I, Heu R, Stranick M, Lavender 5, Zaidel L, Cummins D, Sullivan RJ, Hsueh C, Gimzewski JK (2009) A Breakthrough therapy for dentin hypersensitivity: How dental products containing 8% arginine and calcium carbonate work to deliver effective relief of sensitive teeth. J Clin Dent 20:23-31; Ayad F, Ayad N, Zhang YP, DeVizio W, Cummins D, Mateo LR (2009) Comparing the efficacy in reducing dentin hypersensitivity of a new toothpaste containing 8.0% arginine, calcium carbonate, and 1450 ppm fluoride to a commercial sensitive toothpaste containing 2% potassium ion: An eight-week clinical study on canadian adults. J Clin Dent 20:10-16; Vahid-Golpayegani M, Sohrabi A, Biria M, Ansari G (2012) Remineralization effect of topical novamin versus sodium fluoride (1.1%) on caries-like lesions in permanent teeth. J Dent (Tehran) 9:68-75). However: These attempts have been found not to be sufficient as a protection against the daily mechanical forces towards which the teeth are exposed (Moritz A, Schoop U, Goharldmy K, Aoid M, Reichenbach P, Lothaller MA, Wernisch 5, Sperr W (1998) Long-term effects of Co, laser irradiation on treatment of hypersensitive dental necks: Results of an in vivo study. J Clin Laser Med Surg 16:211-215; Orchardson R, Gilliam D (2006) Managing dentin hypersensitivity. J Am Dent Assoc 137:990-998; Chiang YC, Lin HP, Chang HH, Cheng YW, Tang HY, Yen WC, Lin PY, Chang KW, Lin CP (2014) A mesoporous silica biomaterial for dental biomimetic crystallization. ACS Nano 8:12502-12513).

Application of a new formula that is based on calcium and phosphorus which have been embedded into gelatin-based mesoporous silica biomaterial (Chiang YC, Lin HP, Chang HH, Cheng YW, Tang HY, Yen WC, Lin PY, Chang KW, Lin CP (2014) A mesoporous silica biomaterial for dental biomimetic crystallization. ACS Nano 8:12502-12513).

The state-of-the-art of inorganic polyphosphate (polyP) that is one of the constituents used for the fabrication in the microparticles underlying this invention has been described, for example, in: Schroder HC, Muller WEG, eds (1999) Inorganic Polyphosphates -Biochemistry, Biology, Biotechnology. Prog Mol Subcell Biol 23:45-8 I; Kulaev IS, Vagabov V, Kulakovskaya T (2004) The Biochemistry of Inorganic Polyphosphates. New York: John Wiley & Sons Inc.; and Muller WEG, Tolba E, Schroder HC, Wang NH (2015) Polyphosphate: a morphogenetically active implant material serving as metabolic fuel for bone regeneration. Macromolec Biosci, in press (DOI: 10.1002/mabi.201500100).

PolyP, a nontoxic polymer, exists in a wide range of organisms, from bacteria to human. This linear polymer consists of tens to hundreds of phosphate units which are linked together by energy-rich phosphoanhydride bonds.

In previous studies, the inventor showed that polyP i. is accumulated especially in bone cells (Leyhausen G, Lorenz B, Zhu H, Geurtsen W, Bohnensack R, Muller WEG, Schroder HC (1998) Inorganic polyphosphate in human osteoblast-like cells. J Bone Mineral Res 13:803-812; Schroder HC, Kurz L, Muller WEG, Lorenz B (2000) Polyphosphate in bone. Biochemistry (Moscow) 65:296-303), ii. is hydrolyzed by alkaline phosphatase (ALP) present in human osteoblasts (Lorenz B, Schroder HC (2001) Mammalian intestinal alkaline phosphatase acts as highly active exopolyphosphatase. Biochim Biophys Acta 1547:254-261), stimulates mineralization of bone-forming cells (reviewed in: Wang XH, Schroder HC, Wiens M, Ushijima H, Muller WEG (2012) Bio-silica and bio-polyphosphate: applications in biomedicine (bone formation). Curr Opin Biotechnol 23:570-578; Wang XH, Schroder HC and Muller WEG (2014) Enzymatically synthesized inorganic polymers as morphogenetically active bone scaffolds: application in regenerative medicine. Int Rev Cell Mol Biol 313:27-77), iv. induces the expression of the gene encoding the ALP (Muller WEG, Wang XH, Diehl-Seifert B, Kropf K, Schlol3macher U, Lieberwirth I, Glasser G, Wiens M and Schroder HC (2011) Inorganic polymeric phosphate/polyphosphate as an inducer of alkaline phosphatase and a modulator of intracellular Cat level in osteoblasts (SaOS-2 cells) in vitro. Acta Biomater 7:2661-2671), and v. acts as a morphogenetically active polymer during bone-formation (reviewed in: Wang XH, Schroder HC, Muller WEG (2014) Enzymatically synthesized inorganic polymers as morphogenetically active bone scaffolds: application in regenerative medicine. Int Rev Cell Mol Biol 313: 27-77).

Besides of these properties, polyP displays antibacterial activity (Kulakovskaya TV, Vagabov VM, Kulaev IS (2012) Inorganic polyphosphate in industry, agriculture and medicine: Modem state and outlook. Proc Biochem 47:1-10; Miller WEG, Wang XH, Guo YW, Schroder MC (2012) Potentiation of the cytotoxic activity of copper by polyphosphate on biofilm-producing bacteria: A bioinspired approach. Marine Drugs 10:2369-2387).

In GB1420363.2 (Morphogenetically active calcium polyphosphate nanoparticles; inventor: Muller WEG), the inventor described a new biocompatible, biodegradable and biologically active material that is based on polyP. In previous preparations, polyP has been used as potential scaffold for bone implants after calcination. This treatment causes in degradation of the polyP chain. The size of the microparticles described in GB1420363.2 can be adjusted by a defined Pi: Ca2 molar ratio of 1: I or 1:2 (Muller WEG, Tolba E, Schroder BC, Wang S, GlaBer G, Mutioz-Espi R, Link T, Wang XH (2015) A new polyphosphate calcium material with morphogenetic activity. Materials Lett 148:163-166). The particles formed are amorphous and hence are prone to enzymatic hydrolysis by ALP.

Summary of the invention

In a first aspect, the invention is a s disclosed in the appended claims.

Previously, the inventor disclosed a hard amorphous polyphosphate (polyP)-based material that is produced at ambient conditions in the presence of a defined concentration of CaC1,. -4 -

This material consists of spherical, amorphous particles that are biocompatible and biodegradable. Now the inventor surprisingly found that this material, prepared with a size in the microparticulate range, strongly binds to the HA of tooth enamel, cementum and dentin, exposed at the tooth surfaces or damaged tooth surfaces. This finding was unexpected because it was shown that the calcium salt or complex of polyP does not bind to these surfaces, as demonstrated by electron microscopically and element analytical (EDX) methods. In addition, the inventor found that the calcium polyP microparticles trigger differentiation of precursor osteoblasts to the functionally active, alkaline phosphatase expressing cells, if the diameter of these particles is in a range (300 nm) that is not suitable for receptor-mediated endocytosis (around 50 nm). The method according to this invention can be used for resealing dentinal tubules exposed to the tooth surface and coating of teeth to ameliorate tooth hypersensitivity and to prevent tooth decay (caries formation). A further aspect of this invention is the application of the inventive method for preparation of tooth implants that are able to stimulate the formation of body's own new tooth material (HA) by triggering differentiation of osteoblast precursor cells and activating osteoblasts.

The following patent applications on polyP are deemed relevant: GB1406840.7. Morphogenetically active hydrogel for bioprinting of bioartificial tissue. Inventors: Muller WEG, Schroder HC, Wang XII GB1403899.6. Synergistic composition comprising quercetin and polyphosphate for treatment of bone disorders. Inventors: Midler WEG, SchrOder HC, Wang XH PCT/EP2011/062159. Hydroxyapatite-binding nano-and microparticles for caries prophylaxis and reduction of dental hypersensitivity. Inventors: Muller WEG, Wiens M GB1420363.2. Morphogenetically active calcium polyphosphate nanoparticles. Inventor: Midler WEG GB1502116.5. Synergistically acting amorphous calcium-polyphosphate nanospheres containing encapsulated retinol for therapeutic applications. Inventor: Muller WEG

Detailed description of the invention

In GB1420363.2, a polyP material that is characterized by the following properties was described. The material a) is amorphous b) has an unusual hardness, c) consists of nanoparticles (diameter of about 200-300 nm), and d) can be prepared under mild conditions, in particular at room temperature.

The properties of this material were found to be superior compared to conventional polyP preparations used, for example, for bone regeneration and as a bone replacement material (see, e.g., GB1406840.7. Morphogenetically active hydrogel for bioprinting of bioartificial tissue [Inventors: Muller WEG, SchrOder HC, Wang XH]; and GB1403899.6. Synergistic composition comprising quercetin and polyphosphate for treatment of bone disorders [Inventors: Midler WEG, Schroder HC, Wang XH]).

The inventive method described here relates to innovative uses of this material, in the form of microparticles with a size range of around 300 nm (diameter), for the (re)sealing of dentinal tubules exposed at the tooth surface and the filling of tooth defects (defects in tooth enamel, -5 -cementum and dentin). This invention is based on the unexpected finding by the inventor that these Ca-polyP microparticles are able to form a tightly bound polyP layer onto the HA surface. This finding was surprising because the calcium salt or calcium complex of polyP do not bind to these surfaces. A possible explanation might be the existence of free ionic valencies at the surface of the microparticles that are not saturated by calcium ions and can interact with surface-exposed calcium of the HA material, if a phosphate to calcium ratio of 1 1 has been used during preparation of the particles.

A further aspect of this invention concerns the finding that these calcium polyP microparticles are able to stimulate the differentiation of osteoblast precursor cells to mature osteoblasts (expressing the enzyme alkaline phosphatase which is involved in HA formation).

It was also unexpected that the Ca-polyP microparticles display such bioactivity although their diameter (300 nm) is outside the range allowing receptor-mediated endocytosis (around 50 nm).

The inventive method can be used for resealing dentinal tubules exposed to the tooth surface and coating of teeth to ameliorate tooth hypersensitivity and to prevent tooth decay (application for caries prophylaxis).

The inventive method can also be used for the preparation of tooth implants that trigger the body's own tooth material (HA) formation via induction of differentiation of osteoblast precursor cells and activation of mature osteoblasts.

The polyP layer formed on the tooth surface was demonstrated to have a hardness and elastic modulus similar like natural enamel.

The preferred average size (diameter) of the Ca-polyP microparticles used in the inventive method is in the range of about 50 to about 500 nm, preferably 300 nm.

The preferred composition of the Ca-polyP microparticles used in the inventive method is a weight ratio of 0.1 to 10 (phosphate to calcium), preferably of 0.5 to 1, and most preferred 1 to 1.

The chain lengths of the polyP component of the Ca-polyP microparticles can be in the range 3 to up to 1000 phosphate units. Optimal results are achieved with polyP molecules with an average chain length of approximately 200 to 20, and optimally about 40 phosphate units.

The polyP material is biodegradable and displays superior morphogenetic activity, compared to the Ca-polyP salts prepared by conventional techniques.

A further aspect of the inventive method is the application of this method to ameliorate dental hypersensitivity or for caries prophylaxis.

Another aspect of the inventive method is the application of this method for preparation of tooth implants that stimulate differentiation and activation of odontoblast precursor cells and odontoblasts.

The invention will now be described further in the following preferred examples, nevertheless, without being limited thereto. For the purposes of the present invention, all -6 -references as cited herein are incorporated by reference in their entireties. In the Figures listing, Figure 1 shows the amorphous Ca-polyP microparticles (aCa-polyP-MP) and their proposed interaction with the Ca-phosphate surface of the teeth. (A and B) The aCa-polyP-MP; SEM analysis. (C) Proposed interaction of the microparticles with the hydroxyapatite (HA) enamel of a tooth. Enamel (en) forms the crown around the dentin (de) region and surrounds the dental pulp (pu). The minerals enamel and dentin are composed of HA plates, built mainly of P043-and Ca2+ ions. Into an existing dental cavity (caries, or tooth decay) the aCa-polyP-MP are filled. It is proposed that the Ca2+ ions within the microparticles form a bridging to the HA of the enamel.

Figure 2 shows the coating of teeth specimens from the root region with polyp. Teeth samples were incubated in 10 mg/mL of either Na-polyP [Ca2] (A and C) or aCa-polyP-MP (B and D) for 2 d. Then the samples were taken, subjected to slicing and inspected by light microscopy. Images were taken either from the cut areas (A and B) or the corresponding surfaces (C and D). The different layers, cement (ce) and dentin (de) as well as the polyP layer are marked.

Figure 3 shows the formation of a polyP layer onto the teeth specimens after incubation with aCa-polyP-MP; SEM. In parallel, teeth samples were submersed in Na-polyP [Can or aCapolyP-MP (10 mg/mL each) for 2 d. Then the samples were, after washing, cut and then analyzed by SEM. The images A, C and E were taken from samples that had been exposed to Na-polyP [Can], while those of B, D and F came from teeth samples incubated in aCa-polyPMP. The cement (ce) and dentin (de) layers are seen in all samples, while only in those treated with aCa-polyP-MP the additional polyP layer (polyP) is seen. The dentinal tubules are exposed in the Na-polyP [Can sample (E; ..::dt::..), while no opening from the tubules are seen on the surface of the aCa-polyP-MP sample (F).

Figure 4 shows the kinetics of coating with polyp; SEM. (A) Surface of the dentin with the opening of the dentinal tubules (dt). (B and C) Incubation of the root samples for 30 min with aCa-polyP-MP; the microparticles (Ca-polyP-MP) are accumulating in the openings of the tubules. (D and E) A longer incubation period with aCa-polyP-MP results in an expansion of the polyP deposits under formation of a layer; at higher magnification the individual microparticles can be resolved.

Figure 5 shows the time course of polyP deposition onto the surface of the teeth; EDX analysis. (A) Untreated enamel. The enamel samples were treated for 30 min (B) or 2 d (C) with the aCa-polyP-MP; a strong increase of the signals for P and Ca is seen in the sample after 2 d incubation.

Figure 6 shows the mechanical characteristics of the polyP coating onto enamel. Slices from human teeth were prepared and either measured directly or incubated in a saline solution supplemented with 10 mg/mL of aCa-polyP-MP. Incubation at 25°C was performed for 3 h or 2 d. After the incubation the specimens were dried for 10 min and then measured. A typical load-penetration depth curve for a control sample (solid line) or a polyP treated sample after 2 d (broken line) or 3 h (dotted line) is shown. The indentation loads of 82 mN are given. The following load-penetration stages are marked within the curves: loading stage, dwell period at maximum load and unloading part. -7 -

Figure 7 shows the increase of the levels of ALP transcripts in hMSCs after exposure to polyP. The cells remained either untreated or were exposed to 30 ftg/mL of Na-polyP [Can or aCa-polyP-MP. Samples were collected at day 1, day 3 and day 7. The cells were harvested, RNA was extracted and subjected to qRT-PCR analysis; the expression levels, correlated to the expression of the reference gene GAPDH, were determined for the untreated cells (control; open bars), as well as for the Na-polyP [Cal (Na-polyP; cross-hatched) and the aCa-polyP-MP-treated cultures (Ca-polyP-MP; filled bars). Data are expressed as mean values ± SD for four independent experiments. Differences between the groups were evaluated using the unpaired (-test. *p < 0.05.

Figure 8 represents a summary of the mode of action of the aCa-polyP-MP, which is twofold. The microparticles attach strongly to the surface of the tooth, especially in the exposed openings of the dentinal tubules (dt). Those tubules are located in the dentin (de), which is usually covered by the enamel (en) layer. First mode of action (morphogenes.is): Within the dentinal tubules the microparticles undergo hydrolysis via the enzyme ALP, which is released by the odontoblasts (od). In concert with the odontoblasts, the ALP and the hydrolysis product of Ca-polyP, the ortho-phosphate, form hydroxyapatite (HA) and by that repair the dentinal tubules. Second mode of action (resealing): After termination the microparticles (aCa-polyPMP) form a coat onto the decayed tooth surface.

Examples

In the following examples, the inventive method described only for polyP molecules with a chain length of 40 phosphate units. Similar results can be obtained by using polyP molecules with lower and higher chain lengths, such as between 100 to 20 units.

Incubation of teeth with Na-polyP versus aCa-polyP-MP: light microscopy Human teeth specimens were submersed into a solution/suspension (10 mg/mL) of Na-polyP [complexed with Can or aCa-polyP-MP. After an incubation period for 2 d the samples were taken, sliced inwards through the cement-dentin zones and inspected by light microscopy (Fig. 2). In the specimens, treated with Na-polyP [Ca21 no additional layer was observed on the surface of the cement (Fig. 2A). No difference to sections through control, non-treated samples could be seen (images not shown). In contrast, if from a similar region a section, treated with aCa-polyP-MP, was examined a distinct 50-ftm thick additional layer (polyP layer) was observed on top of the cement (Fig. 2B). Surface inspection of cement layer from the Na-polyP [Can treated samples shows the coarse texture (Fig. 2C), while the surface roughness of the polyP (aCa-polyP-MP)-treated teeth is much finer (Fig. 2D).

Incubation of teeth with Na-polyP versus aCa-polyP-MP: SEM analysis In parallel, the samples were examined by SEM (Fig. 3); they were treated for 2 d with the respective polyP sample. Again those specimens that were treated with 10 mg/mL of NapolyP [Ca21 did not show any additional visible layer on top of the cement (Fig. 3A and C). In regions with only a thin cement layer and viewed in the transversal profile the dentinal tubules are exposed (Fig. 3E). However, if the samples treated with 10 mg/mL of aCa-polyPMP were inspected they were covered by a -----50 pm thick additional polyP layer (Fig. 3B and D). No differences between the control samples, not treated with polyP, or with Na-polyP [Ca21 could be detected (not shown).

The coating of the teeth with polyP during incubation with aCa-polyP-MP is dependent on the incubation period (Fig. 4). In the absence of any polyP preparation (Fig. 4A) or after incubation with Na-polyP [Can some dentinal tubules are seen on the surface of the dentin -8 -region (not shown) at the root part. If those teeth samples are exposed to aCa-polyP-MP for 30 min already all dentinal tubules are filled with the polymer (Fig. 4B); at a higher magnification the microparticles within the openings of those tubules can be visualized (Fig. 4C). If the incubation time is prolonged to 2 d an (almost) homogenous polyP coating can be resolved by SEM at low magnification (Fig. 4D); at a higher resolution the microparticles are visible (Fig. 4E).

EDX analysis of the polyP deposits The technique of EDX spectroscopy was employed to characterize the polyP deposition onto the enamel surface of the root part of the teeth Analyzing the element distribution of the surface of the untreated enamel shows the characteristic signals for 0, P and Ca, especially representing the mineral part of the teeth, while the significant C signal reflects the organic constituents of the teeth. In addition, low amounts of Na and Mg are seen (Fig. 5A). During the short incubation period with 10 mg/mL of aCa-polyP-MP for 30 min only low signals for P and Ca are measured (Fig. 5B); however, after the 2 d incubation period the P and Ca signals substantially increased, reflecting the deposition of polyP from the microparticles (Fig. 5C).

Mechanical properties of the polyP coating Hardness measurements were performed with a triangular Berkovich diamond indenter. Per given value, 30 single measurements were performed onto the (polyP) enamel. A maximum load of 82 mN was applied resulting in a displacement of 1000 nm in maximum. On average the maximum penetration depth of the indents was 250 + 21 nm. Within one group all load-displacement curves showed a similar shape as the one given in Fig. 6. The typical loading, hold and unloading periods in a typical test cycle of a single indent are shown for each of the three series of experiments. The contact stiffness at maximum load was calculated by fitting a power-law function under the unloading segment. In turn, the slope of the obtained function at maximum load was used to calculate the contact depth of an indent. With this parameter the Martens hardness and the reduced elastic modulus were calculated, applying the algorithms described by Oliver and Pharr (Oliver WC and Pharr GM (1992) An improved technique for determining hardness and elastic modulus using load and displacement sensing indentation experiments. J Mater Res 7:1564-1583). For the untreated enamel a Martens hardness of 4.33+0.69 GPa and a reduced elastic modulus of 101.61+8.52 GPa were calculated (average of 30 measurements). Only slightly lower were the respective values for polyP coated enamel samples; after a 3 h incubation [2 d incubation] period a Martens hardness of 3.85+0.64 [4.05+0.59] GPa and a reduced elastic modulus of 94.72+8.54 [85.62+5.33] GPa were calculated.

Functional analysis of the aCa-polyP-MP: ALP expression in hMSC cells The stromal cells, hIVISC, differentiating towards odontoblasts in the presence of conditioned medium were cultivated either in the absence or presence of polyP. As polyP samples both Na-polyP [Ca2+] and aCa-polyP-MP were used at a concentration of 30 ug/mL. In separate assays cells were harvested after 1, 3 or 7 d of incubation for determination of the expression level of the ALI' gene. The results show that in the absence of polyP the steady-state-expression level of ALP remains almost unchanged during the 7-d incubation period with a ratio to the expression of the reference gene GAPDH of approximately 0.02 (Fig. 7). In contrast, if Na-polyP [Ca211] is added to the cultures a significant increase of the expression level to 0.53+0.01 is measured. A stronger effect is seen if the cells were exposed to aCapolyP-MP. Already after a 3 d incubation period, a significant 2.6-fold increase of the level of ALP transcripts was determined, a value which further increased to 7-fold during the total 7 d incubation period.

The new aCa-polyP-MP-based tooth resealing biomaterial The amorphous Ca-polyP microparticles (aCa-polyP-MP) strongly attach to the surface of the teeth and -undergo in the dentinal tubules hydrolysis to ortho-phosphate via the enzyme alkaline phosphatase (ALP). In turn, the products elicit morphogenetic activity during which the gene encoding for the ALP becomes induced; this process contributes to the repair of the hydroxyapatite in the decayed dentinal tubules. Finally the dentinal tubules are resealed by a layer of Ca-polyP (Fig. 8).

Methods Polyphosphate The sodium polyphosphate (Na-polyP of an average chain of 40 phosphate units) used in the Examples has been obtained from Chemische Fabrik Budenheim (Budenheim; Germany).

Preparation of amorphous Ca-polvP microparticl es The microparticles, composed of Ca-polyP (termed aCa-polyP-MP), are prepared as described (Muller WEG, Tolba E, Schroder HC, Wang S, Gla13er G, Muiloz-Espi R, Link T, Wang XH (2015) A new polyphosphate calcium material with morphogenetic activity. Materials Lett 148:163-16) with some modifications. In brief, 10 g of Na-polyP are dissolved in distilled water and added to 14.2 g of CaC12*2H20 at room temperature. During the preparation the pH is adjusted to 10.0. After stirring (4 h), the particles are collected, washed with ethanol and dried at 60°C.

Chemical characterization by FTIR The polymer characteristics of polyP within the microparticles can be verified, for example, by application of Fourier transform infrared spectroscopy; X-ray diffraction analysis can be used prove that the material is amorphous. The average size of the particles is 300 nm and they vary between the size range of 100 to 600 nm (Fig. lA and B).

In vitro incubation of teeth In order to determine the efficacy of the aCa-polyP-MP to reseal the dentin layer and to check for the potency of the microparticles to occlude the dentinal tubules human teeth are used. Prior to use the teeth are mechanically cleaned from soft tissue, treated for 5 h in 3% Nahypochlorite to remove tissue remains and then stored at 4°C in a 100% relative humidity chamber.

Teeth specimens are submersed in saline (0.90% [w/v] NaCI) which contained, as mentioned in the text, either 10 mg/mL of aCa-polyP-MP or Na-polyP, stoichiometrically complexed with Ca2+ (molar ratio of 2:1/phosphate monomer:Ca2-[Muller WEG, Wang XH, Diehl-Seifert B, Kropf K, SchloBmacher U, Lieberwirth I, Glasser G, Wiens M and Schroder HC (2011) Inorganic polymeric phosphate/polyphosphate as an inducer of alkaline phosphatase and a modulator of intracellular Ca2+ level in osteoblasts (SaOS-2 cells) in vitro. Acta Biomater 7:2661-2671]). Incubation is performed at 25°C. Then the samples are sliced by cutting 1-2 mm thick discs inwards to the pulp as described (Wang XH, Schroder HC and Muller WEG (2014) Enzyme-based biosilica and biocalcite: biomaterials for the future in regenerative medicine. Trends Biotechnol 32:441-447); where indicated, the dentin or enamel regions, as well as the cement layer, are included in the measurements.

Microscopic analyses Scanning electron microscopy (SEM) can be performed, for example, with a HITACHI SU 8000 (Hitachi High-Technologies Europe GmbH), equipped with a low voltage (<1 kV; -10 -analysis of near-surface organic surfaces) detector. Tooth samples, after the respective incubation, are washed 3-times in PBS (phosphate-buffered saline). After a short traversing through distilled water the specimens are dried and inspected. Where mentioned the samples have been cut.

Digital light microscopy can be performed, for example, with a VHX-600 Digital Microscope (Keyence) equipped with a VH-Z100 zoom lens.

Energy dispersive X-ray spectroscopy Energy dispersive X-ray (EDX) spectroscopy can be performed, for example. with an EDAX Genesis EDX System attached to a scanning electron microscope (Nova 600 Nanolab) operating at 10 kV with a collection time of 30-45 s. Areas of approximately 10 um are analyzed.

Mechani cal -nanoin dentati on determinations The surfaces of either the untreated or the polyP-coated teeth specimens are evaluated at 25°C by depth-sensing indentation, using, for example, a NanoTest Vantage system (Micro Materials Ltd). A three-sided Berkovich diamond indenter is used to produce triangular-shaped indentation marks on the coating surface; the tip radius measures approximately 50100 mu, A total of 30 single measurements is performed. The loading rate as well as the unloading rate is fixed to 0.5 mN s-1. To determine the "creep-effect" a 30 s dwell period is introduced at a maximum load. In the unloading curve a second dwell period (60 s) at 10% of the maximum load is used to determine the thermal drift of the system. The Martens hardness and the reduced modulus of the specimens are calculated with the unloading data according to the Oliver and Pharr Method (Oliver WC and Pharr GM (1992) An improved technique for determining hardness and elastic modulus using load and displacement sensing indentation experiments. J Mater Res 7 1564-1583). For the calculations the software "NanoTest Platform Four V.40.08 (Micro Materials Ltd)" can be used.

Reverse transcription-quantitative real-time PCR analyses To quantify the expression level of the gene encoding the ALP in the hMSC, the technique of reverse transcription-quantitative real-time polymerase chain reaction (qRT-PCR) is applied. After incubating the cells for 1, 3 and 7 d in the presence of 30 us/mL either of Na-polyP [Ca2-] or of aCa-polyP-MP they are harvested, RNA is isolated and qRT-PCR is performed. The primer pairs, matching with the human ALP gene (accession number NM 000478.4) Fwd: 5'-TGCAGTACGAGCTGAACAGGAACA-3' (SEQ ID NO. 1) [ntii4i to ntii64] and Rev: 5'-TCCACCAAATGTGAAGACGTGGGA-3' (SEQ ID NO. 2) [nti4i8 to nt1395; PCR product length 278 bp] and with the reference gene GAPDH (glyeeraldehyde 3-phosphate dehydrogenme; NM_002046.3) using the primer pair Fwd: 5'-CCGTCTAGAAAAACCTGCC-3' (SEQ ID NO. 3) [ntg45 to nts63] and Rev: 5'-GCCAAATTCGTTGTCATACC-3' (SEQ ID NO. 4) [ntios9 to ntio78; 215 bp], can be used. The amplification can be performed, for example, in an iCycler (Bio-Rad) with the respective iCycler software. After determination of the C, values the expression of the respective transcripts can be calculated (Pfaill MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucl Acids Res 29:2002-2007).

Cultivation of the human multipotent stromal cells The human multipotent stromal cells (hMSC) differentiate into odontoblasts in the presence of conditioned medium from developing tooth germ cells; the conditioned medium is prepared as described (Huo N, Tang L, Yang Z, Qian H, Wang Y, Han C, Gu Z, Duan Y, Jin Y (2010) Differentiation of dermal multipotent cells into odontogenic lineage induced by embryonic and neonatal tooth germ cell-conditioned medium. Stem Cells Dev 19:93-104). The description of the cultivation procedure for the hMSCs has been published (Wang XH, Schroder HC, Grebenjuk V, Diehl-Seifert B, Mailander V, Steffen R, SchloBmacher U and Muller WEG (2014) The marine sponge-derived inorganic polymers, biosilica and polyphosphate, as morphogenetically active matrices/scaffolds for differentiation of human multipotent stromal cells: Potential application in 3D printing and distraction osteogenesis. Marine Drugs 12:1131-1147). The human cells can be obtained, after approval from the responsible ethics committee, from bone marrow aspirations after informed consent of the donors. Incubation was performed in a humidified incubator at 37°C and 5% CO2. The sixth passage is used for the studies. The cells are incubated in a-NIEM (Biochrom), supplemented with 20% fetal calf serum (FCS; Gibco Invitrogen) as well as with 100 units/mL penicillin and 100mg/mL streptomycin. In addition, 5% of conditioned medium is added to the assays.

After the third passage in the presence of the conditioned medium the cells are continued to culture in 48-well plates (Cat. no. 677102; Greiner) either in the absence of polyP or the presence of 30 jtg/mL either of Na-polyP [Cal or of aCa-polyP-MP. Then the cells are harvested for qRT-PCR analysis.

Statistical analysis The results can be statistically evaluated using the paired Student's t-test.

Claims (11)

1. -12 -CLAIMS1. Solid, biocompatible and biodegradable amorphous calcium polyphosphate microparticles that i) form a tightly bound polyphosphate layer onto the HA surface, ii) have a hardness and elastic modulus close to natural enamel, iii) are able to trigger differentiation of precursor cells into odontoblasts, and iv) activate the expression of alkaline phosphatase in precursor odontoblasts for use in sealing dentinal tubules exposed at the tooth surface and filling defects in tooth enamel, cementum and dentin.
2. 2. The amorphous calcium polyphosphate microparticles for use according to claim 1, wherein said calcium polyphosphate microparticles are characterized by a weight ratio of 0.1 to 10 (phosphate to calcium), preferably of 0.5 to 1 (phosphate to calcium), and more preferably of 1 to 1 (phosphate to calcium).
3. 3. The amorphous calcium polyphosphate microparticles for use according to claim 1 or 2, wherein the chain length of the polyphosphate is in the range of about 3 to about 1000 phosphate units, preferably in the range of about 10 to about 100 phosphate units, and most preferred about 40 phosphate units.
4. 4. The amorphous calcium polyphosphate microparticles for use according to any of claims 1 to 3, wherein the average size of the calcium polyphosphate microparticles is in the range of about 50 to about 500 nm, preferably wherein the average size of the calcium polyphosphate microparticles is 300 nm.
5. 5. The amorphous calcium polyphosphate microparticles for use according to any of claims 1 to 4 for resealing dentinal tubules to ameliorate dental hypersensitivity or for caries prophylaxis.
6. 6. A method for producing a tooth implant material that stimulate differentiation and activation of odontoblast precursors cells and odontoblasts, comprising including the solid, biocompatible and biodegradable amorphous calcium polyphosphate microparticles as disclosed according to any of claims 1 to 4 into said tooth implant material.
7. 7. A method for producing a toothpaste that stimulates differentiation and activation of odontoblast precursors cells and odontoblasts comprising including the solid, biocompatible and biodegradable amorphous calcium polyphosphate microparticles as disclosed according to any of claims 1 to 4 into said toothpaste.
8. 8. A method for producing a toothpaste that that reseals the dentinal tubules and, by that, ameliorates hypersensitivity comprising including the solid, biocompatible and biodegradable amorphous calcium polyphosphate microparticles as disclosed according to any of claims 1 to 4 into said toothpaste.
9. 9. Tooth implant material produced according to claim 6 or toothpaste produced according to claim 7 or 8.
10. 10. Tooth implant material or toothpaste according to claim 9 for use in stimulating differentiation and activation of odontoblast precursors cells and odontoblasts.
11. 11. Toothpaste according to claim 9 for use in resealing the dentinal tubules and, by that, ameliorating hypersensitivity.