DENTAL TREATMENT

Abstract

A pharmaceutically acceptable small molecule which inhibits GSK-3 activity, such as BIO, CHIR99021 or tideglusib; are used in the repair or regeneration of dentine. Combinations with matrix materials forming dental implants are also described and claimed.

Claims

1-14. (canceled)

15. A method for repairing or regenerating dentine which comprises administering to a patient in need thereof, a pharmaceutically acceptable small molecule which inhibits GSK-3 activity.

16. The method according to claim 15 wherein the small molecule is applied topically to an area of exposed dentine.

17. The method according to claim 16 wherein the small molecule is administered in association with a matrix material.

18. The method according to claim 17 wherein the matrix material comprises a collagen sponge, which has been impregnated with the small molecule.

19. The method according to claim 17 wherein the matrix material is shaped to fill a cavity in which dentine is exposed.

20. The method according to claim 17 wherein the matrix material is held in place by means of a cap, crown or ionomer.

21. (canceled)

22. The method according to claim 15, which is a method for the treatment of dental caries or for the treatment of dental trauma.

23. The method according to claim 15, wherein the pharmaceutically acceptable small molecule is a thiadiazolidindione, or a pharmaceutically acceptable salt thereof.

24. The method according to claim 15, wherein the pharmaceutically acceptable small molecule is selected from the group consisting of formula (I): ##STR00009## formula (II): ##STR00010## wherein: W is optionally substituted carbon or nitrogen; X and Y are independently selected from the group consisting of nitrogen, oxygen, and optionally substituted carbon; A is optionally substituted aryl or heteroaryl; R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are independently selected from the group consisting of hydrogen, hydroxyl, and optionally substituted loweralkyl, cycloloweralkyl, alkylaminoalkyl, loweralkoxy, amino, alkylamino, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, heteroarylcarbonyl, heteroaralkylcarbonyl, aryl and heteroaryl; R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are independently selected from the group consisting of hydrogen, and optionally substituted loweralkyl; R.sub.6 and R.sub.7 are independently selected from the group consisting of hydrogen, halo, and optionally substituted loweralkyl, cycloalkyl, alkoxy, amino, am inoalkoxy, alkylcarbonylamino, arylcarbonylamino, aralkylcarbonylamino, heteroarylcarbonylamino, heteroaralkylcarbonylamino, cycloimido, heterocycloimido, am idino, cycloamidino, heterocycloamidino, guanidinyl, aryl, biaryl, heteroaryl, heterobiaryl, heterocycloalkyl, and arylsulfonamido; and R.sub.6 is selected from the group consisting of hydrogen, hydroxy, halo, carboxyl, nitro, amino, amido, amidino, imido, cyano, and substituted or unsubstituted loweralkyl, loweralkoxy, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, heteroarylcarbonyl, heteroaralkylcarbonyl, alkylcarbonyloxy, arylcarbonyloxy, aralkylcarbonyloxy, heteroarylcarbonyloxy, heteroaralkylcarbonyloxy, alkylaminocarbonyloxy, arylaminocarbonyloxy, formyl, loweralkylcarbonyl, loweralkoxycarbonyl, am inocarbonyl, am inoaryl, alkylsulfonyl, sulfonamido, am inoalkoxy, alkylamino, heteroarylamino, alkylcarbonylamino, alkylaminocarbonylamino, arylaminocarbonylamino, aralkylcarbonylamino, heteroarylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino cycloamido, cyclothioamido, cycloamidino, heterocycloamidino, cycloimido, heterocycloimido, guanidinyl, aryl, heteroaryl, heterocyclo, heterocycloalkyl, arylsulfonyl and arylsulfonamido; and formula (III): ##STR00011## wherein R.sub.15 is an organic group having at least 8 atoms selected from C or O, which is not linked directly to the N through a C(O) and comprising at least an aromatic ring; and R.sub.16, R.sub.17, R.sub.18, R.sub.19, R.sub.20, R.sub.21 and R.sub.22 are independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocyclyl, COR.sub.23, C(O)OR.sub.23, C(O)NR.sub.23R.sub.24 CNR.sub.23, CN, OR.sub.23, OC(O)R.sub.23, S(O).sub.tR.sub.23, NR.sub.23R.sub.24, NR.sub.23C(O)R.sub.24, NO.sub.2, NCR.sub.23R.sub.24 or halogen; t is 0, 1, 2 or 3; R.sub.23 and R.sub.24 are each independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryloxy, halogen; wherein R.sub.21 and R.sub.22 together can form a group O, and wherein any pair R.sub.21 R.sub.16, R.sub.16 R.sub.17, R.sub.17 R.sub.18, R.sub.18 R.sub.19, R.sub.19 R.sub.20, R.sub.20 R.sub.22, or R.sub.23 R.sub.24 can form together a cyclic substituent; or a pharmaceutically acceptable salt thereof.

25. The method according to claim 24, wherein the pharmaceutically acceptable small molecule is BIO (6-bromoindirubin-3-oxime), CHIR.sub.99021 (6-[[2-[[4-(2,4-Dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2- pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile), or tideglusib (4-benzyl-2-(naphthalen-1-yl)-[1,2,4]thiadiazolidine-3,5-dione).

26. A combination of a matrix material suitable for use in a dental implant, and a pharmaceutically acceptable small molecule which inhibits GSK-3 activity.

27. The combination according to claim 26, wherein the matrix material is biodegradable.

28. The combination according to claim 27, wherein the matrix material is porous.

29. The combination according to claim 28, wherein the pharmaceutically acceptable small molecule is impregnated into the matrix material.

30. The combination according to claim 28, wherein the matrix material is a collagen sponge.

31. The combination according to claim 26, wherein the pharmaceutically acceptable small molecule is a thiadiazolidindione, or a pharmaceutically acceptable salt thereof.

32. The combination according to claim 26, wherein the pharmaceutically acceptable small molecule is selected from the group consisting of formula (I): ##STR00012## formula (II): ##STR00013## wherein: W is optionally substituted carbon or nitrogen; X and Y are independently selected from the group consisting of nitrogen, oxygen, and optionally substituted carbon; A is optionally substituted aryl or heteroaryl; R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are independently selected from the group consisting of hydrogen, hydroxyl, and optionally substituted loweralkyl, cycloloweralkyl, alkylaminoalkyl, loweralkoxy, amino, alkylamino, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, heteroarylcarbonyl, heteroaralkylcarbonyl, aryl and heteroaryl; R.sub.1, R.sub.2, R.sub.3, and R.sub.4, are independently selected from the group consisting of hydrogen, and optionally substituted loweralkyl; R.sub.6 and R.sub.7 are independently selected from the group consisting of hydrogen, halo, and optionally substituted loweralkyl, cycloalkyl, alkoxy, amino, aminoalkoxy, alkylcarbonylamino, arylcarbonylamino, aralkylcarbonylamino, heteroarylcarbonylamino, heteroaralkylcarbonylamino, cycloimido, heterocycloimido, amidino, cycloamidino, heterocycloamidino, guanidinyl, aryl, biaryl, heteroaryl, heterobiaryl, heterocycloalkyl, and arylsulfonamido; and R.sub.6 is selected from the group consisting of hydrogen, hydroxy, halo, carboxyl, nitro, amino, amido, amidino, imido, cyano, and substituted or unsubstituted loweralkyl, loweralkoxy, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, heteroarylcarbonyl, heteroaralkylcarbonyl, alkylcarbonyloxy, arylcarbonyloxy, aralkylcarbonyloxy, heteroarylcarbonyloxy, heteroaralkylcarbonyloxy, alkylaminocarbonyloxy, arylaminocarbonyloxy, formyl, loweralkylcarbonyl, loweralkoxycarbonyl, am inocarbonyl, am inoaryl, alkylsulfonyl, sulfonamido, am inoalkoxy, alkylamino, heteroarylamino, alkylcarbonylamino, alkylaminocarbonylamino, arylaminocarbonylamino, aralkylcarbonylamino, heteroarylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino cycloamido, cyclothioamido, cycloamidino, heterocycloamidino, cycloimido, heterocycloimido, guanidinyl, aryl, heteroaryl, heterocyclo, heterocycloalkyl, arylsulfonyl and arylsulfonamido; and formula (III): ##STR00014## wherein R.sub.15 is an organic group having at least 8 atoms selected from C or O, which is not linked directly to the N through a C(O) and comprising at least an aromatic ring; and R.sub.16, R.sub.17, R.sub.18, R.sub.19, R.sub.20, R.sub.21 and R.sub.22 are independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocyclyl, COR.sub.23, C(O)OR.sub.23, C(O)NR.sub.23R.sub.24 CNR.sub.23, CN, OR.sub.23, OC(O)R.sub.23, S(O).sub.tR.sub.23, NR.sub.23R.sub.24, NR.sub.23C(O)R.sub.24, NO.sub.2, NCR.sub.23R.sub.24 or halogen; t is 0, 1, 2 or 3; R.sub.23 and R.sub.24 are each independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryloxy, halogen; wherein R.sub.21 and R.sub.22 together can form a group O, and wherein any pair R.sub.21 R.sub.16, R.sub.16 R.sub.17, R.sub.17 R.sub.18, R.sub.18 R.sub.19, R.sub.19 R.sub.20, R.sub.20 R.sub.22, or R.sub.23 R.sub.24 can form together a cyclic substituent; or a pharmaceutically acceptable salt thereof.

33. The combination according to claim 32, wherein the pharmaceutically acceptable small molecule is BIO (6-bromoindirubin-3-oxime), CHIR.sub.99021 (6-[[2-[[4-(2,4-Dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrim idinyl]amino]ethyl]amino]-3-pyridinecarbonitrile), or tideglusib (4-benzyl-2-(naphthalen-1-yl)-[1,2,4]thiadiazolidine-3,5-dione).

34. The combination according to claim 26, which further comprises an antibiotic, a transforming growth factor beta (TGF-) agonist, a bone morphogenetic protein (BMP) agonist, or a combination thereof.

35. A kit comprising the combination according to claim 26.

Description

[0069] The invention will now be particularly described by way of example with reference to the accompanying diagrammatic drawings.

[0070] FIG. 1. Drug Titration and Agonist Activation of the Wnt Pathway

[0071] MTT cytotoxity assay for (A) BIO, (B) CHIR.sub.99021, and (C) Tideglusib. (D) Axin2 qPCR for the In vitro assay with the 171IA cell line shows that when 50 nM BIO, 5pm CHIR, and 50 nM

[0072] Tideglusib are in the sponge, Wnt activity increases after 30 minutes of incubation and remains elevated. This elevation is not seen when just media or collagen sponge without the drug are incubated with the cells. (E) Axin2 qPCR for dental pulp cells collected either without injury or after one day of injury and capping with the conditions. BIO, CHIR and Tideglusib shows significant upregulation of Wnt activity when compared with control, MTA or collagen sponge. *P=0.0365, ****P<0.0001.

[0073] FIG. 2. Injury and Direct Tooth Capping

[0074] (A) photograph of upper first molars. (B) A carbide bur cuts the tooth exposing the dentine until the roof of the pulp chamber (red dashed line). (C) Using a needle the dental pulp is exposed indicated by the arrowheads. (D) The collagen sponge is soaked in drug and a small piece of it, indicated by the black dashed line, is removed for the direct capping. (E)

[0075] The injury capped with MTA. (F) The sponge piece condensed inside the exposed pulp area. (G) The tooth is then sealed with glass ionomer until the date of collection. (H) MicroCT image right after capping showing the close contact of MTA (RO area indicated by arrow) with the dental pulp and the glass ionomer sealing. (I) MicroCT image right after capping showing the close contact of the collagen sponge (RL area indicated by arrow) with the dental pulp and the glass ionomer sealing. ED, exposed dentine; EP, exposed pulp; CS, collagen sponge; GI, glass ionomer; RO, radiopaque; RL, radiolucent.

[0076] FIG. 3. MicroCT Analysis of Mineral Deposition

[0077] (A) MTA repair after 4 weeks, note the material (strong RO area at the injury site) at the injury site. (B) Collagen sponge repair after 4 weeks, spaced dentine formation at the injury site. (C) BIO, (D) CHIR, and (E) Tideglusib repairs show mature mineral at the injury site after 4 weeks. (F) MTA repair after 6 weeks still shows material at the injury site (strong RO area at the injury site). (G) Collagen sponge treatment shows injury mildly repaired. (H) BIO and (I) CHIR repair after 6 weeks displays injury site filled with mature dentine. (J) Tideglusib repair after 6 weeks shows mature reparative dentine formed at the injury site almost at the same Radiopacity as the primary/secondary dentine. No external material is seen at the injury site after repair when teeth were treated with signalling modulators in collagen sponge. (K, L) 4 and 6 weeks, respectively, Mineral formation analysis at the injury site shows that teeth treated with small molecules form more mineral than when treated either with collagen sponge or MTA. 4 weeks BIO *P=0.0101, 4 weeks CHIR *P=0.0136, 4 weeks Tideglusib *P=0.0194; 6 weeks BIO *P=0.0101, 6 weeks CHIR *P=0.0194, 6 weeks Tideglusib *P=0.0101.

[0078] FIG. 4. Histology of Reparative Dentine Formation And Pulp Vitality

[0079] (A) 4 weeks MTA repair shows dentine formed underneath where the material was placed. (B) Collagen sponge shows sparse dentine formation in the dental pulp. (C) BIO, (D) CHIR, and (E) Tideglusib repairs show dense dentine formation at the injury site with vital pulp after 4 weeks. (F) 6 weeks MTA repair shows dentine formed underneath where the material was placed. (G) Collagen sponge repair shows little and immature dentine formed at the injury site after 6 weeks. (H) BIO treatment shows new mature dentine formed where the sponge was placed filling the injury site. (I) CHIR treatment shows mature new mature dentine formed where the sponge was placed filling the injury site. (J) Tideglusib treatment shows complete repair with vital dental pulp after 6 weeks.

[0080] FIG. 5. Non-Exposed Pulp Injury Model. (A) CT of sound mouse upper first molar displaying the three cusps and pulp horns. (B) Linear measuring of damage on mouse molar without pulp exposure reveals a dentine band of 0.08 mm between pulp horn and floor of the cavity. (C) 3D reconstruction of damage reveals no pulp exposure; Dotted line indicates the area where the dentine was cut, and the dashed line, the area where the capping material is placed. (D) 3D reconstruction of sealed tooth shows glass ionomer sealing the damage (dashed line). (E) Schematic of damage model ((i)capping material, (ii)sealing material).

[0081] FIG. 6. Masson trichrome staining of wild type (CD1) and mutant mice, 4 weeks after injury without pulp exposure with glass ionomer sealing. (A, B; A, B) CD1 and Wntless mice display reactionary dentine repair with normal tubular structure. (C, C) Axin2 Homozygus mouse molars display increased reactionary dentine secretion within the pulp chamber with irregular tubular structure. Dotted line outlines secreted reactionary dentine. *, Damage site.

[0082] FIG. 7. 4 weeks of repair in wild type mouse molars injured without pulp exposure and capped with TGF- and BMP inhibitors and control (GI only). (A, A) Mouse molars capped with glass ionomer only show normal, tubular reactionary dentine secretion. (B, B) Molars capped with collagen sponge soaked in LY2157299 show atypical globular dentine without tubular reactionary dentine features. (C, C) Molars capped with collagen sponge soaked in Dorsomorphin show tubular reactionary dentine discontinued by globular dentine. (Squares delineate the magnified area). *, Damage site.

[0083] FIG. 8. Wnt responsive cells 1 day after injury in reactionary dentine repair. Immunohistochemistry against GFP reveals increase of TCF/LEF+cells and Axin2+ cells right under the injury in teeth capped with GSK-3 inhibitor (Tideglusib -TG) in collagen sponge (B, D) when compared with collagen sponge alone (A, C). (E) Axin2 Q-PCR for dental pulp collected one day after dentine was injured without exposing the dental pulp and treated with different capping. Tideglusib shows significant upregulation of Wnt activity in comparison to the positive (MTA) and negatives controls (No damage, CS, and DMSO). ****p<0.0001

[0084] FIG. 9. 4 weeks of repair in wild type (CD1) mouse molars capped with GSK-3 inhibitor in vehicle and controls. (A, A) Mouse molar without injury shows where injury was created (Dotted line) and the shape of the middle pulp horn without injury. (B, C) Dotted line delineates middle pulp horn, showing the reactionary dentine formed when capping molars with collagen sponge only or 50 nM Tideglusib respectively, showing larger reactionary dentine secretion when GSK-3 inhibitor is used. This finding was confirmed by pCT (B, C). (B, C) Magnification of squares on images B and C reveal tubular reactionary dentine in both cappings. (E) pCT linear measurement confirms that the distance from the top of the middle pulp horn to the point where the dentine was cut was significantly smaller when molars were capper with collagen sponge only comparison to molars without injury or capped with 50 nM Tideglusib. These results were reflected by the high mineral content in the injured area (D). D, dentine; *, damaged area. (D)**P=0.001; (E)**P=0.0022.

EXAMPLE 1

Effective Concentrations and Cytotoxicity Testing

[0085] 171A4 mouse dental pulp cells were incubated with a range of concentrations of the three small molecule GSK inhibitors,

[0086] BIO, CHIR.sub.99021 and Tideglusib, and cytotoxicity analysed with the MTT assay after 24h in culture. Specifically, 171A4 mouse dental pulp cells were plated in 96 well plates at 20,000 cells/cm.sup.2 and incubated (37 C., 5% CO.sub.2/95% air, 100% humidity) for 24 hours using standard culture medium. Thereafter, the medium was replaced with conditioned (drugs +media) and control media for another 24hrs. (10 l of drug in DMSO+90 l of medium resulting in the following concentrations BIO: 200, 100, 50 nM; CHIR99021: 10, 8, 5 M; Tideglusib: 200, 100, 50nM). For cell metabolic activity, MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide, Sigma) was added after 24hrs. The resulting formazan product was dissolved in 200p1 dimethyl sulfoxide (DMSO, Sigma). A colorimetric plate reader (Thermo Multiskan Ascent 354 microplate reader) was used to read the absorbance at 540 nm with background subtraction at 630 nm.

[0087] The results are shown in FIGS. 1A-C. The highest concentration of inhibitor that was not cytotoxic was used in separate assays with the same cells and levels of Axin2 measured by qPCR in the first 24 hours of culture.

[0088] In vitro drug release from Kolspon sponge was tested. 171A4 cells were plated in 24-well plates and incubated (37 C., 5% CO2/95% air, 100% humidity) for 24 h using standard culture medium. Falcon cell culture inserts for use with 24-well plates (3pm pore size) were placed in the wells carrying 96 mm.sup.2 Kolspon cubes either dry or soaked in 30 l of the drug optimal concentration for 15 and 30 minutes, 1, 6, and 12 hours. The cells were collected with TRIzol and stored at 20 C.

[0089] RNA was extracted from the cells using TRIzol (Thermo Fisher Scientific) as recommended by the manufacturer. RNA was quantified using Nanodrop and reverse transcribed into cDNA. Beta-actin was used as housekeeping gene (Forward-GGCTGTATTCCCCTCCATCG (SEQ ID NO 1), Reverse-CCAGTTGGTAACAATGCCTGT) (SEQ ID NO 2) and Axin2 for Wnt activity (Forward-TGACTCTCCTTCCAGATCCCA (SEQ ID NO 3), Reverse- TGCCCACACTAGGCTGACA (SEQ ID NO 4).

[0090] Increased Axin2 expression was observed after 30 mins reaching a maximum after 1 hr (FIG. 1D). BIO induction of Axin2 expression was 4 greater than both CHIR.sub.99021 and Tideglusib, each of which showed similar levels of induction (FIG. 1D).

EXAMPLE 2

Testing Induction of Axin2 in vivo in mice

[0091] To test the induction of Axin2 in vivo, an injury model was developed. Mice were anaesthetized with a solution of Hypnorm (Fentanyl/fluanisoneVetaPharma Ltd.), water and Hypnovel (MidazolamRoche) in the ratio 1:2:1 at 10 ml/kg by an intraperitoneal injection. Experimental tooth damage was created by drilling and making 0.13 mm holes in mouse maxillary first molars to expose the pulp. A rounded carbide bur FG coupled to a high speed hand piece was used to access the dentine. Once the bur cut exposed the dentine, a 30G needle was used to penetrate the pulp.

[0092] In order to protect the pulp from external contamination and stimulate dentine repair, the injury was capped either with ProRoot Mineral Trioxide Aggregate (MTA) (Maillfer Dentsply), or Kolspon (Fish Collage Type 1Eucare Ltd) alone, or in association with 50 nM BIO (SIGMA), 5 M CHIR.sub.99021 (SIGMA), or 50 nM Tideglusib (SIGMA) dissolved and diluted in DMSO, in contact with the pulp. Pieces of Kolspon were cut to size and soaked in solutions of the three inhibitors before being physically placed into the holes, in contact with the pulp.

[0093] A glass ionomer cement was used to cover the sponge and protect the tooth (FIG. 2G). Specifically, a layer of 3M Ketac-Cem Radiopaque was used as a capping material to seal the injured site. The injury was performed on the two upper first molars. Post-op the mice were given Vetergesic (BuprenorphineCeva) at the rate of 0.3 mg/kg intraperitonially as analgesic. The animals were sacrificed after 1 day.

[0094] Treated teeth were removed after 24 h along with controls consisting of untreated teeth, MTA only and collagen sponge with no inhibitor.

[0095] Pulp collection P21 mice had their superior first molars drilled according to the drilling protocol and tooth pulp tissue collected. Molars were extracted using a 21G needle as an elevator to lift them from the alveolar bone and kept in ice cold PBS. Using a 23 scalpel blade the molars were separated at the crown-root junction, so that the pulp chamber could be visualized. Using a 0.6 mm straight tip tweezer the pulp was gently scraped from the pulp chamber and the root canal. The pulp was then placed into cold Sigma RNAlater and stored at 80 C.

[0096] The extracted cells were tested for expression of Axin2 by qPCR as described in Example 1 (FIG. 1E). Expression of Axin2 was 3 higher in inhibitor treated pulp cells when compared to controls (FIG. 1E). Significantly MTA showed no effect on Axin2 expression suggesting current protocols do not activate Wnt signalling.

[0097] This shows that this experimental model of tooth damage and pulp exposure provides a way of delivering small molecules that were able to affect pulp cell gene expression in a reproducible way.

EXAMPLE 3

Reparative Dentine Formation

[0098] The model described in Example 2 was then used to examine the effect on the formation of reparative dentine. Molars were drilled and sponges inserted and left as described in Example 2, this time for 4-6 weeks before the mice were sacrificed. Micro-computed tomographic (CT) scanning was used to visualise and quantify mineral deposition at the drill site. Mice upper molars were fixed with PFA 4% overnight and scanned using a Bruker Skyscan1272 micro-CT scanner. Microview software programme (GE) was used for visualization and analysis. Two dimensional (2D) images were obtained from micro-CT cross-sectional images of superior first molar internal part, to evaluate the drilling and mineral formation. To assay tissue mineral content a ROI of X=0.2 mm, Y=0.4 mm, and Z=0.2 mm was set as standard for all the samples and mineral analysis performed. T filled with mineral=0.0017 mg.

[0099] Analysis at both 4 and 6 weeks revealed increased mineralisation with all three agonists compared to controls (FIG. 3 KL). These increases were statistically significant at 4 and 6 weeks. Overall the mineralisation with the inhibitors was on average 2 higher than in the sponge alone control and 1.7 higher than with MTA treatment.

[0100] After 4 weeks decalcification in 19% EDTA the teeth were embedded in wax blocks and sectioned using 0.8 m thickness. Sections were stained using Masson's Trichrome to reveal new dentine formation. The sections confirmed the CT data showing that teeth treated with GSK inhibitors had reparative dentine was formed at the injury site than with collagen sponge or MTA (FIG. 4). Moreover, the new dentine formed presented as dense dentine localised centrally to the injury site, revealing no remaining collagen sponge where the dentine was formed. Interestingly, by 6 weeks of treatment, the reparative dentine secreted when teeth were treated with BIO, CHIR, and Tideglusib filled the whole injury site from occlusal to pulp chamber roof (FIG. 4H-J). Most importantly, dental pulp remained vital (FIG. 4 H-J).

EXAMPLE 4

Effect of Wnt Signaling Modulation on Reactionary Dentine Secretion

[0101] To investigate the effect of modulation of Wnt signaling on reactionary dentine formation, a reproducible tooth damage model was established. 6 weeks old, Axin2.sup.CreERT2; Rosa26mTmG (fl/+) and GPR.sub.1777(Wntless).sup.pCAGCreERT2 (fl/fl) mice were injected intraperitoneally with three doses of tamoxifen (2 mg per 30 g mouse, SIGMA), one dose a day. 5 days after the last tamoxifen injection, the height of the middle cusp of mouse maxillary first molars were reduced without exposing the dental pulp, leaving a band of dentine to protect the inner pulp tissue (FIG. 5). Specifically, the mice were anaesthetized with a solution made with Hypnorm (Fentanyl/fluanisoneVetaPharma Ltd.), sterile water and Hypnovel (MidazolamRoche) in the ratio 1:2:1 at the rate of 10 ml/kg intraperitonially. A rounded carbide burr FG coupled to a high-speed hand piece was used to expose the dentine of the mouse superior first molars (left and right side).

[0102] The exposed dentine was capped either with calcium hydroxide (Dycal; Dentsply) or mineral trioxide aggregate (MTA) (ProRoot MTA; Dentsply), or dry collagen sponge (Kolspon-Fish Collage Type 1; Eucare Ltd), or collagen sponge soaked in dimethyl sulfoxide (DMSO; SIGMA), or 50 nM Tideglusib, or 1 M LY2157299, or 1 M Dorsomorphin. All drugs were dissolved and diluted in DMSO. A layer of glass ionomer cement (Ketac-Cem Radiopaque; 3M ESPE) was used as a sealing material. Vetergesic (BuprenorphineCeva) was injected to all mice post-operative at the rate of 0.3 mg/kg by intraperitoneal injection as analgesic. The animals were sacrificed after 1 day and 4 weeks. A total of 14 genetically-modified mice (28 damaged molars) and 26 CD1 mice (52 molars) were used.

[0103] CD1 (wild type) were used as control and to study the effect of small molecules. Mice were collected 1 day and 4 weeks after injury.

[0104] Mice upper molars were dissected, fixed in 4% paraformaldehyde (PFA) for 24-hours at 4 C. and scanned using a Bruker Skyscan1272 micro-CT (pCT) scanner. Microview software program (GE) was used for visualization and analysis. Two-dimensional (2D) images were obtained from pCT cross-sectional images of superior first molar, to evaluate mineral formation. Three-dimensional (3D) reconstructions were used to verify pulp exposure. The dentine thickness was measured using the Line function of the software. For the dentine thickness-analysis the distance between the center of the roof of the middle pulp horn and the floor of the injured dentine margin was measured. In order to assess tissue mineral content a region of interest (ROI) of X=0.2 mm, Y=0.4 mm, and Z=0.2 mm was set as standard for all the samples and the mineral analysis was performed. The region measured comprised only the site of injury. ROI complete filled with mineral=0.0017 mg.

[0105] The model was first tested with the current standard materials used in dentistry, (glass ionomer, MTA, and calcium hydroxide) and showed the formation of tubular reactionary dentine and preservation of tooth vitality. The effect of modulation of Wnt/-catenin signaling activity on reactionary dentine formation was studied using Axin2.sup.LacZ/LacZ and Wntless.sup.cko/cko mice.

[0106] After 4 weeks decalcification in 19% EDTA pH 6, the teeth were embedded in wax blocks and sectioned at 8 m thickness. Sections were histologically stained using Masson's Trichrome. The histology revealed that inhibition of Wnt activity did not prevent reactionary dentine formation or affect its tubular structure, while enhanced Wnt activity lead to a large increase in the amount of reactionary dentine formed that was disorganized and lacked a regular tubular structure (FIG. 6).

EXAMPLE 6

Effect of BMP and TGF- Inhibition on Repair

[0107] Sequestered latent BMP and TGF- proteins present in the dentine matrix have been implicated in tertiary dentine formation following damage, mainly based on results obtained from in vitro experiments. The effect of inhibition of these signalling pathways in was investigated in the in vivo model of reactionary dentine formation described in Example 5 by utilising small molecules to inhibit these signalling pathways. The small molecule LY2157299 is a TGF- type I receptor kinase inhibitor and the small molecule Dorsomorphin is an inhibitor of BMP type I receptors ALK2, ALK3 and ALK6 (Bhola et al. 2013, Yu et al. 2008). Both compounds were first tested for cytotoxicity and effectiveness of signalling pathway blocking in vitro using 17IA4 cells. The first upper molars of CD1 mice were damaged to stimulate reactionary dentine formation and a collagen sponge was soaked in either 1 M LY2157299 or 1 M Dorsomorphin was used as a delivery vehicle. The sponges were placed on the exposed dentine and sealed with a layer of glass ionomer.

[0108] 4 weeks after injury (FIG. 7A), the molars treated with 1 M LY2157299 showed secretion of disorganised (globular) dentine (FIG. 7B) rather than tubular reactionary dentine observed in controls. Molars treated with 1 M Dorsomorphin secreted a mixture of tubular dentine and globular dentine (FIG. 7C). TGF- and BMP signalling following dentine damage appear not to be essential for reactionary dentine formation but are required for modulation of dentine structure.

EXAMPLE 7

Cells under the Injury Site Can Respond to Wnt/-Catenin Signaling

[0109] Since Wnt/-catenin signaling is required for reparative dentine formation we investigated if this pathway plays a role in reactionary dentine formation. The small molecule GSK3 antagonist Tideglusib was delivered on collagen sponges at the site of damage and sealed with glass ionomer. Sponge alone and MTA sealed with glass ionomer were used as controls. To identify whether cells responded to the drug, TCF/Lef:H2B-GFP, Axin2.sup.CreERT2; Rosa26mTmG flox/+ and CD1 wild type mice were used. TCF/Lef:H2B-GFP reporter mice allowed the visualisation of Wnt active cells (ie. Cells receiving a Wnt signal), Axin2.sup.CreERT2; Rosa26mTmG flox/+ mice were used to lineage trace Axin2-expressing cells and gene expression analysis via qPCR (as described in Example 1) was performed on pulp cells from CD1 mouse molars. The first molars of genetically modified mice were collected 1 day after the injury and immunohistochemistry was performed. Deparaffinised sections were retrieved with sodium citrate (pH 6) and incubated with chicken polyclonal anti-GFP antibody (1:500; Abcam, Cambridge, Mass., USA; ab13970) overnight at 4 C. Sections were washed and exposed to appropriate biotinylated secondary antibody, then horseradish peroxidase (HRP)-conjugated streptavidin-biotin antibody and washed with PBST. Immunoreactivity was visualized with MenaPath green chromogen kit (Bio SB). For immunofluorescence, chicken polyclonal anti-GFP antibody (1:1000; Abcam, Cambridge, Mass., USA; ab13970) was added overnight at 4 C. Sections were washed and exposed to secondary antibody (1:500; Thermo Fisher Scientific, Eugene, Oreg., USA; A21449) for 1 hour at room temperature.

[0110] Localisation of GFP showed that in TCF/Lef:H2B-GFP reporter mice, odontoblasts and pulp cells under the injury site were responsive to Wnt signalling and an increased local response to Wnt signalling at the injured pulp horn site could be observed with addition of Tideglusib (FIG. 8A,B). Axin2.sup.CreERT2; Rosa26mTmGflox/+ mice presented a similar pattern of Wnt responsiveness with more GFP-positive cells in the dental pulp when 50 nM Tideglusib was applied compared to the collagen sponge only (FIG. 8 C,D).

[0111] To confirm the elevated Axin2 gene expression in the dental pulp, P21 CD1 molars were damaged and the dental pulp collected and dissociated for qPCR analysis as described in Example 1.

[0112] The results are shown in FIG. 8E. Axin2 expression was 2-fold higher in teeth treated with Tideglusib compared to controls. Notably MTA and collagen sponge showed no effect on early-response Axin2 expression suggesting that current treatment protocols for indirect pulp capping material do not act through this pathway. These results showed that small molecule drugs such as Tideglusib are able to penetrate damaged dentine and exert effects on odontoblasts and pulp cells.

EXAMPLE 8

GSK-3 inhibitor Small Molecules Increase Local Reactionary Secretion

[0113] Having confirmed that Tideglusib can reach to the inner pulp through the remaining dentine band and activate Wnt signaling in odontoblasts and cells, its capacity to modulate reactionary dentine formation was evaluated using the model described in Example 5. Mouse upper first molars were damaged and capped with sponges soaked in 50 nM Tideglusib and left for 4 weeks. Histology of the upper first molars revealed that teeth indirectly capped with 50 nM Tideglusib showed enhanced reactionary dentine formation compared with controls (FIG. 9 A,A-C,C). Importantly, histology also showed normal tubular reactionary dentine formation with the Wnt activator and the dental pulp remained vital (FIG. 9B,C). Moreover, QCT scanning confirmed by mineral content analysis an increase of mature mineral formation under the injury site, when teeth were treated with Tideglusib in comparison to collagen sponge alone (FIG. 9D). In addition, linear measurement analysis revealed that upper first molars treated with the drug presented a thicker mineral band at the site of injury than control molars with the collagen sponge alone (no drug). Compared to non-injured molars, GSK-3 antagonist treated molars showed a similar dentine thickness (FIG. 9E).