JP5519121B2 - Novel RGD-containing peptide and dentin regenerating agent, bone regenerating agent, periodontal tissue regenerating agent - Google Patents

Description

  The present invention relates to a novel RGD-containing peptide, a dentin regenerating agent, a bone regenerating agent, and a periodontal tissue regenerating agent. More specifically, the present invention relates to a novel RGD-containing peptide derived from dentin phosphophorin, a dentin regenerating agent, a bone regenerating agent, and a periodontal tissue regenerating agent using the same.

  So far, dental treatment has been centered on simply removing the teeth and nerves completely without losing them, and filling them with materials such as resin and metal. However, in recent years, with the aging of society, people's interest in health has been increasing. It is changing to medical care that seeks a quality of life. Furthermore, the technique of manipulating the patient's own cells to freely regenerate the teeth and periodontal tissue is considered to be able to solve many problems that have been impossible to treat until now. Under these circumstances, the present inventors have aimed to develop a tissue-inducing material and technique that show biocompatibility and strongly induce bone regeneration in a short period of time.

  Dentin phosphophorin is an acidic protein that accounts for 50% of the non-collagenous protein of dentin. Regarding dentin phosphophorin, there are reports on the high calcification-inducing activity and bone and periodontal tissue-inducing activity of bovine dentin phosphophorin-collagen complex. First of all, the calcification-inducing activity of the phosphophorin-collagen complex was quantified using the interfacial tension between the substrate and crystal during calcification induction, and its height was demonstrated (T. Saito et al. ., Bone 21 (4), 305-311, 1997. (Non-patent document 1)) Since the calcification-inducing ability is lost by dephosphorylation, the phosphate group is essential for calcification by phosphophorin ( T. Saito et al., J Bone Miner. Res., 13 (2), 265-270, 1998. (Non-Patent Document 2), T. Saito et al. J. Bone Miner. Res., 15, 1615- 1619, 2000. (Non-Patent Document 3)) Next, the phosphorophore / collagen complex was transferred to the femoral defect of an animal (D. Iejima, T. Saito, T. Uemura, J. Biomater. Sci. Polymer Ed). , 14, 1097-1103, 2003. (Non-patent document 4)), alveolar bone defect as a periodontal disease model (T. Fujii, Y. Terada, F. Kobayashi, T. Koike, T. Saito, T . Uemura, Bone 36, S103, 2005. (Non-Patent Document 5)) and their high induction activity for tissue regeneration It has been reported that showed. In particular, in the alveolar bone regeneration experiment, not only the alveolar bone but also cementum regeneration, and between the regenerated alveolar bone and cementum, periodontal ligament regeneration, which is a key point for periodontal disease treatment, was observed. It has been reported.

  Furthermore, in a pulp capping experiment in which a porcine phosphophorin / collagen complex was transplanted onto the artificial demyelinating surface of rats and dogs, the formation of dense repaired dentin with a tubule structure was observed at an early stage, and inflammation of the dental pulp was observed. Almost no recognition was found, and it was revealed that the material was a pulp-capping material having both a powerful repairing dentin formation-inducing activity and biocompatibility (Japanese Patent Application Laid-Open No. 2003-235953 (Patent Document 1), WO2005 / 079728 (Patents) Reference 2)).

  It has also been proposed to apply phosphophorin itself to bone and dentin using the cell differentiation promoting action (WO2005 / 016368 (Patent Document 3)).

There are also reports on the treatment of bone disorders including dental use with peptides. For example, it has been proposed to promote good bone and tooth growth by adding peptides with RGD sequence of matrix extracellular phosphoglycoprotein, glycosaminoglycan binding motif and calcium binding motif to toothpaste and mouthwash. (Japanese translations of PCT publication No. 2004-506654 (patent document 4)). Furthermore, dental products such as toothpastes, mouth washes and dental floss containing this bone growth promoting peptide (Japanese translations of PCT publication No. 2004-506002 (Patent Document 5), JP-A-2006-83176 (Patent Document 6)) ) Has also been proposed.
T. Saito et al., Bone 21 (4), 305-311, 1997. T. Saito et al., J Bone Miner. Res., 13 (2), 265-270, 1998. T. Saito et al. J. Bone Miner. Res., 15, 1615-1619, 2000. D. Iejima, T. Saito, T. Uemura, J. Biomater. Sci. Polymer Ed., 14, 1097-1103, 2003. T. Fujii, Y. Terada, F. Kobayashi, T. Koike, T. Saito, T. Uemura, Bone 36, S103, 2005. Japanese Patent Laid-Open No. 2003-235953 WO2005 / 079728 WO2005 / 016368 JP-T-2004-506654 JP-T-2004-506002 JP 2006-83176 A

  However, due to the emergence of the BSE problem from several years ago, the use of biologically-derived proteins described in Non-Patent Documents 1 to 5 and Patent Documents 1 and 2 has a concern about safety, and is a safer tissue regeneration material. Development has become an important issue. There is a similar safety concern when using phosphorophorin itself described in Patent Document 3.

  In addition, the bone growth promoting peptides described in Patent Documents 3 to 5 have the effect of increasing the number of skeletal growth and odontoblasts, and our peptides can promote differentiation into osteoblasts and odontoblasts. it is conceivable that. However, the bone growth promoting effect is not sufficiently satisfactory, and it has been desired to provide a new material having a bone regeneration effect.

  Accordingly, an object of the present invention is to provide a peptide that can be artificially synthesized and that has no bone regeneration effect, without concern about safety inherent in biologically derived materials such as BSE. It is in. Furthermore, this invention is providing the bone regeneration agent, dentin regeneration agent, and periodontal tissue regeneration agent using this new peptide.

The present invention is as follows.
[1]
A peptide having any one of the following amino acid sequences.
SESDNNSSSRGDASYNSDES (1-1)
ESDNNSSSRGDASYNSDES (1-2)
SDNNSSSRGDASYNSDES (1-3)
SESDNNSSSRGDASYNSDE (1-4)
SESDNNSSSRGDASYNSD (1-5)
SESDNNSSSRGDASYNS (1-6)
SESDNNSSSRGDASYN (1-7)
ANSESDNNSSSRGDA (2-1)
NSESDNNSSSRGDA (2-2)
SRGDASYNSDESKD (3-1)
SRGDASYNSDESK (3-2)
DNNSSSRGDASYNSD (4)
[2]
A dentin regenerating agent containing at least one peptide according to [1].
[3]
The dentin regenerating agent according to [2], further comprising a recombinant body of porous hydroxyapatite or type I collagen.
[Four]
A bone regeneration agent containing at least one peptide according to [1].
[Five]
The bone regeneration agent according to [4], further comprising a recombinant body of porous hydroxyapatite or type I collagen.
[6]
A periodontal tissue regeneration agent containing at least one peptide according to [1].
[7]
The periodontal tissue regenerating agent according to [6], further comprising a recombinant body of porous hydroxyapatite or type I collagen.

  According to the present invention, a synthetic peptide comprising a phosphorin RGD sequence retaining hard tissue inducing activity is provided, and this peptide can be used as a new bone regeneration material with high quality and high safety. Furthermore, by using a recombinant body of type I collagen having biocompatibility as a carrier, it is possible to provide a bone regeneration agent, dentin regeneration agent, and periodontal tissue regeneration agent with improved bone regeneration ability.

The present invention relates to a peptide having any of the following amino acid sequences.
SESDNNSSSRGDASYNSDES (1-1) (SEQ ID NO: 1)
ESDNNSSSRGDASYNSDES (1-2) (SEQ ID NO: 2)
SDNNSSSRGDASYNSDES (1-3) (SEQ ID NO: 3)
SESDNNSSSRGDASYNSDE (1-4) (SEQ ID NO: 4)
SESDNNSSSRGDASYNSD (1-5) (SEQ ID NO: 5)
SESDNNSSSRGDASYNS (1-6) (SEQ ID NO: 6)
SESDNNSSSRGDASYN (1-7) (SEQ ID NO: 7)
ANSESDNNSSSRGDA (2-1) (SEQ ID NO: 8)
NSESDNNSSSRGDA (2-2) (SEQ ID NO: 9)
SRGDASYNSDESKD (3-1) (SEQ ID NO: 10)
SRGDASYNSDESK (3-2) (SEQ ID NO: 11)
DNNSSSRGDASYNSD (4) (SEQ ID NO: 12)

  The peptide of the present invention comprises the RGD sequence of dentin phosphophorin and the amino acids before and after it. The peptide sequence of only RGD shows no differentiation promoting action or mineralization promoting action into osteoblasts. The peptide having the amino acid sequence (1-1) has the highest activity, and the peptide having RGD and at least one amino acid on both the N-terminal side and the C-terminal side has high activity. RGD and its N-terminal peptide alone or RGD and its C-terminal peptide only show similar effects, but their activities are considerably reduced. In the peptide of the present invention, amino acids before and after RGD are considered to play an important role in addition to RGD. These peptides can be easily obtained by peptide synthesis by a conventional method.

  The present invention relates to a dentin regeneration agent, a bone regeneration agent, or a periodontal tissue regeneration agent containing at least one of the above peptides. The dentin regenerating agent, bone regenerating agent or periodontal tissue regenerating agent of the present invention contains one or more of the above peptides, and further comprises a porous hydroxyapatite or type I collagen recombinant as a carrier. Furthermore, it is preferable to include. Porous hydroxyapatite or type I collagen recombinant can be used at a mass ratio in the range of 100 to 200 with respect to 100 masses of peptide.

  The dentin regenerative agent, bone regenerative agent or periodontal tissue regenerative agent of the present invention acts by directly touching cells. Thus, it is possible to regenerate dentin and bone, and it is also effective for regenerating periodontal tissue.

  The dentin regenerative agent, bone regenerative agent or periodontal tissue regenerative agent of the present invention uses porous hydroxyapatite or type I collagen which is a sustained release system and is effective as a scaffold for cell differentiation as a carrier for pulp capping. Is preferred. For example, a complex can be prepared by placing 25 mg of carrier in a syringe and dropwise impregnating a peptide adjusted to 5 μg / 30 μl with physiological saline. The pulp covering material is used as a material for promoting restoration dentin formation when a very small range of non-infectious exposed pulp is produced, such as medullary exposure during cavity formation.

  In the peptides described in Patent Documents 4 to 6, only a peptide having two motifs of RGD of the substrate extracellular phosphoprotein and a glycosaminoglycan binding site is supposed to promote skeletal growth and increase in the number of odontoblasts. Peptides containing several amino acids before and after RGD have not found the same effect. In other words, these two motifs are necessary for the peptide, and the other amino acids before and after that are hardly important.

  On the other hand, in addition to the RGD sequence of dentin phosphophorin, the amino acids before and after the peptide of the present invention are involved in the expression of activity. The peptide sequence of only RGD shows no differentiation promoting action or calcification promoting action into osteoblasts. Therefore, although it is a peptide containing the same RGD sequence, it is speculated that there is a great difference in the mechanism of action between the peptide of the present invention and the peptides described in Patent Documents 4-6.

  Hereinafter, the present invention will be described in more detail with reference to examples.

  FIG. 1 shows the primary structure of human dentin phosphophorin and the amino acid sequence of the peptide. A peptide derived from human dentin phosphophorin was commissioned to Invitrogen. RGD-1, RGD-2, RGD-3, RGD-4, and RGD-5 are peptides that contain an RGD sequence and several amino acids adjacent to it at the N- and C-termini. SGXG-1 and SGXG-2 A peptide containing a glycosaminoglycan binding site. Furthermore, RGD and RAD peptide (Biomol) were used in the experiments as controls to clarify the role of the RGD sequence. Furthermore, RAD-1 in which RGD-1 and RGD sequences were changed to RAD sequences was also commissioned to Invitrogen for comparison.

Experiment 1
The effect of peptides on calcification ability of human mesenchymal stem cells was investigated. The results are shown in FIG. The method is as follows.
Human mesenchymal stem cells (BioWhittaker, Walkersville, MD) were cultured under conditions of 37 ° C. and 5% CO ₂ using human mesenchymal stem cell medium (BioWhittaker) supplemented with 10% FBS. After seeding 1 × 10 ⁵ cells in a 24-well plate, changing to serum-free medium 24 hours later and culturing for another 24 hours, the cells were 10% FBS, 50 μg / ml ascorbic acid, 10 mM β-glycerophosphate, 100 nM dexamethasone and 50 μM The cells were cultured for 28 days in a calcification induction medium containing peptides. The cells were fixed with 70% ethanol for 1 hour, washed twice with PBS, stained with 40 mM alizarin red staining solution (pH 4.2, Sigma) for 10 minutes, and washed 3 times with distilled water and PBS, respectively. The calcified nodule was observed with an optical microscope. RGD-1 most promoted calcification of human mesenchymal stem cells. RGD-2 and RGD-3 also promoted calcification but were less effective than RGD-1. SGXG-1 and -2 did not affect calcification.

Experiment 2
The effect of peptides on the proliferation ability of human mesenchymal stem cells was investigated. The results are shown in FIG. The method is as follows.
The proliferation of human mesenchymal stem cells was measured using Cell Counting Kit-8 (Dojindo). Cells were seeded in a 96-well plate at 1 × 10 ³ cells. After 24 hours, the cells were replaced with a serum-free medium and further cultured for 24 hours, and then the cells were cultured in media containing 1% FBS and 50 μM various peptides. 48 hours later, 10 μl of new tetrazolium salt WST-8 was added to each well and incubated for 30 minutes.The absorbance at 450 nm of water-soluble formazan produced by WST-8 reduction by intracellular dehydrogenase was measured using Microplate Reader Model 680 ( Bio-Rad). All peptides did not affect the proliferation of human mesenchymal stem cells.

Experiment 3
The effect of peptides on alkaline phosphatase activity of human mesenchymal stem cells was investigated. The results are shown in FIGS. 4-1 and 4-2. The method is as follows.
After seeding 1 × 10 ⁵ cells in a 24-well plate and replacing the serum-free medium 24 hours later and culturing for another 24 hours, the cells were treated with 10% FBS, 50 μg / ml ascorbic acid, 10 mM β-glycerophosphate, 100 nM dexamethasone and 5, The cells were cultured in a calcification induction medium containing 50 μM various peptides for 7 days, and the alkaline phosphatase (ALP) activity in the cell extract was measured using an ALP-Lab assay kit (WAKO). The ALP activity (International Unit: IU) was calculated as the activity (IU / mg) per total protein amount (mg) in each well measured by Bio-Rad Protein Assay (Bio-Rad). As shown in FIG. 4-1, RGD-1 significantly promoted ALP activity compared to the control, and concentration dependency was also observed. RGD-2 and RGD-3 also significantly enhanced ALP activity, but there was little difference due to concentration. Other SGXG and RGD control peptides had little effect on ALP activity. As shown in FIG. 4-2, RGD-4 significantly promoted ALP activity compared to the control, and concentration dependence was also observed. RGD-5 and RAD-1 also promoted ALP activity, but there was no significant difference from the control.

Experiment 4
The synergistic effect of peptides on alkaline phosphatase activity of human mesenchymal stem cells was investigated. The results are shown in FIGS. 5-1 and 5-2. The method is as follows.
Since Dentonin (Acologix) containing RGD and SGXG sequences has been reported to have an effect of promoting cell differentiation, the effect of the mixed peptide of RGD and SGXG was examined. In addition, a comparative test was performed on Dentonin (Acologix). However, the ALP activity promoting effect by the combination of RGD-1 and SGXG peptide was not recognized. Both RGD-1 and Dentonin significantly promoted ALP activity compared to the control at 5 μM, and RGD-1 was found to have almost the same activity promotion as Dentonin.

Experiment 5
The effects of RGD control peptide and integrin antibody on alkaline phosphatase activity of human mesenchymal stem cells were investigated. The results are shown in FIG. The method is as follows.
After seeding 1 × 10 ⁵ cells in a 24-well plate and replacing the serum-free medium 24 hours later and culturing for another 24 hours, the cells were 10% FBS, 50 μg / ml ascorbic acid, 10 mM β-glycerophosphate, 100 nM dexamethasone and 50 μMRGD or The cells were cultured in a mineralization induction medium containing RAD peptide, 25 μg / ml αvβ3 integrin antibody (Santacruz). After 1 hour, 50 μM RGD-1 was added and further cultured for 7 days, and ALP activity in the cell extract was measured. The pretreatment with RAD hardly affected the ALP activity, but the pretreatment with RGD or integrin antibody significantly suppressed the AGD activity promoting effect of RGD-1. From this result, it became clear that the RGD-1 cell differentiation promoting action is due to the binding of the RGD sequence to the integrin.

Experiment 6
The effects of peptides on osteocalcin production by human mesenchymal stem cells were investigated. The results are shown in FIG. The method is as follows.
After seeding 1x10 ⁵ cells in a 24-well plate and changing to serum-free medium for 24 hours and culturing for another 24 hours, the cells were treated with 10% FBS, 50 μg / ml ascorbic acid, 10 mM β-glycerophosphate, 100 nM dexamethasone and 50 μMRGD. It was cultured for 7 days in the calcification induction medium containing. Replace with serum-free medium (500 μl) containing 50 μg / ml ascorbic acid, 10 mM β-glycerophosphate, 100 nM dexamethasone and 50 μMRGD, and after culturing for 2 days, collect all cultures and use human osteocalcin ELISA kit (Biomedical technologies) Then, the amount of osteocalcin in the medium was measured. The treatment with RGD-1 significantly increased the amount of osteocalcin, about 3 times that of the control. In addition, the RGD-2 and RGD-3 treatments increased about twice. However, the SGXG peptide showed almost no difference from the control.

  The present invention provides materials useful in the field of dentistry.

FIG. 1 shows the primary structure of human dentin phosphophorin and the amino acid sequence of the peptide. The result of having investigated the influence of the peptide on the mineralization ability of a human mesenchymal stem cell is shown. The result of having investigated the influence of the peptide on the proliferation ability of a human mesenchymal stem cell is shown. The result of having investigated the influence of the peptide on the alkaline phosphatase activity of a human mesenchymal stem cell is shown. The result of having investigated the influence of the peptide on the alkaline phosphatase activity of a human mesenchymal stem cell is shown. The result of having investigated the synergistic effect of the peptide on the alkaline phosphatase activity of a human mesenchymal stem cell is shown. The result of having investigated the effect of Dentonin (Acologix) on the alkaline phosphatase activity of a human mesenchymal stem cell is shown. The result of having investigated the influence of the RGD control peptide and the integrin antibody on the alkaline phosphatase activity of a human mesenchymal stem cell is shown. The result of having investigated the influence of the peptide which has on osteocalcin production of a human mesenchymal stem cell is shown.

Claims (2)

A dentin regenerating agent containing at least one peptide consisting of any of the following amino acid sequences.
SESDNNSSSRGDASYNSDES (1-1)
ANSESDNNSSSRGDA (2-1)
SRGDASYNSDESKD (3-1) 2. The dentin regenerating agent according to claim 1, further comprising a recombinant body of porous hydroxyapatite or type I collagen.