DEEP-LAYER REMINERALIZATION OF HYDROXYLAPATITE

Abstract

The present invention relates to the use of Ca.sub.5(PO.sub.4).sub.3(OH) (hydroxyapatite; HAP) and a dental care composition comprising Ca.sub.5(PO.sub.4).sub.3(OH) for the deep-layer remineralization of demineralized teeth, in particular for the deep-layer remineralization of demineralized dental enamel. Ca.sub.5(PO.sub.4).sub.3(OH) used according to the invention and the dental care composition used according to the invention can be applied in the treatment and/or prevention of various diseases affecting the teeth, in particular caries.

Claims

1-10. (canceled)

11. A method for the remineralization of teeth, said method comprising administering a composition comprising Ca.sub.5(PO.sub.4).sub.3(OH) (hydroxyapatite; HAP) to a surface of one or more teeth, wherein administration of the composition provides deep-layer remineralization to the one or more teeth.

12. The method of claim 11, wherein the composition is free of fluoride.

13. The method of claim 11, wherein the composition comprises Ca.sub.5(PO.sub.4).sub.3(OH) in an aggregated form.

14. The method of claim 11, wherein the composition comprises Ca.sub.5(PO.sub.4).sub.3(OH) in an microaggregated form.

15. The method of claim 11, wherein the composition comprises Ca.sub.5(PO.sub.4).sub.3(OH) in an amount of 0.1 to 50.0 wt.c/o in relation to the total weight of the composition.

16. The method of claim 11, wherein the composition comprises Ca.sub.5(PO.sub.4).sub.3(OH) in an amount of 0.1 to 20.0 wt.c/o in relation to the total weight of the composition.

17. The method of claim 11, wherein the one or more teeth are remineralized up to a depth of 200 μm.

18. The method of claim 11, wherein the one or more teeth are remineralized up to a depth of 100 μm.

19. The method of claim 11, wherein the composition is administered to improve a cosmetic or medical condition of the one or more teeth.

20. A method for treating a disease, said method comprising administering a composition comprising Ca.sub.5(PO.sub.4).sub.3(OH) (hydroxyapatite; HAP) to a surface of one or more teeth of a subject in need thereof, wherein the disease comprises caries, tooth erosion, tooth wear, tooth abrasion, bruxism, molar incisor hypomineralization (MIH), amelogenesis imperfecta, dentinogenesis imperfecta, or fluorosis, and wherein administration of the composition provides deep-layer remineralization to the one or more teeth.

21. The method of claim 20, wherein the caries is a code 3 or code 4 caries according to the International Caries Detection and Assessment System (ICDAS).

22. The method of claim 20, wherein the composition comprises Ca.sub.5(PO.sub.4).sub.3(OH) in an aggregated form.

23. The method of claim 20, wherein the composition comprises Ca.sub.5(PO.sub.4).sub.3(OH) in an microaggregated form.

24. The method of claim 20, wherein administration of the composition remineralizes the one or more teeth up to a depth of 200 μm.

25. The method of claim 20, wherein administration of the composition remineralizes the one or more teeth up to a depth of 100 μm.

26. The method of claim 20, wherein the composition comprises Ca.sub.5(PO.sub.4).sub.3(OH) in an amount of 0.1 to 50.0 wt.c/o in relation to the total weight of the composition.

27. The method of claim 20, wherein the composition comprises one or more pharmaceutical or cosmetic ingredients and/or is free of fluoride.

28. A dental care composition for the remineralization of teeth, wherein said composition comprises Ca.sub.5(PO.sub.4).sub.3(OH) (hydroxyapatite; HAP), one or more pharmaceutical or cosmetic ingredients, and is free of fluoride, and wherein the dental care composition is able to provide deep-layer remineralization to the teeth.

29. The dental care composition of claim 28, the composition comprises Ca.sub.5(PO.sub.4).sub.3(OH) in an amount of 0.1 to 50.0 wt.c/o in relation to the total weight of the composition.

30. The dental care composition of claim 28, wherein the dental care composition is able to remineralize teeth up to a depth of 200 micrometers.

Description

FIGURES

[0121] FIG. 1 shows a flow chart having the step-by-step methodology. This is a crossover study, so the 30 test subjects who completed the study received the two procedures in a crossover design as phase I & II.

[0122] FIG. 2 shows representative microradiographic images of healthy dental tissue before (A) and after (B) inter-oral exposure to demineralization, while the test subject uses a children's toothpaste (Karex™) containing 10% hydroxyapatite in the form of microclusters.

[0123] FIG. 3 shows representative microradiographic images of healthy tooth tissue before (A) and after (B) inter-oral exposure to demineralization while the test subject uses a children's toothpaste (Elmex) containing 500 ppm fluoride as amine fluoride.

[0124] FIG. 4 shows representative microradiographic images of lesions lying below the surface on the tooth enamel (initial carious lesions) before (A) and after (B) in situ remineralization by treatment of a children's toothpaste (Karex™) containing 10% hydroxyapatite in the form of microclusters.

[0125] FIG. 5 shows representative microradiographic images of lesions lying below the surface on the tooth enamel (initial carious lesions) before (A) and after (B) in situ remineralization by treatment of a children's toothpaste (Elmex) containing 500 ppm fluoride as amine fluoride.

NATURE OF THE STUDY

[0126] In a randomized, monocentric, in situ controlled, crossover double-blind study, two children's toothpaste compositions containing either 10% hydroxyapatite in the form of microclusters or 500 ppm fluoride provided as amine fluoride (AMF) were compared to one another with respect to their ability to induce rem ineralization and prevent the development of initial carious lesions.

[0127] Test Subject Selection

[0128] The study included 32 people of various ethnic origins, ages 18 to 60, who were not taking antibiotics or medications that negatively affect salivation, had at least 20 natural, uncapped teeth, and who had a history of caries but who had no clinically active caries at the beginning of the study. In addition, the persons had no dental and/or gum disease, were not pregnant or breastfeeding, and did not smoke tobacco products.

[0129] Creation of Artificial Initial Caries and Production of the In Situ Device

[0130] 32 freshly extracted human deciduous teeth without caries, cracks, or enamel defects were selected and cleaned. Four tooth blocks were produced from the buccal and lingual surfaces of each of the selected teeth, wherein each of the blocks was approximately 2 mm in length, 2 mm in width, and 1.5 mm in depth. Two blocks were retained as healthy blocks for the assessment of the demineralization prevention, while artificial initial caries was induced in the two blocks designated for the assessment of the remineralization assessment. All sides of each block were coated in two coats of acid-resistant nail polish except for the buccal and lingual surfaces, respectively, on which an initial caries lesion (demineralization) was created by subjecting the exposed surface to an acidified gel system (0.1 M lactic acid, 0.1 M sodium hydroxide, 60% w/v hydroxyethyl cellulose, pH 4.5) for seven days. The nail polish was then carefully removed using acetone. A tooth section approximately 150 μm thick was cut from each tooth block to measure the baseline mineral loss (Δz.sub.1) and lesion depth (LD.sub.1) of each induced initial carious lesion and to select the lesions suitable for remineralization evaluation. The sections were prepared for transverse microradiography as follows. Both sides of the sections were polished using a lapping film in an MultiPrep™ precision polishing machine from Allied High Tech to create plane-parallel surfaces and reduce the thickness of the sections to 100 μm. Sections were then microradiographed on a Type 1A high-resolution glass X-ray plate from Microchrome Technology, CA, USA using a Philips X-ray generator having the suitable settings. The plates were exposed to radiation for 10 minutes at an anode voltage of 20 kV and a tube current of 10 mA and then further processed, wherein the further processing consisted of a five-minute development in a Kodak HR developer, a fifteen-minute fix using a fixing agent (Kodak Rapid-fixer), and a thirty-minute wash. After drying, the microradiographs were examined under an optical microscope (Leica DMR) connected to a PC via a camera (Sony; model XC-75 CE CCTV). Using software for image analysis (TMR2006 version 3.0.0.11; Inspector Research Systems, Amsterdam), the magnified image of the microradiographs was analyzed under standard conditions with respect to light intensity and magnification together with the image of a step wedge described in the literature. Then the images were only used to select the lesions suitable for the comparison experiment. Only the samples that had caries-like surface lesions that displayed a reasonably uniform width along their length were selected for the remineralization process. Their blocks were used for the device of the in situ application.

[0131] As mentioned above, the four blocks from each tooth were distributed as follows: two blocks having lesions for the assessment of the remineralization and two blocks for the assessment of the demineralization inhibition. These blocks were used to fabricate the in situ device as follows. Each block was covered with a polyester gas (Bard Peripheral Vascular, Inc. Tempe, Ariz., USA) and mounted in a fitted orthodontic fixture. The device consists of an orthodontic molar pad with retaining mesh lining (American Orthodontics Corp., Sheboygan, USA) having a ring of 0.7 mm orthodontic wire bent so that the ring snugly encloses each test block. Each device was sterilized using gamma radiation prior to delivery to the test subject.

[0132] Carrying Out the Study

[0133] The study was conducted in two different treatment phases, in which the test subjects were subjected to one of the following two treatments in a randomized, crossover comparison: (A) Toothpaste containing 10% hydroxyapatite in the form of microclusters (Kinder Karex™, Dr. Kurt Wolff GmbH & Co. KG, Bielefeld, Germany and (B) toothpaste containing 500 ppm fluoride provided as amine fluoride (AMF) (Elmex children's toothpaste, GABA GmbH, Hamburg, Germany). A one-week rinsing phase was followed by a four-week treatment phase, which consisted of two-week phases during which each test subject applied his/her assigned treatment under the following conditions: the first two-week phase for the test subjects wearing the healthy enamel block in situdevice and the two-week phase for the subjects wearing the in situdevice having enamel block with lesions.

[0134] For the one-week rinsing phase, the test subjects who met the inclusion criteria were given a specially made rinsing toothpaste that contained neither hydroxyapatite nor fluoride for two minutes of use twice a day (morning and evening).

[0135] After the rinsing phase, the test subjects were assigned to either the group using hydroxyapatite or the group using amine fluoride by the coordinator, who assigned random numbers generated by a computer program. However, to ensure that both the people conducting the experiment and the test subjects were blind to the product, all toothpaste tubes were identically packaged and coded by the producing/packaging company. After randomization, the four block-bearing in situdevices derived from one tooth were assigned to one test subject. Then, the first of the four assigned devices was fastened on the buccal surface of the selected lower molar by a qualified dentist in accordance with accepted principles of orthodontic practice. To fasten the device, the buccal surface of the selected tooth was gently etched for 30 seconds, washed with water, dried for 30 seconds, and isolated using cotton rolls. The lower side of the device was coated with Transbond™ XT easy curing adhesive paste (3M Unitek, Monrovia, Calif., USA) and carefully placed. The excess material emerging from the sides was used to cover the sides and the adhesive paste was cured for 20 seconds using an Ortholux XT (3M Unitek, Monrovia, Calif., USA). After fastening the device, each test subject was given his/her appropriate test toothpaste and a special soft toothbrush. Subjects were instructed to continue their routine of brushing their teeth twice a day for two minutes using only 10 milliliters of water for rinsing. In addition, special instructions were given for dispensing the toothpaste, and test subjects were asked not to brush the device directly, not to eat or drink for at least 30 minutes after brushing, and not to use other oral hygiene products (e.g., mouthwash, chewing gum etc.). As a control, a diary was provided to each test subject to record the time of each brushing phase, and the weight of the toothpaste tubes was determined. After two weeks, without using the test toothpaste that morning, the test subjects had the device removed and sent to the laboratory for analysis. The device for the second two-week treatment phase was fastened. Upon completion of the second two-week treatment phase, the second device was removed from the test subjects and they were given a rinse toothpaste and soft-bristled toothbrush, so that they could undergo a seven-day rinsing phase without the device to prepare for phase 2 of the study. After completing this rinsing phase, the phase 1 procedure was repeated to complete the second two-week treatment, so that each test subject had passed through both arms of the study.

[0136] Post-Study Procedure and Study Exit

[0137] After inter-oral exposure, a section approximately 150 μm thick was cut from each healthy and lesion-containing tooth block and processed for microradiography as previously described for baseline control sections. Although the lesion-containing control sections were microradiographed to select appropriate lesions, they were microradiographed again together with post-test sections to quantify Az and LD of the lesions, as with the baseline sections. This step allows control and test sections from the same block to be microradiographed and analyzed under the same conditions. For the lesion-containing sections, this process yielded the mineral loss (Δz.sub.1) and lesion depth (LD.sub.1) before the test, the mineral loss (Δz.sub.2) and lesion depth (LD.sub.2) after the test, and the microradiograms for the lesions before and after the test. For the healthy sections, this process yielded the mineral loss (Δz) and lesion depth (LD) after the test and the microradiograms before and after the test. Using the microradiographs, the pattern and extent of remineralization in each lesion which was produced by the treatment by each treatment arm was examined by comparing the images before and after the test. For each test subject, the mineral loss after treatment was subtracted from the mineral loss before treatment, and then standardized among the test subjects by dividing this difference by the mineral loss before treatment to obtain the remineralization in percent. The depth of lesions before and after treatment were managed in the same way to obtain the reduction in lesion depth in %. The two toothpastes used were compared using these values.

[0138] Analysis and Calculation of Sample Size

[0139] Sample size calculations were performed using nQuery Advisor software (Statistical Solutions, Cork, Ireland). Based on previous studies in which the mean percent remineralization was 30.3 with a standard deviation of 16.3 and assuming that each of the two toothpaste compositions promotes remineralization and a reduction in lesion depth significantly greater than zero, an effective sample size of 30 test subjects has an informative power of 0.95 with a one-tailed 0.05 significance level. A difference between a hypothetical mean of zero and a sample mean of remineralization equal to or greater than 10% can be determined hereby using a two-tailed t-test of two independent means. To provide a 5% failure, however, 32 test subjects were included.

[0140] Statistical Analysis

[0141] Three endpoints were determined in each case to determine mineral loss and lesion depth (1). The mean amount of remineralization and mean amount of lesion depth reduction was determined for the hydroxyapatite-containing toothpaste (Karex™) as a respective percentage of mineral loss before treatment and lesion depth before treatment. These percentages were compared to a value of 0, which is the expected value of a toothpaste with no effect. The statistical test used for this purpose was a one-tailed t-test of a group mean. (2) In the same way, the mean amount of remineralization and the mean amount of lesion depth reduction for the amine fluoride-containing toothpaste (Elmex) were determined and also compared to 0. (3) The primary endpoint was taken using the two-tailed t-test of two independent means to compare the mean of the hydroxyapatite-containing toothpaste (Karex™) to the mean of the amine fluoride-containing toothpaste (Elmex). Equivalence was established when the difference between the two toothpaste compositions was considered clinically irrelevant and Δ≤20% for each measurement method, wherein the statistical package R, version 3.5.0 was used for the analysis.

[0142] Results

[0143] As can also be seen from FIG. 1, two test subjects dropped out of the study, one during/after the rinsing phase and one in the first two-week treatment phase while wearing the device. The study was completed by 30 subjects (19 female and 11 male) of various ethnic origins having an average age of 39.5 years.

[0144] The mean rate of rem ineralization and lesion depth reduction is shown in Table 1 below.

TABLE-US-00002 TABLE 1 Mean rates of remineralization and lesion depth reduction in % for each toothpaste 500 ppm p-value, 10% HAP amine fluoride 2 mean Measurement (Karex ™) (Elmex) values remineralization (%) 55.8 56.9 0.81 (SD 13.8) (SD 14.9) p-value, a group <0.0001 <0.0001 lesion depth reduction 27.1 28.4 0.68 (%) (SD 10.6) (SD 9.8) p-value, a group <0.0001 <0.0001

[0145] As can be seen from the table above, each of the toothpastes shows a remineralization greater than 50% and a lesion depth reduction of more than 25%. For both toothpastes, the mean remineralization and mean lesion depth reduction was statistically significantly greater than 0. When compared with one another, there was no statistically significant difference in remineralization (p=0.81) or lesion depth reduction (p=0.68). The 95% confidence interval of the difference between HAP (KarexTM) and amine fluoride (Elmex) for the mineralization was −8.8% to 6.5% and the 95% confidence interval of the difference between HAP (Karex™) and amine fluoride (Elmex) for lesion depth reduction was -6.8% to 4.1%. Consequently, this study confirms that a HAP-containing toothpaste is not inferior in effectiveness to a fluoride-containing toothpaste.

[0146] Upon analysis of the healthy tooth blocks evaluated for the ability of the two toothpastes to prevent demineralization of healthy tooth surfaces, there was no evidence of demineralization in any of the tooth blocks after intra-oral exposure to either toothpaste, as shown in FIGS. 2A & 2B as well as FIGS. 3A & 3B. In a differentiated comparison of the microradiographs of the lesion-containing specimens which were exposed during the treatment to the toothpastes having HAP (FIGS. 4A & 4B) and amine fluoride (FIGS. 5A & 5B), to the corresponding control microradiograms, the following could be established. While the HAP-containing toothpaste caused a more homogeneous remineralization throughout the thickness of the lesion lying under the surface (FIG. 4B), the remineralization caused by the amine fluoride-containing toothpaste was denser in the outer half (surface region), so that in FIG. 5B two regions having different density can be clearly seen. Overall, no incidents of adverse effects were reported by the test subjects or clinically noted.

[0147] In summary, it can be stated that a HAP-containing toothpaste is on a par with a fluoride-containing toothpaste in terms of remineralization and lesion depth reduction, but without the mentioned negative side effects that can be associated with the use of a fluoride-containing toothpaste.

[0148] In addition, unlike a fluoride-containing toothpaste, which prevents demineralization and causes remineralization in the surface region to about 30 μm, a HAP-containing toothpaste can be used to prevent demineralization and also to remineralize subsurface regions, for example in the shown case of approximately 100 μm. It was thus shown under conditions of the oral cavity (in situ) that, in particular, deeper demineralized/carious tooth enamel areas are homogeneously rem ineralized. This can improve the resistance of the teeth to dental diseases.