JPH06340555A - Pulp-capping agent enabling formation of dentine - Google Patents

Abstract

PURPOSE:To obtain a dental pulp-capping agent used for therapies of dental caries, especially for the repairing therapy of teeth, utilizing the biological reactions of bone-forming proteins, and having a dentine-regenerating ability on the therapies. CONSTITUTION:This dental pulp-capping agent enabling the formation of dentine contains a purified bone-forming protein as an active ingredient and, if necessary, a carrier having biodegradability, tissue compatibility and sustained releasability. When the pulp-capping agent is charged on the dental pulp-cut surface of a tooth cavity after the removal of a carious part, a large volume of dentine is formed on the dental pulp-cut surface to perfectly protect the left dental pulp, thereby enabling to provide a remarkable therapeutic method capable of keeping the original functions of teeth.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a pulp capping agent for treating dental caries teeth, particularly for treating dental caries, which has the ability to promote dentin formation.

[0002]

2. Description of the Related Art Conventionally, as a method for treating carious teeth, when caries is limited to enamel and dentin, the part is shaved off and repaired with metal or resin. When caries progresses to the pulp, the affected area may be scraped off, calcium hydroxide may be applied to the exposed pulp as a pulp capping agent, and cement may be layered and repaired with metal or resin.

However, even if the above method is applied, since calcium hydroxide does not have a dental pulp cell growth promoting action and an odontoblast inducing action, a necrotic layer due to a strong alkali is formed on the dental pulp surface in contact with calcium hydroxide. However, regeneration of the pulp part cannot be expected. In addition, even if dentin formation occurs, it is only a small amount underneath the necrotic layer, and the pulp is not completely blocked. Therefore, it often re-infects and needs re-treatment as the years go by. Therefore, a pulp extraction procedure for completely removing the dental pulp is often performed.

However, there are many problems to be solved in this treatment method. For example, the supply of nutrients and water to the teeth is cut off, and dentin becomes only mineral, leading to weakening of the teeth. Since the nerve system that conveys pain disappears at the same time when pulp is removed, subjective symptoms associated with caries progression may not be obtained and it may be too late. After the pulp removal, the root canal is filled with a cutter percha to prevent bacterial infection, but due to the anatomical complexity of the root canal, it is almost impossible to completely fill it. It is possible that Therefore, in later years, it becomes necessary to treat the root canal again (infectious root canal treatment) or to remove the tooth.

Since preservation of dental pulp is very important in terms of prolonging the life of the tooth, there is a strong demand for development of a pulp capping agent having a pulp cell proliferation promoting action and an odontoblast differentiation inducing action.

[0006]

SUMMARY OF THE INVENTION When caries reaches the pulp, there is a long-felt need for a surgical method that preserves the pulp as much as possible and brings it closer to a healthy tooth. As a surgical method for that purpose, there are a direct pulp capping method in which the exposed surface is covered with a pulp capping agent and a living pulp cutting method in which the root pulp is cut and removed and the root pulp is covered with a capping agent. There are challenges. Therefore, it is necessary to develop a biological pulp capping agent having an anti-inflammatory action, a dental pulp cell growth promoting action, and an odontoblast differentiation inducing action in place of calcium hydroxide.

As one of the methods, a method of applying the biological activity of a physiologically active substance can be considered. The existence of substances with osteoinductive activity in dentin has been suggested (Butler
W. T. et al. J. ofDent. Res. ，
Vol, 56, p228-232, 1977, Cono
ver M. A. et al. , Procedings
of the 1st International
Confencence Chemistry an
d Biology of Mineralized
Connective Tissue, p597-60
6, Northnwestern University
yNeW York, 1982, Mera K. et al. J.
Jpn. Stomat. Soc. Vol. 37, p38
9-399, 1988, Katz et al, Bio.
chem. Biopys. Res. Commun. , V
ol. 157, 1253-1257, 1988, Kaw
aiTet al, J .; Dent. Res. Vol. 6
8, p1069-1074,1989). Especially Bess
sho et al. have purified a protein having a molecular weight of 20 kD from a tooth matrix using ectopic osteoinductive activity in rats as an index (J. of Dent. Res. Vol. 70, p17).
1-175, 1991).

However, the biological activity is seen as osteoinductive activity, and the activity in the original vital tooth is not mentioned. Therefore, it is clear from these reports that factors existing in the tooth matrix and restoring the original function of the tooth are not disclosed, and in order to solve these, it is necessary to induce dentin formation derived from the tooth matrix. Identification of factors is essential.

On the other hand, it has been attempted to apply a protein having an osteoinductive activity derived from a dentin matrix to a cut surface of living pulp to completely preserve the residual pulp and form a large amount of dentin on the cut surface. Was successfully formed, but the protein factor has not been purified yet (Nakashima).
M. , Archs Oral Biol. , Vol.
35, p277-281, 1989).

Further, it is known that a crude extract from dog bone matrix promotes the formation of dentin, but the formation of dentin is partial and not completely satisfactory (Nakas).
Hima M. , Archs Oral Biol. ，
Vol. 35, p493-497, 1990). That is,
There are many possible factors in the crude extract, including the possibility of the presence of inhibitors of growth factor activity that promote dentin formation, and other growth factors acting alone or synergistically. It is unclear what growth factors specifically promote the formation of dentin in the extract.

In one example, various growth factors, namely platelet-delivered grow, are present in the bone matrix.
th factor (PDGF), irsulin-1
ikegrawth factor (IGF), tra
nsforming growing factor (T
GF) -β, fibroblast growfa
ctor (FGF) and the like are included, and it is fully conceivable that these may be effective alone or in combination.

In fact, PDGF and IGF-1 promote the division of dental pulp cells, and acidic-FGF, IGF-1, and I
GF-II increases the extracellular matrix proteoglycan, TGF-β, PDGF, acidic-FGF, b
It has been reported that asic-FGF may regulate the differentiation of dental pulp cells into odontoblasts (Nakas).
Hima M. , Archs Oral Biol. ，
Vol37, p231-236, 1992). Therefore B
It is not difficult to imagine that these factors, which are contained in a larger amount than MP, may be responsible for the above activity.

Furthermore, one of the reasons why dentin formation is not sufficient is that there is no scaffold for the activity of odontoblasts.
That is, this experimental system (Nakashima M, Arch
s. Oral Biol. , Vol35 p496-4
97, 1990) in which dentin repaired without using a carrier, pulp cells adhered to bone-like dentin and differentiated into odontoblasts using it as a scaffold, resulting in secondary formation of tubular dentin. It is speculated that it was done. Therefore, factors involved in dentin formation have not been identified at this stage, and it cannot be concluded from these that BMP is involved in dentin formation.

[0014]

Therefore, the present inventor tried to search for substances having such activity among known growth factors. Various growth factors are now available by genetic recombination. Among them, the present inventors focused on an osteogenic factor as a substance having a possibility of promoting differentiation of dental pulp cells (as described above, it is known that there is a factor having an osteoinductive activity in the tooth matrix). Bone morphogenetic protei
n, hereinafter referred to as BMP) was not known to promote the formation of dentin.

BMP is a bone matrix (Wozney J. et.
al. , Science, Vol. 242, p152
It was isolated as a proteinaceous factor showing ectopic osteoinductive activity using 8-1534, 1988, Table 2-500241) or mouse osteosarcoma (JP-A-5-84081) as a starting material. Its action is considered to have a function of acting on undifferentiated mesenchymal cells and differentiating into osteoblasts / chondrocytes, and its application is focused on repair of defective bone.

On the other hand, it is considered that dentin is formed after odontoblasts lined up in a row on the outside of dental pulp cells are differentiated and undergo a series of calcification processes. So far, little is known about how BMP acting on bone affects pulp cells.

This time, using highly purified BMP, i
We investigated how it works for dentin regeneration of teeth in n vivo. As a result, by applying the pulp capping agent containing BMP to the canines of experimental animals that had undergone living pulp cutting, dentin regeneration as vigorous as that of the dentin matrix-derived substance was observed, and BMP formed bone. Not only this, but it became clear that it is also involved in the formation of teeth, and the present invention was completed.

In summary of the gist of the present invention, (1) the pharmacological activity was significantly improved by the combined use of a suitable carrier. In the pulp capping agent of the present invention, as a result of mixing an inactivated tooth matrix for the purpose of providing a scaffold for the activity of dental pulp cells,
The formation of more tubule dentin was observed. (2) Until now, the technical application fields of BMP were mainly related to bone repair, and even in the field of dentistry, it was only related to repair of dental cartilage or jaw bone, but regarding tissue repair of the tooth itself in vivo. Is characterized by the fact that the present invention can be applied to the application for the first time without any knowledge until now, and it is clear that the present invention is not merely a combination of known techniques.

Unlike the conventional pulp pulp cutting method using calcium hydroxide, the pulp capping agent of the present invention preserves the remaining pulp without forming a necrotic layer, and the cartilage causes dentin due to the action of the pulp capping agent. Solved the development of an ideal pulp capping agent in which dental pulp cells proliferate in the cavity where the quality or pulp has been lost, differentiate into odontoblasts and a large amount of new dentin is reconstructed, and the original function can be restored. To do.

As BMP, ectopic bone formation in mice (so-called ectopic bone formate)
A BMP that is positive in the reaction assay is not particularly specified, but a human-derived BMP (hBM
P) is desirable. The amount of BMP added is preferably 100 ng or more per 1 mg carrier.

The carrier of BMP is not particularly specified as long as it has biodegradability, histocompatibility, and sustained release. For example, inactivated dentin matrix (4M guanidine extraction residue of dentin matrix, It alone lacks the ability to regenerate dentin), or a tissue adhesive (such as Tissile,
(Nippon Organ Co., Ltd.), beriplast (Berlink-Hoechst Co., Ltd.), purified type I collagen (cell matrix LA, Nitta Gelatin Co., Ltd.) and the like are preferable.
The carrier content is not particularly specified as long as the BMP / carrier ratio is constant.

Other desirable compounds as additives are
Chondroitin sulfate can be mentioned, and Tris is used as a buffer.
-HCl (pH 8.0) or phosphate buffer (pH
7.2) It is also possible to add physiological saline.

As an example of the method of using the pulp capping agent in the present invention, a BMP preparation is dissolved in hydrochloric acid, mixed with a carrier (for example, an inactivated dentin substrate), adjusted to be neutral, and freeze-dried. After applying it on the cut surface of the living pulp, the upper part is covered with dental cement, and further molded and filled with plastic. After a predetermined period of time, the construction of dentin is confirmed using a dental X-ray device, and a prosthetic treatment is performed on normal vital teeth.

[0024]

The present invention will be described in detail below with reference to examples. The BMP that can be used in the present invention is not particularly specified as described above, but an example using hBMP will be shown in the following examples.

Example 1 Preparation of Recombinant BMP Regarding expression of hBMP (r-hBMP2, r-hBMP4) which is a gene recombinant (hereinafter referred to as r-) using a molecular biology technique, Japanese Patent Application No. 5-8519. In detail. The outline will be described below.

HBMP4 cDNA, hBMP2 cDNA
Wang et al. (Science, Vol 242, 1
528-1534, 1988), a primer was prepared based on the report, and RNA prepared from human osteosarcoma subcultured using nude mice was used as a template.
image chain reaction (hereinafter P
(Abbreviated as CR) method.

HBMP2 cDNA, hBMP4 cDNA
To pUC vector, and pUC-hBMP2,
pUC-hBMP4 was prepared. Next, the gene transfer site of pBm4, which is a transfer vector for the silkworm polyhedrosis virus,
PUC -hBMP2 and pUC-hB were respectively added to the modified pBm4 plasmids digested with Nru I to generate blunt ends.
Introducing MP4, pBm4-hBMP2 and pBm4
-HBMP4 was obtained.

Subsequently, the silkworm polygon virus gene DNA
And pBm4-hBMP2 plasmid DNA, or p
HBMP2 and hBM by simultaneously introducing Bm4-hBMP4 plasmid DNA into silkworm cultured cells BoMol5AIIc using the calcium phosphate coprecipitation method.
A recombinant virus incorporating the P4 gene was prepared.

Example 2 Preparation of silkworm body fluid A virus solution for inoculating silkworm was prepared . That is, BoMo was subjected to a flat culture for 2 days at an initial culture concentration of 1 × 10 ⁶ cells
10 ml of a BoMol5AIIc cell culture solution containing the recombinant virus obtained in Example 1 was added to the 15AIIc cells, and the mixture was further cultured at 25 ° C. for 14 days. 1 culture
After centrifugation at 000 rpm for 5 minutes, the obtained centrifugation supernatant was used as a recombinant virus solution for inoculation.

A liquid containing a recombinant virus was diluted 10-fold into the silkworm larvae on the 5th day on the 1st day to give 50
After injecting μl, the mixture was fed with commercially available artificial breeding (Katakura Kogyo, Morus) for 4 days at 25 ° C., and the body fluid was collected in an ice-cooled plastic tube to obtain a supernatant after centrifugation.
500 silkworms were used per lot.

Example 3 Purification of BMP (1) Purification of r-hBMP2 Urea was added to the silkworm body fluid obtained in Example 2 to a final concentration of 8 M, and a supernatant was obtained by centrifugation. This sample was added to 6 M urea, 20 mM Tris-HCl (pH 7.
4) equilibrated with heparin-Sepharose
It was added to CL-6B (Pharmacia). After removing the non-adsorbed fraction with the initial buffer, 6M urea, 20mM T
ris-HCl (pH 7.4) -1.5M NaCl,
6M urea, 20mM Tris-HCl (pH 7.4)
Elution was carried out by the linear concentration gradient method.

Fractions showing activity were collected and collected with 6 M urea, 20 m
Dialysis was performed against M Tris-HCl (pH 7.4). This sample was added with 6M urea and 20mM Tris-HC.
heparin-Sep equilibrated with 1 (pH 7.4)
Harose CL-6B was added again. After removing the non-adsorbed fraction with the initial buffer, the same procedure was repeated with 1.5M NaCl.
Elution was carried out by the linear concentration gradient method.

Heparin-Sepharose C
The L-6B elution fraction was adjusted to pH 2.6 with 1N-HCl and added to a TSK-octadedecyl 4PW column (Tosoh Corporation, diameter 7.5 mm × 15 cm).
5 minutes after adding the sample, 24% acetonitrile, 0.
It was washed with 1% trifluoroacetic acid (hereinafter abbreviated as TFA), and thereafter, up to 35 minutes, 24 to 48% acetonitrile,
Elution was performed with a linear concentration gradient method of 0.1% TFA. The osteoinductive activity of the obtained peak was measured.

(2) Purification of r-hBMP4 Urea was added to the silkworm body fluid obtained in Example 2 to a final concentration of 8 M, and the supernatant was obtained by centrifugation. This sample was added with 6 M urea and 20 mM Tris-HCl (pH 7.4).
Heparin-Sepharose C equilibrated with
It was added to L-6B (Pharmacia). After removing the non-adsorbed fraction with the initial buffer, elution was carried out by the linear concentration gradient method to 1.5 M NaCl. Collect the fractions showing activity,
6M urea, 20mM Tris-HCl (pH 7.4)
After dialysis against heparin-Seph
Rechromatography with arose CL-6B was performed.

Fractions showing activity were collected and collected with 6M urea, 20m
Dialysis was performed against M Tris-HCl (pH 7.4). Ammonium sulfate was added to this sample so as to be 30% saturated, and 6 M urea and 20 mM Tris-HCl (pH 7.
4) Pheny equilibrated with buffer solution saturated with 30% ammonium sulfate
Added to l-Sepharose CL-4B. After removing the non-adsorbed fraction with the initial buffer, elution was performed by a linear concentration gradient method to 0% ammonium sulfate and 50% ethylene glycol. The active fraction was purified by HPLC in the same manner as in the case of r-hBMP2.

R-hBMP2, r-h after HPLC fractionation
The analysis results of BMP4 by SDS polyacrylamide gel electrophoresis are shown in FIG. r-hBMP2 showed a single band under reducing conditions, and r-hBMP4 showed mainly three bands.

Example 4 Canine Tooth Performed Vaginal Amputation
For the dentin formation experiment r-hBMP2 and r-hBMP4, the lyophilized preparation (20 μg each) was dissolved in 100 μl of 50 mM HCl. A dentin matrix prepared from bovine teeth was used as a carrier. That is, fresh bovine teeth (1 kg) were crushed with a grinder, washed with PBS, and then suspended in 4 M guanidine hydrochloric acid (50 ml used per 5 g of powder). Using a magnetic stirrer, the mixture was vigorously stirred (48 hours) and then centrifuged to collect the extraction residue (precipitation portion), which was washed again with PBS and lyophilized to give a powder (inactivated ivory substrate, in the figure below). Abbreviated as M).

The pulp capping agent used in the experiment was prepared as follows.
2 μg of r-hBMP on 20 mg of inactivated dentin matrix
2 or 2 μg of r-hBMP4 is mixed, and further 0.5 m
l chondroitin sulfate (1 mg / ml) and 0.5 m
1 type I collagen (500 μg / ml) was added.
After left at room temperature for 30 minutes, a freeze-dried powder was obtained again using a speed back concentrator.

For the animal test, male dogs (Nakajima Experimental Animal Co., Ltd.) aged 7 to 8 months were used. Under anesthesia by intravenous administration of Nembutal, the canine tooth was perforated with a dental diamond round bar to a diameter of about 2 mm and a depth of about enough to expose the pulp. After that, the dental pulp was cut with an engine round bar. Alternately wash with sodium hypochlorite and hydrogen peroxide solution, then wash with physiological saline and pressurize to stop bleeding.Add sample to cavity to prevent excessive pressure on exposed pulp. Covered with dental cement. After further etching, it was molded and filled with a dental resin. As a negative control, a bovine dentin matrix residue treated in the same manner without adding BMP was used.

After surgery, the animals were fed a normal diet. Two months later, the treated tooth was extracted, and the dentin regeneration activity was evaluated from the soft X-ray photograph image and tissue specimen image. The histology of an example of a control (bovine dentin matrix residue) and an r-hBMP2 addition group is shown (FIG. 2, FIG. 3).

In FIG. 3, it can be clearly seen that neodentin formation is observed around the filled dentin matrix on the cut surface of living pulp, and odontoblasts are partially left or mixed with dentin. Was given. At the top, there is also an image of proliferated dental pulp cells forming a matrix.

The r-hBMP4 group also has r-hBMP
Although it was weaker than that of Group 2, obvious dentin formation was observed in contact with the cut surface.

[0043]

EFFECTS OF THE INVENTION The dentinogenic pulp capping agent of the present invention provides a method of treating dental caries without removing the dental pulp and a material used therefor even in the case of caries having a considerably advanced degree. In the treatment of dental caries, which has been so advanced that there was only a method for treating pulp extraction, it is expected that by applying this dentin-forming pulp capping agent, regeneration of dentin of the tooth can be expected while the pulp is preserved. It is a major contributor in the therapeutic field.

[Brief description of drawings]

FIG. 1 shows purified r-hBMP2 and r-hBMP.
The silver dyeing image concerning 4 is shown. 2ME indicates 2 mercaptoethanol.

FIG. 2 relates to a histological image two months after the inactivated demineralized dentin matrix was filled in a living pulp cutting site, and shows a hematoxylin-eosin stained image. The arrow in (a) indicates a section of living pulp. In (b), dentin matrix (M) surrounding the cavity is filled with eosinophil infiltration and red blood cells. In (c), in the cavity near the cut surface, the matrix surrounding the matrix is filled with new dental pulp (P), and the cells in contact with the matrix are differentiated into odontoblasts, but the formation of new anterior dentin matrix is extremely small. Few.

[Fig. 3] Fig. 3 shows a histological image two months after the living pulp cutting site was filled with r-hBMP2-inactivated dentin matrix.
The arrow in (a) indicates a section of living pulp. In (b), infiltration of fibroblasts is observed around the matrix (M) in the upper part of the cavity, and some of them adhere to the matrix (M).
In (c), in the middle part of the cavity, the periphery of the matrix is filled with new dental pulp (P), and bone-like odontoblasts are partially seen. In (d), the cavity near the cut surface is filled with the tubular dentin (TD) formed by odontoblasts, and some bone-like dentin (OD) is also seen.

Claims (4)

[Claims] 1. A dentinogenic pulp capping agent containing an osteogenic factor as an active ingredient. 2. The dentinogenic pulp capping agent according to claim 1, wherein the osteogenic factor is a human-derived osteogenic factor. 3. The dentinogenic pulp capping agent according to claim 1, which contains a carrier having biodegradability, histocompatibility, and sustained release. 4. The pulp capping agent according to claim 3, wherein the carrier is an inactivated tooth matrix.