JP5757612B2 - Enamel regeneration kit - Google Patents

Description

The present invention relates to error lick quality playback kit.

In dental practice, the degree of caries is classified into C0, C1, C2, C3, and C4, and initial caries classified as C0 is characterized by enamel demineralization and enamel surface roughening. It is said. Decalcification is a dissolution reaction by an acid produced by a caries-causing bacterium and is represented by the following chemical reaction formula.

Since initial dental caries has extremely small tooth defects, it is not the usual treatment for caries that removes the affected tooth and fills with artificial materials. However, if the initial caries is left untreated, it will lead to more serious caries. Because of the high risk of progression, the following treatments are generally given:
(1) Treatment for the purpose of sterilization (brushing, Professional Tooth Cleaning (PMTC), sterilization with ozone, etc.)
(2) Therapies aimed at protecting the affected area (such as sealants)
(3) Therapies aimed at supplying ions (fluoride application, application of CPP-ACP, etc.)

  Application of these treatments is said to promote remineralization of decalcified enamel. Remineralization means remineralization of hydroxyapatite crystals, which are the main inorganic components constituting enamel.

  In addition, since the main component of enamel is hydroxyapatite crystals, hydroxyapatite crystals, or caries treatment kits containing phosphates and calcium salts that can form hydroxyapatite crystals are disclosed (for example, Patent Document 1).

  Patent Document 1 includes a hydroxyapatite particle-containing composition and a phosphorylated saccharide calcium-containing composition, wherein the caries are C1 or C2 stage caries and root caries, and the phosphorylated saccharide calcium is a sugar. Disclosed are kits and compositions comprising a moiety and a calcium salt of a phosphate group, wherein the sugar moiety is a glucan having a degree of polymerization of 2-8 and the number of phosphate groups is 1-2.

JP 2009-167135 A

  The conditions for remineralization are not clear, and it cannot be said that remineralization can be reliably achieved even in the in vivo environment using the above-described treatment method or the kit of Patent Document 1. Although remineralization can be said to be a natural tooth restoration function, it depends greatly on environmental factors and does not always occur.

The present invention has been made in view of the above matters, and it is an object also cause remineralization of enamel in vivo, are useful d lick quality reproduction kit for the treatment or caries prevention of early caries Is to provide.

Enamel playback kit according to the present onset Ming,
A first solution containing calcium ions;
A second solution containing amelogenin and phosphate ions,
It is characterized by that.

  The total amount of the first solution and the second solution is 1.6 to 5.0 mg / mL for the amelogenin, 0.25 to 4.0 mM for the calcium ion, and 0 for the phosphate ion. It is desirable to contain 125-4.0 mM.

  The amelogenin is preferably contained in an amount of 2.0 to 3.0 mg / mL with respect to the total amount of the first solution and the second solution.

  The first solution preferably contains an ionic stabilizer.

  Moreover, it is desirable that the second solution contains a thickener.

  Moreover, it is desirable that the first solution and the second solution each contain a pH adjuster that maintains the pH in the vicinity of neutrality.

The enamel regeneration liquid obtained by mixing the first solution and the second solution is provided with a coating material that holds the enamel surface of the teeth, and the coating material is filled with the enamel regeneration liquid. You may have a recessed part .

In the enamel regeneration kit according to the present invention, amelogenin accumulates on the enamel surface to form a space. Accumulation of calcium ions and phosphate ions occurs in this space, and local supersaturation promotes the reaction between calcium ions and phosphate ions to grow hydroxyapatite crystals. As described above, the action of amelogenin can cause remineralization of enamel from the surface of the enamel even in the living body, which is useful for treatment of initial caries and prevention of caries.

(A) and (B) are photographs of natural enamel (hydroxyapatite crystal) (Source: J Tissue Eng Regen Med. 2007 May-Jun; 1 (3): 185-91. New observations of the hierarchical structure of human. enamel, from nanoscale to microscale.Cui FZ, Ge J.). (A), (B) are photographs of artificial hydroxyapatite crystals (Source: Biomaterials. 2004 May; 25 (12): 2331-9. CaO--P2O5--Na2O-based additives additives for hydroxyapatite (HAp) ceramics. Kalita SJ, Bose S, Hosick HL, Bandyopadhyay A.). (A) is a perspective view of a shell-shaped covering material, and (B) is a schematic view showing a state in which the enamel surface of a tooth is covered with a shell-shaped covering material. It is a schematic diagram which shows the state which coat | covered the enamel surface of the tooth | gear with the coating | covering material which covers the whole tooth | gear. (A) is a perspective view of a film-shaped coating material, and (B) is a schematic diagram showing a state in which the surface of a tooth enamel is coated with a film-shaped coating material. 6 is a graph showing the measurement results of the surface roughness (Ra) of a natural enamel sample in Experimental Example 1. (A), (B), and (C) are atomic force microscope (AFM) images of the surface of the natural enamel sample in Experimental Example 1, respectively. 4 is a graph showing the measurement results of the surface roughness of an enamel sample after acid treatment in Experimental Example 1. (A), (B), and (C) are AFM images of the surface of the enamel sample after acid treatment in Experimental Example 1, respectively. It is an AFM image of a reference example, (A) is Ca / P: 2.0, Ca ²⁺ concentration: 2 mM, (B) is Ca / P: 1.67, Ca ²⁺ concentration: 2 mM, (C) is Ca / P P: 1, Ca ²⁺ concentration: 2 mM, (D) is Ca / P: 0.5, Ca ²⁺ concentration: 2 mM, (E) is Ca / P: 0.25, Ca ²⁺ concentration: 0.5 mM, (F ) Is an AFM image of Ca / P: 0.125, Ca ²⁺ concentration: 0.25 mM. It is an AFM image of Example 1, (A) is Ca / P: 2.0, Ca ²⁺ concentration: 2 mM, (B) is Ca / P: 1.67, Ca ²⁺ concentration: 2 mM, (C) is Ca / P: 1, Ca ²⁺ concentration: 2 mM, (D) is Ca / P: 0.5, Ca ²⁺ concentration: 2 mM, (E) is Ca / P: 0.25, Ca ²⁺ concentration: 0.5 mM, ( F) is an AFM image of Ca / P: 0.125, Ca ²⁺ concentration: 0.25 mM. It is a graph which shows the measurement result of the surface roughness (Ra) in Example 1, (A) is a graph whose Ca / P is 2.0, (B) is a graph whose Ca / P is 1.67, (C) Is a graph with a Ca / P of 1.0, (D) is a graph with a Ca / P of 0.5, (E) is a graph with a Ca / P of 0.25, and (F) is a graph with a Ca / P of 0.125. It is a graph of. It is a digital stereomicroscope image of the enamel in Example 2, (A) is a 250 times image before processing, (B) is a 1250 times image before processing, (C) is 250 times after 8 hours processing. (D) is a 1250 times image after 8 hours processing, (E) is a 250 times image after 16 hours processing, and (F) is a 1250 times image after 16 hours processing. It is an AFM image in Example 3, (A) is before processing, (B) is after acid treatment, (C) is an AFM image after reaction.

  The enamel regeneration solution and enamel regeneration kit according to the present embodiment will be described in detail below.

  The enamel regeneration solution according to the present embodiment contains amelogenin, calcium ions, and phosphate ions. When the enamel regeneration solution is treated on the dental enamel, calcium ions and phosphate ions are accumulated and combined by the action of amelogenin, and crystal formation of hydroxyapatite, the main component of enamel, that is, remineralization is achieved. Be guided.

  Amelogenin is the most abundant enamel protein secreted during the formation of teeth in the living body, and has a function of inducing crystals of hydroxyapatite, which is the main component of enamel. In vivo hard tissue formation by special proteins such as amelogenin is called biomineralization.

  Details of the biomineralization process in enamel formation are unclear, but are thought to be as follows. Amelogenin has a hydrophobic region on the N-terminal side and a hydrophilic region on the C-terminal side, and has a typical amphiphilic protein amino acid sequence. Form. Hydroxyapatite crystals, the main component of enamel, are obtained by accumulating amelogenin on the tooth enamel surface to form an enamel form, and by accumulating calcium ions and phosphate ions in the space and becoming supersaturated. Is thought to grow.

  In this way, amelogenin mediates the chemical reaction between calcium ions and phosphate ions, thereby achieving hydroxyapatite crystal formation under moderate conditions in the living room at normal temperature and normal pressure. It is thought that the form is also controlled.

  Fig. 1 shows enamel crystals of human natural teeth (hydroxyapatite crystals), and Fig. 2 shows artificial hydroxyapatite crystals. In the process of enamel biomineralization, hydroxyapatite produced by enamel proteins such as amelogenin. The crystal growth rate, morphology, and growth direction are controlled, and it is considered that a fine crystal structure unique to tooth enamel that is completely different from artificial hydroxyapatite crystals is constructed.

  That is, since the enamel regeneration solution according to the present embodiment contains amelogenin, which is an enamel protein, the crystal structure similar to that of natural enamel hydroxyapatite crystals from the surface of the enamel is obtained by the action of amelogenin. To grow.

  Various amelogenins are used as amelogenins, and examples thereof include recombinant human amelogenins. Recombinant human amelogenin can be prepared and used as described in the examples below. Amelogenins that are amphipathic and can participate in the growth of hydroxyapatite crystals include amelogenins of various molecular weights ranging from about 6.8 kDa (LRAP (Leucin-rich amelogenin protein)) to about 20 kDa (full-length amelogenin). It preferably contains a material having a large molecular weight. Moreover, it is preferable that amelogenin contains 1.6-5.0 mg / mL. If the concentration is lower than 1.6 mg / mL, enamel regeneration takes time. Moreover, when the concentration is higher than 5.0 mg / mL, dissolution is difficult.

  It is desirable to contain calcium ions and phosphate ions with a relatively low degree of supersaturation. In an aqueous solution containing supersaturated calcium ions and phosphate ions, since the dissolved substance has a high chemical potential, precipitation due to formation of crystal nuclei easily occurs, while many crystal nuclei are formed in the solution by homogeneous nucleation. Is formed, and the dissolved substance component is precipitated in the form of particles as an amorphous precipitate. Moreover, when protein, a peptide, etc. are mixed in the aqueous solution containing a supersaturated calcium ion and phosphate ion, a crystal | crystallization will arise by coprecipitation.

  If calcium ions and phosphate ions are contained at a relatively low degree of supersaturation, and if amelogenin is further contained, calcium ions and phosphate ions will accumulate locally on the enamel surface. From this, it becomes possible to grow hydroxyapatite crystals. For the above reasons, calcium ions are preferably contained in an amount of 0.25 to 4.0 mM. Moreover, it is preferable that the phosphate ion contains 0.125-4.0 mM.

  Further, it preferably contains an ion stabilizer. It is suppressed that calcium ions and phosphate ions react and precipitate unnecessarily, and calcium ions and phosphate ions stably maintain a dissociated state.

As an ion stabilizer, calcium ions and phosphate ions can be contained either as long as they maintain a stable dissociated state in the solution and do not impair the efficacy of the enamel regeneration solution. Examples include potassium chloride (KCl), sodium chloride (NaCl), magnesium chloride (MgCl ₂ ), sodium hydrogen carbonate (NaHCO ₃ ), potassium carbonate (K ₂ CO ₃ ), and the like.

  Moreover, in order to keep pH of a solution near neutrality, it is preferable to contain a pH adjuster. Amelogenin accumulates well in the vicinity of neutrality, constructs a larger three-dimensional structure (nanosphere), the function of nanosphere is likely to be an important factor in the mechanism of enamel formation, and the formation of enamel is promoted It is. The pH adjuster may contain any one as long as the pH of the enamel regeneration solution is kept near neutral (pH 6.0 to 8.0) and the efficacy of the enamel regeneration solution is not inhibited. As Tris-HCl.

  Further, it preferably contains a thickener. The thickener increases the viscosity of the enamel regeneration solution, so that the enamel regeneration solution tends to stay on the surface of the tooth enamel. Any thickener may be contained as long as it does not inhibit the efficacy of the enamel regenerating solution, and examples thereof include propylene glycerol alginate (PEG) and hyaluronic acid (HA). Moreover, the form which added the thickener in large quantities and became gelatinous may be sufficient.

  The enamel regeneration solution is obtained by adding calcium salt, phosphate, and amelogenin to water (pure water). Examples of the calcium salt include general calcium salts such as calcium nitrate, calcium hydroxide, calcium acetate, calcium carbonate, calcium chloride, calcium citrate, calcium lactate and the like. Examples of the phosphate include general phosphates such as potassium hydrogen phosphate, phosphoric acid, ammonium phosphate, sodium phosphate, and potassium phosphate. Furthermore, you may add the above-mentioned ion stabilizer, ion stabilizer, pH adjuster, and thickener.

  The enamel regeneration solution described above is prepared as an enamel regeneration kit prepared by dividing it into two or more solutions each containing different components, and each solution is mixed before remineralization of enamel. The form which treats the enamel reproduction | regeneration liquid to a dental enamel may be sufficient. The enamel regeneration kit will be described below.

  The enamel regeneration kit includes a solution containing calcium ions (hereinafter referred to as solution Ca) and a solution containing phosphate ions and amelogenin (hereinafter referred to as solution P).

  The solution Ca and the solution P are mixed and used before remineralization. Since calcium ions and phosphate ions are stored separately, the deposition of unnecessary hydroxyapatite crystals that occur before use is suppressed.

  The calcium ions in the solution Ca are preferably contained so as to be 0.25 to 4.0 mM when mixed with the solution P. Moreover, it is preferable that the phosphate ion in the solution P is contained so that it may become 0.125-4.0 mM when it mixes with the solution Ca. Moreover, it is preferable that the amelogenin in the solution P is contained so that it may become 1.6-5.0 mg / mL when it mixes with the solution Ca.

  Moreover, the solution Ca and the solution P may each contain a pH adjuster that keeps the pH in the vicinity of neutrality. The solution Ca preferably contains an ion stabilizer. Furthermore, the solution P preferably contains a thickener.

  Since the ion stabilizer and the thickener are the same as those in the enamel regeneration solution, description thereof is omitted.

  Furthermore, the enamel regeneration kit may include a coating material that holds the enamel regeneration solution obtained by mixing the solution Ca and the solution P described above on the surface of the tooth enamel.

  For example, as shown to FIG. 3 (A), the shell-shaped coating | covering material 10 is mentioned. The covering material 10 includes a concave portion 10a that can be filled with an enamel regeneration solution and a peripheral edge portion 10b that is a portion to be bonded to a tooth.

  As shown in FIG. 3 (B), the enamel regeneration solution 20 obtained by mixing the solution Ca and the solution P is filled in the recess 10a, and the edge of the covering material 10 using a dental adhesive material or the like. What is necessary is just to adhere | attach and seal the part 10b on the enamel surface 30 of a tooth | gear. Thereby, since the enamel reproduction | regeneration liquid 20 is hold | maintained at the enamel surface 30, enamel can be remineralized efficiently.

  The covering material 10 is preferably a material that does not harm the human body, such as polycarbonate. Moreover, regarding the adhesion between the edge portion 10b and the enamel surface 30, the edge portion 10b can be made of a soft material or a shrinkable material and sealed.

  Moreover, as shown in FIG. 4, the coating | covering material 11 of the form which covers the whole tooth | gear may be sufficient.

  Furthermore, the film-shaped coating | covering material 12 in which one surface shown to FIG. 5 (A) has adhesiveness may be sufficient. As shown in FIG. 5 (B), enamel regeneration is performed by covering the surfaces of the teeth 30 with a gel-like enamel regeneration solution 21 and further covering with a film-like coating material 12 from above. The liquid can be held on the surface of the tooth 30.

(Experiment 1)
(Verification of surface roughness of enamel before and after caries)

First, the enamel was cut in a 2 × 2 mm square from the center of the crown of the extracted human tooth. Hereinafter, this is referred to as a natural enamel sample. The conditions of the natural enamel sample used are described below.
1) Healthy teeth with no gross caries, coloring, or significant damage.
2) Observed with a digital stereomicroscope at a magnification of 1250, and no abnormalities are observed on the enamel surface.

  The natural enamel sample was washed with water and dried, and then an arbitrary region (10 μm × 12 μm) was scanned with an atomic force microscope (AFM: KEYENCE, VN-8000). The inspection site was the central part of the natural enamel sample.

  Three AFM tests were performed per natural enamel sample. The surface roughness (average surface roughness (Ra value)) of the obtained three-dimensional image was measured using dedicated observation application software (VN Viewer, KEYENCE). The measurement was performed on arbitrary three cross sections in the scan region, and the average value was used as measurement data. For the surface roughness, the arithmetic average roughness (Ra) of each part subjected to AFM inspection at random from the target natural enamel sample was measured.

  The surface of the natural enamel sample was acid-treated for the purpose of imitating mild caries as uniformly as possible. 65% phosphoric acid (Sun Medical Co., Ltd.) was uniformly applied to the entire surface of the enamel sample, and was treated three times in 60 seconds. This is hereinafter referred to as an acid-treated enamel sample.

  Thereafter, in the same manner as the natural enamel sample, an AFM inspection of the enamel sample after acid treatment was performed, and the surface roughness was measured.

  A healthy natural enamel sample (86 samples) had a surface roughness (Ra) of 44.1 ± 52.9 nm, as shown in FIG. Among these, the measurement values within 1 SD (Ra value 0 to 97.1 nm) accounted for 90.7% of the total with 78 samples. Even if the measured value is 1SD or more, it is clinically sound tooth, and it is thought that a large number was detected due to scratches or fine cracks on the enamel surface, but the experimental conditions were uniform. These were excluded in order to

  In the observation of the surface of the natural enamel sample by the AFM image, as shown in FIGS. 7 (A), (B), and (C), even on the tooth surface that looks smooth under the naked eye or by observation with a stereoscopic microscope, The undulation is confirmed. Furthermore, the trabecular structure of the hydroxyapatite crystals constituting the enamel is observed.

  On the other hand, in the AFM inspection of the acid-treated enamel sample (86 samples), the surface roughness (Ra) was 266.8 ± 103.8 nm as shown in FIG. Among these, those whose measured values were within 1 SD (Ra value 163.0 to 370.7 nm) accounted for 70.9% of 61 samples. Among those whose measured values exceeded 1 SD, those within 1 SD (Ra value 0 to 97.1 nm) before acid treatment were slightly included, but this was considered to be due to individual differences in acid sensitivity. It was.

  In the AFM image, a part of the hydroxyapatite crystal was dissolved by the acid treatment, and the enamel trabecular end depression and the band-like structure peculiar to the enamel were clearly confirmed (FIG. 9 (A), ( B), (C)).

Example 1
After the acid treatment in Experiment 1 above, the enamel sample was treated with the enamel regeneration solution to verify the remineralization of the enamel.

(Enamel sample after acid treatment used)
From the results of Experiment 1, an acid-treated enamel sample that satisfies the following criteria was used in Example 1.
(1) The measured value is within 1 SD before the acid treatment (Ra value 0 to 97.1 nm)
(2) Measured value within 1 SD after acid treatment (Ra value 163.0-370.7 nm)

(Preparation of used human recombinant amelogenin and solution)
First, the human recombinant amelogenin used was prepared according to the method of DenBesten et al. (DenBesten PK, et al. (2002). Arch Oral Biol 47 (11): 763-70.). That is, amelogenin cDNA (Gl: 6715562 in Genebank) was introduced into Escherichia coli (BL21-competent E. coli) using a vector to express the protein, followed by purification. After cell disruption, the amelogenin protein component was separated using 20% ammonium sulfate precipitation and C4 stationary phase. The amelogenin protein was freeze-dried and stored frozen and used in the experiment.

Three types of solutions (solution (1), solution (2), and solution (3)) were prepared and prepared. The composition of each solution is as follows.
Solution (1): 1 to 128 mM (final concentration of 0.25 to 4 mM when the solutions (1) to (3) are mixed) calcium nitrate [Ca (NO ₃ ) ₂ .4H ₂ O] and 1200 mM (solution (Final concentration 150 mM when mixing (1) to (3)) Potassium chloride [KCl] was dissolved in Tris buffer [Tris-HCl, pH 7.4] adjusted to 200 mM, pH 7.4.
Solution (2): 4 mg / mL (final concentration of 3 mg / mL when the solutions (1) to (3) are mixed) A human recombinant amelogenin solution is dissolved in pure water.
Solution (3): 1 to 256 mM (final concentration 0.125 to 32 mM when the solutions (1) to (3) are mixed) Tris buffer in which potassium phosphate [KH ₂ PO ₄ ] is adjusted to 200 mM, pH 7.4 Dissolved in [Tris-HCl, pH 7.4].

  First, after the acid treatment, the enamel sample was washed with pure water and dried, and then allowed to stand in a 96-well culture container with the enamel surface facing up.

  The solution (1) was dropped onto the enamel of the enamel sample after 25 μL acid treatment.

  Subsequently, 150 μL of the solution (2) was added.

  Subsequently, 25 μL of the solution (3) was added.

  And it left still for 16 hours in a 37 degreeC incubator.

  After standing, the acid-treated enamel sample was washed with pure water, dried, and then subjected to AFM inspection and surface roughness measurement in the same manner as in Experimental Example 1.

  The final concentration ratio of calcium ions / phosphate ions (hereinafter referred to as Ca / P) was variously combined. The combinations of Ca / P, calcium ion concentration and phosphate ion concentration of the enamel regeneration solution used in the experiment are as follows.

  As a reference example, the same procedure as described above was performed except that the same amount of pure water was added instead of the solution (2).

(Verification of AFM image)
In the reference example not containing amelogenin, as shown in FIGS. 10 (A) to (F), the structure of the enamel surface layer roughened by acid treatment in the AFM image was clearly confirmed without change. In other words, enamel trabecular end depressions and band-like structures peculiar to enamel can be seen as they are, and the formation of new hydroxyapatite crystals and the change in enamel surface structure depend on which calcium ion concentration and phosphate. It was not observed in the combination of ion concentrations.

  On the other hand, in Example 1 containing amelogenin, as shown in FIGS. 11 (A) to (F), although the frequency-like structure on the surface of the enamel can be confirmed slightly, the recess is a hydroxyapatite crystal-like structure. Satisfied and improved surface roughness was observed. Moreover, the structure similar to the natural enamel surface before acid treatment shown to FIG. 7 (A)-(C) was recognized.

(Verification of surface roughness (Ra))
The Ra value before processing of all the samples used (60 samples in total (Example 1: 30 samples + Reference example: 30 samples)) was 281.6 ± 69.0 nm.

  In the reference example, no significant change was observed in the Ra value as compared to before the treatment. In each condition where the calcium ion concentration and the Ca / P ratio were changed, no significant change (p <0.01) was observed in the Ra value compared to the control group. The Ra value after the treatment for all conditions was 296.7 ± 33.8 nm.

  On the other hand, in Example 1 to which amelogenin was added, a decreasing tendency of the Ra value depending on the calcium ion concentration and the phosphate ion concentration was observed as compared with the reference example. In examinations with different calcium ion and phosphate ion concentration conditions, it was revealed that the respective concentrations and ratios (Ca / P ratio) change the Ra value. It was 2 mM calcium ion and 2 mM phosphate ion (Ca / P ratio 1.0) that the decrease in Ra value was obtained most. When the Ca / P ratio of 1.0 was maintained, a low Ra value was observed even under other concentration conditions, but a tendency for the Ra value to increase was observed regardless of whether the Ca concentration was higher or lower.

  On the other hand, if the calcium ion concentration is too high, no significant change occurs in the Ra value. Further, the limit value of the calcium ion concentration also varies depending on the phosphate ion concentration, that is, the Ca / P ratio. If the Ca / P ratio is 1.0 or 0.5, the Ra value does not change significantly at a calcium ion concentration of 4 mM or more. Further, if the Ca / P ratio is 0.25, the Ra value does not change significantly at a calcium ion concentration of 2 mM or more. Furthermore, when the Ca / P ratio is 0.125, the Ra value does not change significantly at a calcium ion concentration of 0.5 mM or more.

  On the other hand, the exact limit value of the calcium ion concentration at which no significant change occurs in the Ra value is not clear under this experimental condition. In particular, when the Ca / P ratio is 1.67 or less, there is a high possibility that a significant therapeutic result can be obtained even when the calcium ion concentration is 0.25 mM or less. When the Ca / P ratio is 2.0, there is a significant difference, but the change in the Ra value is small, and when the calcium ion concentration is further reduced, the difference is likely to disappear.

In summary, the optimum conditions for the calcium ion concentration and phosphate ion concentration in this example are as follows.
Ca / P: 0.125 to 0.25: calcium ion concentration 0.25 mM, phosphate ion concentration 1 to 2 mM
Ca / P: 0.25 to 0.5: calcium ion concentration 0.25 to 1 mM, phosphate ion concentration 0.5 to 4 mM
Ca / P: 0.5-1: calcium ion concentration 0.25-2 mM, phosphate ion concentration 0.25-4 mM
Ca / P: 1-2: calcium ion concentration 0.25-4 mM, phosphate ion concentration 0.125-4 mM
Among these, the most effective is Ca / P: 1 to 2 with a calcium ion concentration of 2 mM and a phosphate ion concentration of 1 to 2 mM.

(Example 2)
(Regeneration experiment of enamel using extracted teeth)
Using human extracted tooth enamel, an enamel regeneration experiment for initial caries was conducted.

  The surface of the unextracted human extracted tooth enamel (hereinafter referred to as a pre-treatment sample) was subjected to surface observation using a digital stereomicroscope.

Subsequently, after washing the pre-treatment sample, it was immersed in an enamel regeneration solution to induce hydroxyapatite crystals. The reaction was carried out at 37 ° C. for 8 hours. The composition of the enamel regeneration solution used is shown below.
Calcium nitrate 1.8 mM
Potassium hydrogen phosphate 8.3 mM
Potassium chloride 150 mM
Tris-HCl, pH 7.4 50 mM
Human recombinant amelogenin 2.0mg / mL
Propyleneglycerol alginate 1mg / mL

  Thereafter, it was thoroughly washed with pure water and completely dried at room temperature. Hereinafter, this is referred to as an 8-hour processed sample. About this 8-hour process sample, the surface observation of the same location as the sample before a process was performed using the digital stereomicroscope.

  The surface-treated 8-hour treated sample was further immersed in a new enamel regeneration solution having the above composition and reacted at 37 ° C. for 8 hours. After the reaction, it was thoroughly washed with pure water and completely dried at room temperature. Hereinafter, this is referred to as a 16-hour processing sample. About this 16-hour processing sample, the surface observation of the same location as the sample before a process was performed using the digital stereomicroscope.

  Compared to the pre-treatment samples shown in FIGS. 13A and 13B, the 8-hour post-treatment samples shown in FIGS. 13C and 13D show that the enamel surface scratches and dents are reduced. . Furthermore, it can be seen that the 16-hour processing sample added with the 8-hour processing has a substantially uniform surface as shown in FIGS. 13 (E) and 13 (F). It can be seen that enamel remineralization was caused by the treatment with the enamel regeneration solution.

(Example 3)
(Enamel regeneration experiment using a beagle dog)
In Example 1 and Example 2, the extracted tooth was verified, but it was verified that enamel regeneration was possible even in vivo.

  An enamel regeneration experiment was conducted on the upper canine teeth of an 8-month-old female beagle dog. First, the upper canine teeth of the subject were observed using an atomic force microscope (AFM).

  Subsequently, an acid treatment was performed on the enamel to reproduce the initial caries. The conditions for the acid treatment are the same as in Experimental Example 1. After acid treatment, observation was performed using AFM.

  In order to create a reaction space, a coating material was prepared by molding dental thermoplastic polycarbonate to match the teeth. The enamel regeneration solution was put into this covering material, and after being attached to the maxillary canine, it was fixed to the maxillary canine with a dental adhesive and the margin was sealed. The composition of the enamel regeneration solution used is the same as in Example 2.

  And it was made to react in the living body for 16 hours. Thereafter, the maxillary canine was removed, sufficiently washed with water and dried, and then observed using AFM.

  FIGS. 14A, 14B, and 14C show the state of the enamel surface before treatment, after acid treatment, and after reaction. It can be seen that the surface of the enamel surface is roughened by the acid treatment, and the unevenness of the enamel surface is changed to a smooth surface with less unevenness by the enamel regeneration liquid. Therefore, it was proved that the enamel regeneration solution causes remineralization of enamel even in vivo.

  Since the enamel regeneration solution and the enamel regeneration kit cause enamel remineralization in vivo by the action of amelogenin, treatment of initial caries and the like is possible. In addition, since the enamel layer can be thickened, it is effective in preventing caries. Therefore, utilization in the dentistry industry is expected.

DESCRIPTION OF SYMBOLS 10 Coating | covering material 10a Recessed part 10b Edge part 11 Coating | coated material 12 Coating | coated material 20 Enamel reproduction | regeneration liquid 30 Enamel surface

Claims (7)

A first solution containing calcium ions;
A second solution containing amelogenin and phosphate ions,
Enamel regeneration kit characterized by that. The total amount of the first solution and the second solution is 1.6 to 5.0 mg / mL of the amelogenin, 0.25 to 4.0 mM of the calcium ions, and 0.125 of the phosphate ions. Containing ~ 4.0 mM,
The enamel regeneration kit according to claim 1 . Containing 2.0-3.0 mg / mL of the amelogenin with respect to the total amount of the first solution and the second solution,
The enamel regeneration kit according to claim 2 . The first solution contains an ion stabilizer;
The enamel regeneration kit according to any one of claims 1 to 3 . The second solution contains a thickener;
The enamel regeneration kit according to any one of claims 1 to 4 , wherein the enamel regeneration kit is provided. Each of the first solution and the second solution contains a pH adjusting agent that keeps the pH in the vicinity of neutrality.
The enamel regeneration kit according to any one of claims 1 to 5 , wherein: A coating material for holding an enamel regeneration solution obtained by mixing the first solution and the second solution on the surface of the tooth enamel ;
The dressing that having a recess in which the enamel regenerant is filled,
The enamel regeneration kit according to any one of claims 1 to 6 .