BMP9 to be used in regenerating dental tissue or periodontal tissue

Abstract

The present invention provides a dental composition comprising BMP9 to be used in regenerating dental tissue or periodontal tissue, or a method for regenerating dental tissue or periodontal tissue, comprising administering BMP9 to a subject requiring the regeneration of dental tissue or periodontal tissue. The dental tissue can be dentin, and the periodontal tissue can be cementum, alveolar bone, or periodontal ligament. BMP9 according to the present invention is effective, as exhibited by experimental results, in promoting dentin regeneration and periodontal tissue regeneration for teeth.

Description

BMP9 for use in regenerating dental tissue or periodontal tissue

[0001] The present invention relates to dental uses of BMP9, and more particularly to the use of BMP9 in regenerating dental tissue or periodontal tissue. The dental tissue may be dentin, and the periodontal tissue may be cementum, alveolar bone, or periodontal ligament.

[0002] Bone morphogenetic protein (BMP) was first discovered as a substance that promotes bone growth in muscle. Since Dr. Urist reported in 1965 that BMP can induce bone growth in muscle, many subsequent studies have confirmed that BMP functions in various physiological and pathological processes. BMP is an important member of the TGF-β superfamily, a group of highly conserved signaling proteins that play important roles in embryogenesis, organogenesis, cell proliferation, and stem cell differentiation. See the literature [Zhu L et al., Frontiers in Bioengineering and Biotechnology 10 (2022): 810880].

[0003] Through repeated research by numerous researchers, more than 20 types of BMP proteins have been identified, and among them, BMP2, BMP4, and BMP7 are known to exhibit outstanding functions that can induce bone formation. Since the US FDA approved recombinant BMP2 and BMP7 for use in spinal fusion surgery, an orthopedic surgery, in 2001 and 2002, their applications have expanded steadily. BMP2 and BMP7 were also approved for use in dentistry in 2007.

[0004] BMP2 is primarily used in orthopedics for bone regeneration purposes, and while some applications in dentistry have been approved, its use is limited due to side effects. Since 2001, cases of edema, airway obstruction, and fatal side effects have been reported in clinical applications of BMP2, leading the FDA to issue a 2008 adverse event report regarding BMP2 use. Concerns about side effects persist in dentistry, hindering its widespread clinical use. Furthermore, BMP2 is water-soluble and has a short half-life in the body, necessitating frequent injections at high concentrations.

[0005] [Prior Art Literature]

[0006] (Non-patent literature)

[0007] (Non-patent Document 1) Zhu L et al., Frontiers in Bioengineering and Biotechnology 10 (2022): 810880

[0008] (Non-patent literature 2) Brown MA, et al., Journal of Biological Chemistry 280.26 (2005): 25111-25118

[0009] The periodontium is a complex organ comprising both soft and hard tissues, including the gingiva (gingiva), cementum, periodontal ligament, and alveolar bone. While the gingiva is a simple connective tissue that supports the teeth and participates in mastication, the periodontal ligament is a highly differentiated cellular connective tissue located between the teeth and the alveolar bone, attaching and supporting the teeth to the jaw bone. It resists mechanical stress from various directions, such as masticatory forces, and also has a sensory function. The periodontal ligament is composed of various cells, including osteoblasts and osteoclasts on the alveolar bone side; fibroblasts, epithelial cells of Malassez, macrophages, undifferentiated mesenchymal cells, neural elements, and vascular endothelial cells on the central side; and cementoblasts on the root side. These cells are known to be involved in the repair and regeneration of alveolar bone, the tooth root, and the cementum.

[0010] The gums are part of the tooth-supporting tissue that we can see inside the mouth, and this is where the disease (gingivitis) begins. As the disease worsens and spreads deeper into the supporting tissue, the periodontal ligament that connects the tooth root surface and the bone tissue surrounding the tooth is destroyed, and the alveolar bone surrounding the tooth is also destroyed, causing periodontal disease.

[0011] The incidence of periodontal disease increases with age. When periodontal disease is limited to the gums, it is called gingivitis. When this inflammation spreads to the gums and surrounding bone, it is called periodontitis. As periodontitis progresses, it damages the periodontal ligament and even the alveolar bone. An example of alveolar bone damage is alveolar bone osteodystrophy, which is further subdivided into alveolar bone osteoporosis, alveolar bone osteomalacia, and alveolar bone osteopenia. Damage to the alveolar bone ultimately leads to tooth loss.

[0012] Currently, periodontal disease is primarily treated with surgical methods such as non-surgical or surgical scaling, root planing, gingival curettage, and periodontal tissue regeneration. However, these surgical treatments are not only painful and cumbersome for patients, but are also applied only when the disease has progressed to a certain extent, rather than preventing it. In particular, true periodontal tissue regeneration requires the complete regeneration of all three components: alveolar bone, periodontal ligament, and cementum. However, the success rate in this field is very low with current medical technology. Therefore, there is a pressing need to develop treatment methods that achieve more complete periodontal tissue regeneration and restore normal tooth function.

[0013] The inventors of the present invention have revealed for the first time that BMP9 is effective in regenerating teeth and periodontal tissues (periodontal ligament and cementum).

[0014] Accordingly, the present invention provides a dental composition comprising BMP9 for regenerating dental tissue or periodontal tissue based on the excellent regenerative ability of BMP9 for dentin regeneration, alveolar bone regeneration, and periodontal tissue regeneration of teeth. The regeneration of the dental tissue or periodontal tissue involves the formation of new dentin or new cementum. That is, the dental composition comprising BMP9 according to the present invention is characterized by causing the formation of new dentin or new cementum. The dental tissue may be dentin, and the periodontal tissue may be cementum, alveolar bone, or periodontal ligament.

[0015] The present invention also provides a method for regenerating dental tissue or periodontal tissue, comprising administering BMP9 to a subject in need of regeneration of dental tissue or periodontal tissue. The regeneration of dental tissue or periodontal tissue involves the formation of new dentin or new cementum. That is, the method for regenerating dental tissue or periodontal tissue using BMP9 according to the present invention is characterized by causing the formation of new dentin or new cementum. The dental tissue may be dentin, and the periodontal tissue may be cementum, alveolar bone, or periodontal ligament.

[0016] The novel BMP9-based treatment according to the present invention offers stable and superior efficacy in regenerating dental and periodontal tissues, a level unmatched by existing BMP2-based treatments. Furthermore, it minimizes the side effects reported with BMP2. This novel treatment can maximize therapeutic efficacy in the dental field, shorten patient recovery times, and improve the quality of treatment.

[0017] The present invention provides a dental composition comprising BMP9 for use in regenerating dental tissue or periodontal tissue, or a method for regenerating dental tissue or periodontal tissue, comprising administering BMP9 to a subject in need of regeneration of dental tissue or periodontal tissue. The dental tissue may be dentin, and the periodontal tissue may be cementum, alveolar bone, or periodontal ligament. Experimental results show that BMP9 according to the present invention is effective in promoting regeneration of dental dentin and periodontal tissue. In particular, it induces the formation of new cementum accompanied by effective regeneration of Sharpie fibers, thereby enabling complete regeneration of periodontal tissue.

[0018] Figure 1 is an ALP expression graph showing that when osteoblasts are treated with BMP9, the expression of alkaline phosphatase (ALP), a differentiation marker of osteoblasts, increases.

[0019] Figure 2 shows the results of Western blotting showing that when odontoblasts were treated with BMP9, the phosphorylation of AKT, which is involved in intracellular signaling in odontoblasts, increased. "NT" indicates the untreated group.

[0020] Figure 3 is a qPCR graph showing that when cementoblasts were treated with BMP9, the expression of Runx2, Osterix (Osx), Collagen type 1 (Col1), and Cementum attachment protein (CAP), which are markers related to cementum differentiation, increased.

[0021] Figure 4 is a bar graph (Figure 4b) showing that bone density increases in a concentration-dependent manner when BMP9 is treated in a rabbit calvarial defect model (Figure 4a). "CS" stands for collagen sponge.

[0022] Figures 5 to 7 are Micro-CT 3D images (Figure 5), histological staining images (Figure 6), and bar graphs (Figure 7) showing that bone regeneration (new bone formation) occurred when BMP9 was treated in an adult dog implant placement model. "None" represents an untreated control group, and "Collagen Sponge" (or "CS") represents a control group treated only with a collagen sponge that did not absorb the test substance.

[0023] Figure 8 is a histological staining image of the soft tissue surrounding dentin when treated with BMP9 in an adult dog periodontal tissue regeneration model. "JE" shown in Figure 8 indicates the junctional epithelium region, "CT" indicates the connective tissue region, and "NB" indicates the new bone region, i.e., the newly formed alveolar bone region. The area outlined by the dotted line on the left indicates dentin.

[0024] Figures 9 and 10 are bar graphs showing changes in the thickness of the junctional epithelium, connective tissue, new bone, and cementum obtained in the same experiment, where Figure 9 shows changes in the junctional epithelium, connective tissue, and new bone, and Figure 10 shows changes in the cementum. In Figures 9 and 10, “NT” refers to the group in which no collagen sponge was inserted after the defect.

[0025] Figure 11 is a histological staining image at a magnification of more than 800 times of the junction area between dentin and surrounding soft tissue, similar to the boxed area in Figure 8, showing that Sharpey's fiber regeneration occurred effectively when BMP9 was treated. The area marked with an asterisk (*) indicates the end of the Sharpey's fiber, and it was confirmed that, unlike in other treatment groups, when BMP9 was treated, the Sharpey's fiber connecting the cementum and new bone was well formed as a horizontal and functional linear bundle structure.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In general, the terms used herein are those well known and commonly used in the art.

[0027] The present invention provides a dental composition comprising BMP9.

[0028] BMP9 is an abbreviation for bone morphogenetic protein 9, also known as GDF2. For the sequence and structure of BMP9, see [Brown MA, et al., Journal of Biological Chemistry 280.26 (2005): 25111-25118].

[0029] The BMP9 used in the present invention may have the following amino acid sequence or a conservative amino acid substitution thereof.

[0030] 서열번호 1

[0031] MAWVWTLLFLMAAAQSIQAKPLQSWGRGSAGGNAHSPLGVPGGGLPEHTFNLKMFLENVKVDFLRSLNLSGVPSQDKTRVEPPQYMIDLYNRYTSDKSTTPASNIVRSFSMEDAISITATEDFPFQKHILLFNISIPRHEQITRAELRLYVSCQNHVDPSHDLKGSVVIYDVLDGTDAWDSATETKTFLVSQDIQDEGWETLEVSSAVKRWVRSDSTKSKNKLEVTVESHRKGCDTLDISVPPGSRNLPFFVVFSNDHSSGTKETRLELREMISHEQESVLKKLSKDGSTEAGESSHEEDTDGHVAAGSTLARRKRSAGAGSHCQKTSLRVNFEDIGWDSWIIAPKEYEAYECKGGCFFPLADDVTPTKHAIVQTLVHLKFPTKVGKACCVPTKLSPISVLYKDDMGVPTLKYHYEGMSVAECGCR

[0032] The term "conservative amino acid substitution" refers to the replacement of an amino acid residue by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially change the functional properties of the protein. When two or more amino acid sequences differ from each other by conservative substitutions, the degree of sequence identity or similarity may be adjusted upward to compensate for the conservative nature of the substitution. Means for performing such adjustments are well known to those skilled in the art [see, e.g., Pearson (1994) Methods Mol. Biol. 24: 307-331]. Examples of groups of amino acids having side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartate and glutamate; and (7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conservative amino acid substitution groups are as follows: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative substitution can be any change having a positive value in the PAM250 log-likelihood matrix described in the literature [Gonnet GH, et al, Science 256.5062 (1992): 1443-1445].

[0033] The BMP9 used in the present invention preferably has a sequence identical to that of human BMP9 or is a conservative substitute thereof.

[0034] The BMP9 used in the present invention may be a protein derived from any naturally occurring organism, or may be a protein manufactured through artificial methods such as genetic recombination or artificial synthesis. Genetically recombinant BMP9 may be expressed and purified from bacteria such as Escherichia coli or eukaryotic cells. Examples of eukaryotic cells include, but are not limited to, yeast, fungi, insect cells, plant cells, or animal cells. For example, the BMP9 used in the present invention may be manufactured using a CHO cell line.

[0035] A dental composition comprising BMP9 according to the present invention can be used to regenerate dental tissue or periodontal tissue.

[0036] A dental composition comprising BMP9 according to the present invention may be used to regenerate dental tissue.

[0037] A dental composition comprising BMP9 according to the present invention may be used to treat a defect in tooth tissue.

[0038] The term “dental tissue” as used herein refers to the tissue that makes up the tooth, including enamel and dentin.

[0039] "Enamel" is the hard, grayish-white, translucent substance that covers the tooth crown and is the visible white part of the tooth.

[0040] Dentin is the hard, softer portion of the tooth, surrounding the pulp, which contains blood vessels and nerves. It is formed by odontoblasts, which develop from ectomesenchymal cells.

[0041] The anatomical location and meaning of the above dental tissues are well known to those skilled in the art.

[0042] The dental composition comprising BMP9 according to the present invention can be used to regenerate dentin among dental tissues. For example, the dental composition comprising BMP9 according to the present invention can be used to regenerate dentin after treatment of dental caries (cavities).

[0043] The dental composition comprising BMP9 according to the present invention can be used to treat dentin caries or defects in dental tissue. For example, the dental composition can be used to treat dental caries accompanied by dentin caries or defects.

[0044] A dental composition comprising BMP9 according to the present invention may be used to regenerate periodontal tissue.

[0045] A dental composition comprising BMP9 according to the present invention may be used to treat a defect in periodontal tissue. The defect in periodontal tissue may be caused as a result of periodontal disease.

[0046] Periodontal tissue regeneration using BMP9 according to the present invention may be part of periodontal disease treatment.

[0047] The term "periodontal tissue" as used herein refers to the tissues that make up the periodontium, including cementum, periodontal ligament, and alveolar bone.

[0048] Cementum is the hard tissue that covers the dentin of the tooth root. It serves to anchor the tooth to the alveolar bone. Periodontal disease can cause the periodontal ligament and cementum to degenerate, leading to tooth looseness. Cementum is formed by cementoblasts, cells derived from ectodermal mesenchymal cells.

[0049] The periodontal ligament is the connective tissue that surrounds the tooth root and connects the tooth to the alveolar bone. The most important component of the periodontal ligament is the main fiber, which is composed of bundles of collagen fibers. One end of this fiber is attached to the alveolar bone and the other end is embedded in the cementum. The portion of the main fiber attached to or embedded in the alveolar bone or cementum is called Sharpey's fiber. Periodontal tissue regeneration involves the synthesis of new alveolar bone to the height of the pre-damaged alveolar bone. Cement that has been necrotic and removed due to inflammation is replaced by healthy new cementum to re-cover the root surface. The periodontal ligament is newly synthesized between the regenerated alveolar bone and cementum, and Sharpey's fibers, which form the main fiber bundles, are inserted into the alveolar bone and cementum surfaces. In other words, the well-formed Sharpey's fibers embedded in the cementum indicate successful periodontal tissue regeneration.

[0050] The "periodontal ligament" is the connective tissue that supports the tooth between the cementum of the tooth and the alveolar bone.

[0051] "Alveolar bone" is the bone where the root of the tooth is located.

[0052] The anatomical location and meaning of the above periodontal tissues are well known to those skilled in the art.

[0053] A dental composition comprising BMP9 according to the present invention can be used to regenerate cementum in periodontal tissue. For example, a dental composition comprising BMP9 according to the present invention can be used to regenerate cementum after treatment of dental caries.

[0054] A dental composition comprising BMP9 according to the present invention can be used to treat cementum caries or defects in periodontal tissue. For example, the dental composition can be used to treat dental caries involving cementum caries or defects.

[0055] The dental composition comprising BMP9 according to the present invention can be used to regenerate periodontal ligament among periodontal tissues. For example, it can be used to treat periodontal diseases including periodontitis and gingivitis. The BMP9 according to the present invention can be particularly useful for regenerating a portion of the periodontal ligament connected or in contact with cementum. As used herein, the term "connected cementum and periodontal ligament" means that the cementum and periodontal ligament are connected via fibrous cells. It has been confirmed that the BMP9 according to the present invention is more effective than other proteins known to be useful for periodontal tissue regeneration in promoting the regeneration of Sharpie fibers that connect new periodontal ligament and cementum.

[0056] A dental composition comprising BMP9 according to the present invention can be used for periodontal tissue regeneration to treat periodontal ligament defects in periodontal tissue. BMP9 according to the present invention may be particularly useful for promoting regeneration of the periodontal ligament region connected to the cementum.

[0057] The dental composition comprising BMP9 according to the present invention can be used to regenerate alveolar bone among periodontal tissues. Examples of alveolar bone damage include alveolar osteogenesis disorders, which include alveolar osteoporosis, alveolar osteomalacia, and alveolar osteopenia. The dental composition according to the present invention can be used to regenerate alveolar bone damage accompanying alveolar osteogenesis disorders, such as alveolar osteoporosis, alveolar osteomalacia, and alveolar osteopenia. The BMP9 according to the present invention can be particularly useful for regenerating alveolar bone connected or in contact with cementum. As used herein, the connection between cementum and alveolar bone means that the cementum and alveolar bone are connected via fibrous cells such as Sharpie fibers. It has been confirmed that the BMP9 according to the present invention induces the regeneration of Sharpie fibers that connect new alveolar bone and cementum.

[0058] The dental composition comprising BMP9 according to the present invention can be used to treat alveolar bone defects in periodontal tissue. For example, the dental composition according to the present invention can be used to treat alveolar bone disorders accompanied by alveolar bone damage, such as alveolar osteoporosis, alveolar osteomalacia, or alveolar osteopenia. BMP9 according to the present invention may be particularly useful for promoting regeneration of the alveolar bone region connected to the cementum.

[0059] A dental composition comprising BMP9 according to the present invention can be used for the treatment of periodontal disease.

[0060] "Periodontal disease" refers to an inflammatory disease that occurs in the periodontal tissue. When periodontal disease is limited to the gums, it is called "gingivitis." When it progresses beyond the gums and extends to the alveolar bone below the gums, it is called "periodontitis." As inflammation progresses and damages more tissue, periodontal pockets form. The more severe the periodontitis, the deeper the periodontal pockets become. Deepening periodontal pockets are known to cause inflammation of the periodontal ligament, ultimately leading to bone loss. The fundamental treatment for periodontal disease is to restore damaged periodontal ligament connective tissue, cementum, and alveolar bone. This requires not only regeneration of the periodontal ligament supporting the alveolar bone, but also regeneration of the alveolar bone and cementum to which the periodontal ligament attaches. The inventors of the present invention have surprisingly discovered that BMP9, which has rarely been discussed for dental purposes in the past, is effective in regenerating the periodontal ligament, alveolar bone, and cementum.

[0061] The term "periodontal disease" as used in the present invention includes not only gingivitis and periodontitis, but also symptoms such as gingival pain, gingival bleeding, severe bad breath, gingival discoloration, tooth looseness, tooth movement, gingival recession, gingival edema, periodontal pocket formation, pericoronitis, and periodontal abscess.

[0062] In the present invention, the periodontal disease is not particularly limited as long as the dental composition according to the present invention exhibits a therapeutic effect, but may be, for example, gingivitis or periodontitis.

[0063] The dental composition comprising BMP9 according to the present invention can be used to regenerate periodontal tissue lost as a result of periodontal disease. The periodontal disease may be, for example, gingivitis or periodontitis, but is not limited thereto. The periodontal tissue may be, for example, cementum, alveolar bone, or periodontal ligament.

[0064] The term "regeneration" as used herein includes, but is not limited to, the growth, creation, or reconstruction of new cell types or tissues after BMP9 treatment according to the present invention. For example, it may be an increase in the volume or thickness (including but not limited to) of bone-forming cells (e.g., osteoblasts, osteocytes), connective tissues constituting dental tissue or periodontal tissue, cementum, etc., or an increase in the volume or thickness (including but not limited to) of new bone (including but not limited to) in dental tissue or periodontal tissue. Such "regeneration" can be confirmed through conventional techniques such as immunohistochemical staining of dental tissue or periodontal tissue, CT imaging, etc.

[0065] The dental composition comprising BMP9 according to the present invention may be in the form of a gel, powder, chip, sheet, block, soft or hard sponge, matrix, solution or membrane, alone or in combination with collagen, polymer or bone graft material (xenograft, allograft or allograft), but is not limited thereto. In this respect, the present invention provides a dental composition comprising BMP9 according to the present invention and further comprising collagen, polymer or bone graft material. The polymer may be a polymer scaffold. The bone graft material may be selected from the group consisting of xenograft, allograft and synthetic bone.

[0066] In another aspect, the present invention provides a dental implant comprising BMP9 according to the present invention, and further comprising collagen, a polymer, or a bone graft material, and having the form of a gel, powder, chip, sheet, block, soft or hard sponge, matrix, solution, or membrane. The polymer may be a polymer scaffold. The bone graft material may be selected from the group consisting of xenograft, allograft, and synthetic bone. The dental implant may be composed solely of BMP9, or may comprise BMP9 together with materials commonly used as materials for bone graft materials, such as a polymer, a bio-derived bone mineral powder, a calcium phosphate compound, apatite (including hydroxyapatite), collagen, and the like.

[0067] The term "bone graft" as used herein refers to a material for regenerating bone that has been absorbed or destroyed due to factors such as disease, trauma, or aging. Bone grafts can be classified into autogenous bone, allogenic bone, heterogeneous bone, and alloplastic bone grafts depending on their origin. Autogenous bone grafts utilize bone tissue harvested from the recipient. Allogeneic bone grafts utilize bone tissue harvested from a genetically identical species (e.g., bone from a deceased person). Xenogeneic bone grafts utilize bone tissue harvested from an individual of a different species. Synthetic bone utilizes artificially created bone tissue. Various components, including calcium phosphate ceramics, can be used as synthetic bone grafts, and those skilled in the art are well aware of the various materials that can be used as synthetic bone grafts.

[0068] The BMP9 according to the present invention, or the material (graft material) including a polymer, collagen, or bone graft material, may be used by coating the surface of an implant. In this regard, the present invention provides a dental implant coated with a dental composition comprising the BMP9 according to the present invention. The materials for the implant may be those described as usable in the art at the time of filing of the present application, and those skilled in the art of dental implant technology are well aware of which materials can be used to manufacture implants.

[0069] The present invention also provides a method for regenerating dental tissue or periodontal tissue, comprising delivering BMP9 to a subject in need of such regeneration. Furthermore, the present invention provides a method for treating a dental disease or periodontal disease involving tooth or periodontal defects using BMP9. The regeneration of dental tissue or periodontal tissue is as described herein. The subject may be an animal having teeth, and is preferably a human.

[0070] The present invention also provides a method for treating periodontal disease, comprising delivering BMP9 to a subject in need of such treatment. The term "periodontogenic disease" is as described herein. The subject may be an animal having teeth, and is preferably a human.

[0071] The term "treatment" as used herein refers to a therapeutic treatment aimed at reducing (alleviating) or eliminating a dental disease or periodontal disease involving tooth loss or periodontal loss as described herein.

[0072] The present invention may relate to (1) to (25) below based on the above-described contents, but is not limited thereto.

[0073] (1) A dental composition for use in regenerating tooth tissue or periodontal tissue containing BMP9, or a method for regenerating tooth tissue or periodontal tissue using the dental composition.

[0074] (2) A dental composition or method according to (1), wherein the BMP9 protein has the amino acid sequence of sequence number 1 or a conservative amino acid substitution thereof.

[0075] (3) A dental composition or method for causing the formation of new dentin or new cementum in (1) or (2).

[0076] (4) A dental composition or method for regenerating Sharpie fibers in any one of (1) to (3).

[0077] (5) A dental composition or method for use in regenerating tooth tissue according to any one of (1) to (4).

[0078] (6) (5), a dental composition or method in which the tooth tissue is dentin.

[0079] (7) (6) A dental composition or method for use in regenerating dentin after treatment of dental caries.

[0080] (8) A dental composition or method for use in regenerating periodontal tissue according to any one of (1) to (4).

[0081] (9) (8), a dental composition or method in which the periodontal tissue is cementum.

[0082] (10) (9), a dental composition or method in which the periodontal tissue is alveolar bone.

[0083] (11) In (10), the dental composition or method is an alveolar bone portion that is in contact with or connected to cementum.

[0084] (12) (8), a dental composition or method in which the periodontal tissue is periodontal ligament.

[0085] (13) In (12), the dental composition or method is a periodontal ligament portion that is in contact with or connected to cementum.

[0086] (14) A dental composition for use in the treatment of periodontal disease comprising BMP9 protein or a method for treating periodontal disease using the dental composition.

[0087] (15) (14) A dental composition or method wherein the BMP9 protein has the amino acid sequence of sequence number 1 or a conservative amino acid substitution thereof.

[0088] (16) A dental composition or method for causing the formation of new dentin or new cementum in (14) or (15).

[0089] (17) A dental composition or method for regenerating Sharpie fibers in any one of (14) to (16).

[0090] (18) A dental composition or method according to any one of (14) to (17), wherein the periodontal disease is gingivitis or periodontitis.

[0091] (19) A dental composition or method for regenerating periodontal tissue lost as a result of periodontal disease, wherein the BMP9 protein is in any one of (14) to (18).

[0092] (20) (19), a dental composition or method in which the periodontal tissue is cementum.

[0093] (21) (19), a dental composition or method in which the periodontal tissue is alveolar bone.

[0094] (22) In (21), the dental composition or method is an alveolar bone portion in contact with or connected to cementum.

[0095] (23) (19), a dental composition or method in which the periodontal tissue is periodontal ligament.

[0096] (24) In (23), the dental composition or method is a periodontal ligament portion in contact with or connected to cementum.

[0097] (25) A dental composition or method according to any one of (1) to (24), wherein the dental composition further comprises a bone graft material, polymer, or collagen selected from the group consisting of xenograft bone, allograft bone, and synthetic bone.

[0098] (26) A dental implant comprising a dental composition as described in any one of (1) to (25), and having the form of a gel, powder, chip, sheet, block, soft or hard sponge, matrix, solution or membrane.

[0099] (27) A dental implant coated with a dental composition as described in any one of (1) to (25).

[0100] Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended only to illustrate the present invention and the present invention is not limited thereto.

[0101] Example 1: Confirmation of the osteoblast differentiation ability of BMP9

[0102] To evaluate the effect of BMP9 on osteoblast differentiation, an ALP activity test was performed. Specifically, osteoblasts (MC3t3-E1, ATCC, CRL-2593) were cultured in osteogenic differentiation medium (LIFELINE, LM-0023) and treated with BMP2 (Peprotech, 120-02) and BMP9, respectively. BMP9 was manufactured in-house by Naibec Co., Ltd. using the CHO DG44 cell line. Specifically, to manufacture BMP9 protein, RCB cells were cultured for 2 weeks in a bioreactor (Sartorius), and the protein was purified from the culture medium using FPLC (Fast protein liquid chromatography, Cytiva) equipment. The BMP9 has the amino acid sequence shown in SEQ ID NO: 1. The protein concentrations for all treatment groups were set to 1 ng / ml, 10 ng / ml, and 100 ng / ml, and treatment was performed every other day for 5 days. The vehicle test group was treated with the same amount of PBS (Welgene, LB004). ALP staining was performed on cultured osteoblasts on the 5th day using an ALP staining kit (Thermo, EEA002). The stained ALP was dissolved in DMSO (Sigma, D8418) and the absorbance was confirmed at a wavelength of 405 nm.

[0103] As a result, as can be seen in the graph in Figure 1, it was confirmed that the ALP expression level increased in a concentration-dependent manner in the BMP9-treated group. In addition, it was confirmed that the ALP expression level was higher in the BMP9-treated group than in the BMP2-treated group at the same concentration. BMP2 is a substance that has already been reported to induce osteoblast differentiation. The results of this experiment show that BMP9 has the effect of promoting osteoblast differentiation and that its osteoblast differentiation ability is superior to that of BMP2.

[0104] Example 2: Confirmation of the ability of BMP9 to activate odontoblast signaling.

[0105] To evaluate the effect of BMP9 on signal transduction in odontoblasts, the phosphorylation level of AKT was determined. Specifically, odontoblasts (MDPC-23) were cultured in MEM alpha medium (Welgene, LM008-01) and treated with BMP9 as described in Example 1. For all treatment groups, cells were harvested 10 minutes, 1 hour, and 5 hours after treatment, and the phosphorylation level of AKT was analyzed by Western blotting.

[0106] As a result, as shown in Figure 2, it was confirmed that the level of p-AKT increased in odontoblasts when treated with BMP9. It is known that when cells are treated with the BMP2 protein, which is known to induce osteogenic differentiation of odontoblasts, AKT is phosphorylated and the signaling pathway that causes osteogenic differentiation is activated. The above experimental results demonstrate that BMP9 can induce osteogenic differentiation of odontoblasts and contribute to dentin production, and that its effect is superior to that of BMP2.

[0107] Example 3: Confirmation of the differentiation potential of BMP9 into cementum cells

[0108] To evaluate the effect of BMP9 on the differentiation of cementum cells, qPCR was performed to determine the mRNA expression levels of Runx2, Osterix (Osx), Collagen type 1 (Col1), and Cementum Adhesion Protein (CAP), which are markers related to cementum differentiation. Specifically, cementum cells (OCCM-30, Thermo) were treated with BMP2 and BMP9, used in Example 1, at concentrations of 100 ng / ml each. Twenty-four hours after treatment, cDNA was obtained, and qPCR was performed using primers with the following sequences.

[0109]

[0110] As a result, as can be seen in Fig. 3, it was confirmed that the mRNA expression levels of Runx2, Osterix, collagen type 1, and cementum attachment protein all increased in the groups treated with BMP2 or BMP9 compared to the untreated group. In particular, the mRNA expression levels were higher in the BMP9 treated group than in the BMP2 treated group, indicating that BMP9 has a superior ability to differentiate cementoblasts and produce cementum compared to BMP2.

[0111] Example 4: Confirmation of the bone regeneration ability of BMP9 in a rabbit calvarial defect model.

[0112] The bone regeneration potential of BMP9 was evaluated using a rabbit calvarial defect model. Specifically, a defect measuring 8 mm in diameter was created in the rabbit calvarial region. A collagen sponge was used as a scaffold, and liquid BMP2 or BMP9 was absorbed into the scaffold at doses of 10 μg / defect and 20 μg / defect, respectively. The protein concentrations used were set at 10 μg / site and 20 μg / site, respectively. The test substances were applied to the defect site, and the experimental animals were sacrificed at 4 weeks, and bone regeneration potential was evaluated. Bone regeneration potential was assessed by quantifying bone mineral density.

[0113] As a result, as shown in Figure 4, bone regeneration was observed in a concentration-independent manner in the BMP2 treatment group, whereas bone regeneration was observed in a concentration-dependent manner in the BMP9 treatment group. In particular, more effective bone regeneration was observed at higher concentrations. Meanwhile, the degree of ectopic bone formation was lower in BMP9 treatment than in BMP2 treatment. Ectopic bone formation is a side effect to be considered when using BMP proteins for therapeutic purposes.

[0114] Example 5: Evaluation of Bone Regeneration Potential of BMP9 in an Adult Dog Implant Placement Model

[0115] The bone regeneration ability of BMP9 was evaluated using an implant placement model in an adult dog. Specifically, P2, P3, and P4 teeth were extracted from the mandibular premolar region of a Beagle dog, and a healing period of 1 month was allowed. Then, a defect measuring 10 mm x 3 mm x 4 mm was created, and a Bone Level Tapered Implant (ø 3.3 x 8.0 mm; BLT SLActive®, Roxolid®) was implanted into the defect. After implantation, a collagen sponge absorbed with liquid BMP2 and BMP9 at a dose of 150 μg / site was placed in the remaining defect. After a 4-week healing period, the degree of new bone regeneration was evaluated by 3D imaging using Micro-CT (Skyscan).

[0116] As a result, as can be seen in Figures 5 and 6, both the BMP9 treatment group and the BMP2 treatment group showed significant regeneration of new bone (alveolar bone) compared to the control group. In Figure 5, the increased thickness of the tissue on both sides of the defect compared to the control group indicates that new bone was regenerated. As can be seen in Figure 7, the bone density and bone volume / tissue volume ratio (BV / TV) were highest in the BMP9 treatment group and showed similar levels to BMP2.

[0117] Example 6: Evaluation of periodontal tissue regeneration by BMP9 in an adult dog periodontal tissue regeneration model.

[0118] The periodontal tissue regeneration ability of BMP9 was evaluated using a periodontal tissue regeneration model of adult dogs. Specifically, the mandibular premolar P3 of a Beagle dog was extracted, and after a 4-week healing period, a defect was created by artificially removing the cementum, periodontal ligament, and part of the alveolar bone in the distal part of the mandibular P2 and the mesial part of the P4. (1) Collagen sponge, (2) collagen sponge absorbed with PDGF, (3) collagen sponge absorbed with BMP2, and (4) collagen sponge absorbed with BMP9 were injected into the defect site, and after 6 weeks of observation, the animals were sacrificed, and mandibular tissue was extracted and periodontal tissue regeneration was confirmed through immunohistochemical staining (H&E) (Fig. 8). PDGF (GEM21S (Lynch Biologics)) is a substance conventionally used for periodontal tissue regeneration and was used as a comparative example.

[0119] As a result, as can be seen in Figures 9 and 10, it was confirmed that the connective tissue around the dentin was stably adhered and new bone was formed in the BMP9 treatment group. Because the connective tissue and new bone were regenerated, it was confirmed that the length of the junctional epithelium was shorter than that of the control group. Specifically, when BMP9 was treated, the thickness of the connective tissue was equivalent to that when BMP2 or PDGF was treated, and in particular, the degree of new bone formation was superior to that of the BMP2 treatment group or the PDGF treatment group. Above all, when BMP9 was treated, the surface area of ​​the newly regenerated cementum and the number of cementum cells per cementum area were significantly higher than those of the BMP2 treatment group or the PDGF treatment group.

[0120] In addition, it was confirmed that the regeneration of Sharpie fibers connecting the newly regenerated cementum and new bone was significantly observed in the BMP9 treatment group. The part marked with an asterisk (*) in Fig. 11 indicates the end of the Sharpie fibers, and unlike in other treatment groups, it was confirmed that when BMP9 was treated, the Sharpie fibers connecting the cementum and new bone were well formed as a horizontal and functional linear bundle structure. Although Sharpie fibers were observed in the collagen treatment group, PDGF treatment group, and BMP2 treatment group, they did not show a horizontal and functional linear structure compared to the BMP9 treatment group, and were arranged irregularly or vertically.

[0121] This example demonstrates that BMP9 is effective in promoting the regeneration of periodontal tissue (specifically, cementum, periodontal ligament, and alveolar bone) in a periodontal tissue regeneration model of an adult dog, and in particular, can promote the formation of new cementum.

Claims

1. A dental composition for use in regenerating tooth tissue or periodontal tissue containing BMP9.

2. A dental composition according to claim 1, wherein the BMP9 protein has the amino acid sequence of sequence number 1 or a conservative amino acid substitution thereof.

3. A dental composition for regenerating Sharpie fibers according to claim 1 or 2.

4. A dental composition for use in regenerating tooth tissue according to claim 1 or 2.

5. A dental composition in claim 4, wherein the tooth tissue is dentin.

6. A dental composition for use in regenerating dentin after treatment of dental caries, according to claim 5.

7. A dental composition for use in regenerating periodontal tissue according to claim 1 or 2.

8. A dental composition in which the periodontal tissue is cementum in the 7th paragraph.

9. A dental composition in paragraph 7, wherein the periodontal tissue is alveolar bone.

10. A dental composition according to claim 9, wherein the alveolar bone is an alveolar bone portion in contact with or connected to cementum.

11. A dental composition according to claim 7, wherein the periodontal tissue is periodontal ligament.

12. A dental composition according to claim 11, wherein the periodontal ligament is a periodontal ligament portion in contact with or connected to cementum.

13. A dental composition for use in the treatment of periodontal disease comprising BMP9 protein.

14. A dental composition according to claim 13, wherein the BMP9 protein has the amino acid sequence of sequence number 1 or a conservative amino acid substitution thereof.

15. A dental composition for regenerating Sharpie fibers according to claim 13 or 14.

16. A dental composition according to claim 13 or 14, wherein the periodontal disease is gingivitis or periodontitis.

17. A dental composition according to claim 13 or 14, wherein the BMP9 protein regenerates periodontal tissue lost as a result of periodontal disease.

18. A dental composition according to claim 17, wherein the periodontal tissue is cementum.

19. A dental composition in claim 17, wherein the periodontal tissue is alveolar bone.

20. A dental composition in claim 19, wherein the alveolar bone is an alveolar bone portion in contact with or connected to cementum.

21. A dental composition according to claim 17, wherein the periodontal tissue is periodontal ligament.

22. A dental composition according to claim 20, wherein the periodontal ligament is a periodontal ligament portion in contact with or connected to cementum.

23. A dental composition according to any one of claims 1 to 21, further comprising a bone graft material, polymer, or collagen selected from the group consisting of xenograft bone, allograft bone, and synthetic bone.

24. A dental implant comprising a dental composition as described in any one of claims 1 to 22, and having the form of a gel, powder, chip, sheet, block, soft or hard sponge, matrix, solution or membrane.

25. A dental implant coated with a dental composition according to any one of claims 1 to 22.

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