WO2019143070A1 - Use of cpne7 in regeneration of periodontal ligament - Google Patents

Abstract

The present application discloses a periodontal ligament regenerating composition comprising CPNE7 and a periodontal ligament regenerating method. A composition and a method according to the present application can regenerate the periodontal ligament by expediting the formation of cementum and periodontal ligament through the promotion of differentiation and calcification of periodontal ligament fibroblasts into cementoblasts. The composition and the method according to the present application can promote the attachment of the cementum and alveolar bone of the newly formed periodontal ligament and repress tooth ankylosis, which induces the atrophy of tooth tissues, thus finding useful applications in the fundamental treatment of disorders that requires the restoration of damaged connective tissues, such as periodontal disorders and luxation injuries of teeth.

Description

CPNE7 Periodontal Ligament Recycling Purpose

The present invention relates to periodontal ligament regeneration technology using CPNE7.

Periodontium is a complex organ composed of epithelium, soft connective tissue and calcified connective tissue. It is structurally composed of gingiva, periodontal ligament (PDL), cementum and alveolar bone.

The double periodontal ligament is a special connective tissue that is located between the cortex and the alveolar bone covering the root with special bonding soft tissue. The bundle of periodontal ligament fibers runs between the teeth and the bone. Both ends of the fiber bundle are embedded in the cementum or alveolar bone. Not only does it function important to maintain and maintain the structural properties of the hard tissue, but another important function is to act as a sensory receptor. These periodontal ligaments contain a variety of mesenchymal cells. These cells form not only periodontal ligaments but also osteoblasts or cementoblasts that can form adjacent alveolar bone and cementum in vivo by appropriate stimulation And is known to be involved in the formation of alveolar bone and cementum.

Periodontal disease refers to the inflammatory disease that occurs in the gingiva, the periodontal ligament, and the alveolar bone, which are the surrounding tissues of the teeth that maintain teeth. Gingivitis and periodontal disease are known to cause a discomfort that significantly deteriorates the quality of life, and it is difficult to maintain proper cleansing of the oral cavity, thereby causing plaque deposition, thereby causing dental caries and periodontal disease. The ultimate result of treating periodontal disease is the restoration of injured periodontal ligament connective tissue, cementum and alveolar bone. For this, it is necessary not only to regenerate periodontal ligament supporting alveolar bone, but also to treat alveolar bone and cementum Playback is required.

When an external force is applied to a tooth or periodontal tissue, tooth and periodontal tissue may be destroyed due to the difference in direction and size. Tooth trauma can be divided into fracture trauma and degenerative trauma. Deformed trauma refers to injury to the periodontal ligament and can be divided into shaking, subluxation, delusional dislocation, lateral dislocation, complete dislocation, and penetration. It can occur in all age groups, but especially in children between 8 and 12 years . The success of this completely dislocated tooth depends on the regeneration of the periodontal ligament attached to the fully dislocated tooth or the remaining periodontal ligament in the missing tooth. If regeneration of the periodontal ligament does not occur, absorption of the cementum and dentin of the tooth root takes place, resulting in ankylosis, which is replaced by alveolar bone.

The ultimate result of treating periodontal disease or degenerated foreign teeth is to restore damaged connective tissues or bones. For this, regenerating or differentiating periodontal ligaments supporting bone is essential, but until now, periodontal ligament No methods have been reported to directly regenerate or differentiate.

Korean Patent Laid-Open Publication No. 2015-0145470 relates to a method, a composition, and a composition for the differentiation of a marine mesenchymal stem cell comprising a CPNE7 protein into an epithelial cell, and a pharmaceutical composition for regenerating a dermal tissue regeneration and dentin hypersensitivity using the same. The dentin cells are regularly arranged adjacent to the dentin in the dimension, and the dentin matrix contains dentin protuberances. In this case, CPNE7 protein interacts with nucleolin, a receptor on the cell surface of undifferentiated mesenchymal stem cells present in the dimension, to increase the expression of DSPP gene, which is a marker of dAb differentiation. As a result, mesenchymal stem cells Differentiate and form dentin, and the regeneration of the periodontal ligament is irrelevant as described below.

The present invention provides a technique which can fundamentally treat the periodontal ligaments due to periodontal disease by implantation or regeneration of the periodontal ligaments, by implanting and regenerating the traumatized teeth.

In one aspect, the present invention provides a composition or method for regenerating periodontal ligament comprising a CPNE7 protein or gene. Periodontal ligament regeneration in compositions according to the present invention involves the formation of cementum through the promotion of differentiation of periodontal ligament fibroblasts into cementoblasts and the attachment of the regenerated periodontal ligaments to the cementum.

In another embodiment, the use of CPNE7 protein or gene for periodontal ligament regeneration is provided.

In one embodiment, the composition, method, or use according to the present application may be used for the treatment of periodontal disease or degenerated foreign teeth comprising gingivitis and periodontitis, or prevention of tooth-osseointegration by the degenerated trauma, It does not.

In one embodiment, in compositions, methods, or uses according to the present invention, the formation of cementum is due to the promotion of cementum attachment protein (CAP) expression in the cementoblast from said cementoblast and said cementoblast by said CPNE7, It does not.

In one embodiment, the composition, method, or use according to the present application may be administered to, but is not limited to, periodontal ligament or alveolar bone requiring periodontal ligament regeneration.

In another aspect, the present invention also provides compositions, methods, or uses for the differentiation of periodontal ligament fibroblasts into cervical cells comprising a CPNE7 protein or gene.

In another embodiment, the present invention provides a method of promoting the differentiation of periodontal ligament fibroblasts into cementoblasts, comprising the step of treating CPNE7 protein or gene in periodontal ligament fibroblasts in an invivo or an invitro including human or animal do. In one embodiment, it is treated in an animal or in vitro.

In another embodiment, the present invention provides a cell therapy agent for regenerating periodontal ligament, comprising the periodontal ligament fibroblast overexpressing CPNE7, or a cementoblast or white cell differentiated from the fibroblast, as an active ingredient, or a cell therapy agent for regenerating periodontal ligament Comprising administering to the subject a therapeutically effective amount of a periodontal ligament.

In another aspect, the invention provides a method of regenerating periodontal ligament in a subject comprising administering to a subject in need thereof a therapeutically effective amount of a CPNE7 protein or gene, or a periodontal ligament comprising the protein or gene.

Regeneration of periodontal ligaments in the method according to the present invention involves the formation of cementum by promoting the differentiation of periodontal ligament fibroblasts into cementoblasts and the attachment of the regenerated periodontal ligaments to the cementum.

The CPNE7-containing composition and method for regenerating periodontal ligament of the present invention are useful for promoting regeneration and adhesion of periodontal ligaments and for reusing periodontal tissue after surgical treatment of dislodged teeth and periodontal disease .

Particularly, according to the composition and method of the present invention, the formation of periodontal ligaments and the formation of cementum to which newly formed periodontal ligaments can be attached are simultaneously promoted, and dental ankylosis, which induces degeneration of tooth tissues, And can be usefully used for the fundamental treatment of diseases requiring restoration of damaged connective tissues such as periodontal disease and degenerated traumatic teeth.

The composition and method according to the present invention can also induce attachment of the formed periodontal ligament to the cementum or alveolar bone by inducing CAP (Cementum attachment protein) gene expression, thereby treating and preventing periodontal disease or fundamentally effectively treating the disorganized traumatic tooth .

1 shows the mRNA levels of (A) OC (Osteocalcin), (B) Bsp (Bone sialoprotein) and (C) Cap (cementum attachment protein) genes expressed in CW cells according to treatment with CPNE7 recombinant protein . This shows that the CPNE7 recombinant protein can promote the differentiation of cementoblasts.

FIG. 2 shows the effect of transfection of the Cpne7 gene overexpressing vector on the cell cycle of cementobacterium and the influence of the mRNA level of Cap gene, a marker of cementoblast differentiation, on the level of mRNA. (A) shows the results of transfection of the Cpne7 overexpressing gene into the cervical cell line, and (B) shows the change in Cap mRNA level due to Cpne7 overexpression. These results indicate that Cpne7 not only promotes cementoblast differentiation but also significantly promotes the expression of CAP gene, which plays an important role in promoting adhesion of periodontal ligament fibers to cementum.

FIG. 3 shows the effect of CPNE7 recombinant protein on (a) CAP and (b) CP23 expression in the dental follicle cells (DF) of cementoblast. These results suggest that CPNE7 can promote the differentiation of dentigerous cells into cementoblast-like cells that are easy to adhere to periodontal ligaments. .

FIG. 4 shows the results of immunohistochemical staining for the location of Cpne7 protein expression in the molar tissue of the 2 week old mouse. AB means alveolar bone, D means dentin, PDL means periodontal ligament, and P means dimension. Size bar 200 um. This indicates that Cpne7 protein is strongly expressed in the periodontal ligament bundles, suggesting that Cpne7 may play a role in the formation of periodontal ligaments and adhesion of periodontal ligaments.

FIG. 5 is a photograph of a tissue stained with hematoxylin / enosine of teeth implanted at 4 weeks, where A and B represent positive control, C and D represent negative control, and E and D represents the experimental group treated with CPNE7 recombinant protein, AB represents alveolar bone, De represents dentin, CE represents cementum, NPD represents newly formed periodontal ligament-like tissue, NCE represents newly formed cementum Refers to similar tissues, PC refers to a dental steel, and BV means blood vessels. B, D, and F are enlarged photographs of boxes A, C, and E. Arrows D indicate Ankylosis. This indicates that the CPNE7 recombinant protein can promote the adhesion and regeneration of periodontal ligament without osseointegration.

FIG. 6 is a photograph of a tissue stained with hematoxylin / eosin of a tooth implanted at 8 weeks, in which A and B represent positive control, C and D represent negative control, and E and D CE represents cementum, NPD represents newly formed periodontal ligament-like tissue, NCE represents newly formed cortical similarity, CP represents a dentate nucleus, Tissue, PC refers to the dimension river, and BV means blood vessel. B, D, and F are enlarged photographs of boxes A, C, and E. Arrows D indicate Ankylosis. This result shows that CPNE7 recombinant protein can promote the attachment and regeneration of periodontal ligaments and the formation of existing cementum without osseointegration.

FIG. 7 is a graph showing specific findings in the control region of the experimental group treated with CPNE7 recombinant protein. FIG. A and B are the results for the 4th week, and C, D and E are the results for the 8th week. B, D, and E are enlarged photographs of boxes in A and C. The arrows indicate the presence of a newly formed periodontal ligament bundle in newly formed neocortical cortex, suggesting that CPNE7 recombinant protein can promote cement formation and adhesion and regeneration of periodontal ligaments .

Figure 8 shows that at 8 weeks after implantation, newly formed periodontal ligament-like tissues contained in the root of the experimental group treated with positive control, negative control or CPNE7 recombinant protein, tooth-osseointegration with alternative absorption and newly formed cementum , Indicating that CPNE7 plays an important role in promoting adhesion and regeneration of periodontal ligaments and promoting cement formation.

Fig. 9 schematically shows the structure of teeth including periodontal ligament, cementum, and the like.

The present invention is based on the discovery that CPNE7 promotes regeneration and attachment of periodontal ligaments and can regenerate damaged connective tissue of periodontal disease or degenerated foreign teeth.

The subject matter used in this section is for convenience only and should not be construed as limiting the invention. Unless otherwise defined herein, the scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

Justice

The term " CPNE7 protein " refers to one of the copine families, a calcium-dependent membrane binding protein, encoded by the CPNE7 gene. The protein contains two N-terminal C2 domains and one von Willebrand factor A domain. In order to achieve the object of the present invention, various origins and / or forms of CPNE7 gene and / or protein may be used as long as the effect of the present invention is attained. A gene in which a part of the base sequence is artificially modified so as to favor characteristics such as expression in a cell or stability of the protein as well as a wild type sequence and a gene in which a part of the naturally found base sequence is modified or a fragment thereof . Modifications of the gene sequence may or may not involve modification of the corresponding amino acid, and in the case of accompanied by modification of the amino acid, the gene in which such modification occurs is one in which at least one amino acid in the encoded protein is substituted, deleted, And / or an inserted amino acid sequence, which comprises mutants, derivatives, alleles, variants and homologues thereof. In the case where the mutation of the gene sequence does not involve the modification of the amino acid in the protein, for example, there is a mutation in the axis, and such degeneracy mutants are also included in the gene of the present invention.

Modifications of artificial gene sequences can be performed using methods well known to those skilled in the art, for example, site directed mutagenesis (Kramer et al, 1987), error induced PCR (Cadwell, RC and GF Joyce. , 2: 28-33.), Point mutation method (Sambrook and Russel, Molecular Cloning: A Laboratory Manual, 3rd Ed. 2001, Cold Spring Harbor Laboratory Press). Proteins used herein can be prepared using methods known in the art. In one embodiment, the method of producing a protein is by using a gene recombination technique. For example, a vector containing the corresponding gene encoding the protein may be transferred to a prokaryotic or eukaryotic cell such as an insect cell or a mammalian cell for expression, followed by purification. The plasmid can be used, for example, by cloning the gene into an expression vector such as pET28b (Novagen), transferring the gene to the cell line, and then purifying the expressed protein, but is not limited thereto. The synthesized protein was purified by column chromatography including precipitation, dialysis, ion exchange chromatography, gel-permeation chromatography, HPLC, reversed-phase HPLC, SDS-PAGE for free, and affinity column using anti-screening protein antibody Can be separated and purified.

In one embodiment according to the present invention, the CPNE7 gene and protein are derived from mammals such as apes, humans and the like, in particular from humans. The CPNE7 gene and protein may be used in its entirety or as long as it achieves the effect of the present invention. The full-length sequence may be, for example, NM170684 (amino acid), NP733785 (nucleic acid) in the case of a mouse with the GenBank accession number, NM014427 (Amino acid), NP055242 (nucleic acid), but is not limited thereto.

In one embodiment according to the present invention, the gene described above may be provided in the form of a vector operably linked to a promoter such that expression of the gene in the cell in which the composition of the present invention is used.

As used herein, the term " periodontium " is a complex organ composed of epithelium, soft connective tissue and calcified connective tissue, and is structurally composed of gingiva, periodontal ligament (PDL), cementum, And an alveolar bone.

The term " periodontal ligament (PDL) " as used herein refers to a connective tissue fibrous membrane, which is also called rhizome, and binds the cortex and alveolar bone wall of the root in mammals. The periodontal ligament is composed of a main fiber which is a collagen fiber extending parallel or obliquely with respect to a longitudinal axis of the tooth, and a bundle of Sharpey fibers having both ends are continuously embedded in a hard tissue. The periodontal ligament, Respectively. The periodontal ligament not only buffers the pressure generated when chewing food, but also is rich in blood vessels and nerves, and is known to be involved in nutrition or sensation. The periodontal ligament contains various cells such as fibroblasts, undifferentiated mesenchymal cells, and epithelial cells. The periodontal ligament fibroblasts can differentiate into osteoblasts or cementoblasts by appropriate stimulation. However, periodontal ligament fibroblasts can be differentiated into cementoblasts of periodontal ligament fibroblasts The differentiation mechanism and the differentiation promoting protein are not known. Bone sialoprotein (BSP), osteocalcin (OC), and cementum attachment protein (CAP) are known to be the markers of cementoblast differentiation. CAP is known to play an important role in adhesion of periodontal ligament fibers to cementum. The CPNE7 protein according to the present invention promotes the regeneration of periodontal ligaments by increasing the expression of CAP in periodontal ligaments and allowing the periodontal ligaments to adhere well to the cementum.

These periodontal ligaments are distinguished from the ones whose dimensions are located inside the teeth, which means connective tissues including dentin-forming dentin cells and nerve vessels, whereas periodontal ligaments connect the teeth and jaws (alveolar bone) It is an organization to connect, completely differentiated in terms of location, organization and function.

The term " cementum " as used herein is a calcified tissue covering the tooth root (root) of a mammal. The cementum fixes the tooth to the alveolar bone by fixing the periodontal ligament. Therefore, when germs are infected with gums, dense cement around the teeth occurs, and the denatured cementaceous teeth are shaken because the bundles of periodontal ligament fibers, which connect the teeth to the alveolar bone, do not stick together. The treatment of these denatured cementum requires the formation of new cementum.

As used herein, " periodontal ligament regeneration " includes both the formation of cementum and the formation of periodontal ligament, whereby the periodontal ligament formed is attached to the cementum.

As used herein, the term " periodontal ligament fibroblast " is a major cellular constituent of soft tissue connective tissue of periodontal tissue with gingival fibroblast. The periodontal ligament fibroblasts are not only known to form periodontal ligaments but also to repair and regenerate adjacent alveolar bone and cementum in vivo, but there is no known substance capable of promoting them, Gingival fibroblasts are involved. The periodontal ligament fibroblasts can differentiate into osteoblasts or cementoblasts by appropriate stimulation, but the mechanism of differentiating periodontal ligament fibroblasts into cementoblasts is not clearly known. Bone sialoprotein (BSP), osteocalcin (OC), and cementum attachment protein (CAP) are known to be the markers of cementoblast differentiation. CAP is known to play an important role in adhesion of periodontal ligament fibers to cementum. In this study, we demonstrated that CPNE7 protein promotes regeneration of periodontal ligaments by increasing CAP expression in periodontal ligaments and allowing periodontal ligaments to adhere well to cementum.

Therefore, the substances promoting the formation of cementooblasts and cementum in the periodontal ligament fibroblast can be effectively used for the treatment of periodontal disease.

As used herein, " Cementoblast " is a cementogenic cell present in the root. The CPNE7 protein according to the present invention can promote the differentiation into cementoblasts present in periodontal tissue and the formation of cementum.

As used herein, " promoting differentiation " means that cells that have already been differentiated into cementoblasts (differentiation into cementoblasts even without CPNE7 treatment) are able to differentiate into cementoblasts more rapidly, . In other words, induction of differentiation means that undifferentiated stem cells capable of differentiating into various cells are differentiated into cementoblasts by appropriate stimulation.

The term " periodontal disease " as used herein refers to an inflammatory disease that occurs in the gingival tissues surrounding the teeth, the periodontal ligament, and the alveolar bone. It means a disease that is infected with bacteria in the gaps between the gums (gums) and the teeth and damages the periodontal ligaments and adjacent tissues. Gingivitis and periodontitis are classified according to the severity of the disease. The periodontal pocket is formed as the inflammation progresses and more tissue is damaged. The more the periodontal pocket is formed, the deeper the depth of the periodontal pouch becomes, the deeper the periodontal pouch becomes inflamed, and eventually the bone loss is induced It is known. In addition to regenerating periodontal ligaments supporting the alveolar bone, it is necessary to regenerate alveolar bone and cementum that can be attached to periodontal ligaments. need.

The term " de-structured trauma " herein refers to a condition in which an external force is applied to a tooth or periodontal tissue, the tooth and periodontal tissue are destroyed due to the difference in direction and size, Sexual dislocation, lateral dislocation, complete dislocation, and intrusion. Therefore, the fundamental treatment of degenerative trauma also requires the regeneration of injured periodontal ligaments. In particular, the success of complete dislocations depends on the regeneration of the periodontal ligament attached to the fully dislocated tooth or the remaining periodontal ligament in the missing tooth. If regeneration of the periodontal ligament does not occur, absorption of the cementum and dentin of the tooth root takes place, resulting in ankylosis, which is replaced by alveolar bone.

As used herein, the term " treating " is intended to include, for example, reducing, alleviating, alleviating, alleviating, or alleviating the symptoms, symptoms, or conditions of an injury, Including, without limitation, any objective or subjective parameters, including, but not limited to, delaying the rate of deterioration of the symptom or pathological condition of the subject, causing injury, disease or disorder, or improving the quality of life of the patient, mentally or physically, Or alleviation or treatment of a symptom or pathological condition of the disease. The treatment or amelioration of the symptom or pathological condition of such an injury, disease, or disease can be judged based on various indicators and imaging results associated with the disease. In one embodiment, the method according to the present invention includes treatment of periodontal disease, reduction in the incidence of disease incidence, reduction of disease severity and / or alleviation of symptoms of periodontal disease.

As used herein, the term " cell therapeutic agent " refers to an agent capable of regenerating autologous, allogenic, and xenogenic cells in vitro or in vitro, or otherwise altering the biological properties of cells , Which is used for the purpose of treatment, diagnosis and prevention through a series of methods. Since 1993, the United States has been administering cell therapy products as medicines since 2002. Such cell therapy agents include, but are not limited to, stem cell therapy agents for tissue regeneration or recovery of specific organ function.

Composition for regenerating periodontal ligament

In one embodiment, the present invention relates to a composition for regenerating periodontal ligament comprising a CPNE7 protein or gene.

In one embodiment, the composition according to the present application is used as a pharmaceutical composition for the treatment of diseases requiring periodontal ligament regeneration and attachment.

Treatment that requires regeneration of the periodontal ligament includes, but is not limited to, periodontal disease and degenerative trauma as previously mentioned.

Regeneration of periodontal ligaments involves the formation of cementum and formation of periodontal ligaments and attachment of periodontal ligaments to the cementum as mentioned above.

The CPNE7 protein or gene according to the present invention can promote the differentiation of periodontal tissue into cementoblasts and the formation of cementum. The CPNE7 protein or gene can also allow periodontal ligament fibroblasts to form periodontal ligaments so that the formed periodontal ligaments can attach to the formed cortex, thereby enabling effective regeneration of the periodontal ligament.

In one embodiment, the expression of cementum attachment protein (CAP) in the cervical and white cells is promoted by CPNE7 according to the present invention, so that the resulting periodontal ligament can be effectively attached to the cementum.

The composition according to the invention may also be used for the treatment of degenerative trauma. Deformed trauma refers to injury to the periodontal ligament and can be divided into shaking, subluxation, delusional dislocation, lateral dislocation, complete dislocation, and penetration. It can occur in all age groups, but especially in children between 8 and 12 years . The success of this completely dislocated tooth depends on the regeneration of the periodontal ligament attached to the fully dislocated tooth or the remaining periodontal ligament in the missing tooth. If regeneration of the periodontal ligament does not occur, absorption of the cementum and dentin of the tooth root takes place, resulting in ankylosis, which is replaced by alveolar bone. According to one embodiment of the present invention, the present CPNE7 recombinant protein can promote adhesion and regeneration of periodontal ligaments and formation of existing cementum without osseointegration, as disclosed herein in Examples 5 to 8 and the like.

Therefore, the composition for regenerating periodontal tissue according to the present invention can be effectively used for the fundamental treatment of periodontal disease and degenerated trauma.

In this respect, the present invention relates to a pharmaceutical composition for regenerating periodontal ligament comprising a CPNE7 protein or gene, or a pharmaceutical composition for treating periodontal disease or degenerative trauma comprising CPNE7 protein or gene.

Pharmaceutical composition

The composition of the present invention may further contain one or more active ingredients which exhibit the same or similar functions in addition to the active ingredients, or a compound which maintains / increases the solubility and / or absorbency of the active ingredients. The pharmaceutical compositions of the present invention can also be used alone or in combination with methods for the treatment or prevention of periodontal disease or degenerated foreign teeth, or using surgery, drug therapy and biological response modifiers.

In addition to the above-mentioned active ingredients, the composition of the present invention may further comprise at least one pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be a mixture of saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, seeding and at least one of these components. Other conventional additives such as buffers, bacteriostats and the like may be added. In addition, it can be formulated into injection formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like by additionally adding diluents, dispersants, surfactants, binders and lubricants, Specific antibody or other ligand can be used in combination with the carrier. Can further be suitably formulated according to the respective disease or ingredient according to the appropriate method in the art or using the methods disclosed in Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA have.

The method of administration of the composition of the present invention is not particularly limited, and known administration methods can be applied, and in one embodiment according to the present application, topical administration can be performed around the pathologically damaged tissue requiring periodontal ligament regeneration. The dosage may vary widely depending on the patient's body weight, age, sex, health condition, diet, time of administration, administration method, excretion rate, and severity of the disease. For protein preparations, including CPNE7 gene or polypeptide, parenteral administration may be preferred, but not excluding other routes and means. For typical drugs, the dosage unit comprises, for example, from about 0.01 mg to 100 mg, but does not exclude the following ranges and ranges above. The daily dose may be from about 1 μg to 10 g, and may be administered once or divided into several times a day.

COMPOSITION AND METHOD FOR IMPRODUCING DIFFERENTIATION OF CERVICAL CELL FROM PERIODONTAL

In another aspect, the present invention relates to a composition for promoting differentiation from periodontal tissue to cervical cells comprising a CPNE7 protein or gene.

The composition according to the present invention promotes the differentiation of cementoblasts and promotes the formation of cementum. If the differentiation into cervical cells can be promoted, it is possible to attach the generated periodontal ligament, which is advantageous in that periodontal ligament regeneration can be more effectively performed. Promoting differentiation means differentiation into cementoblasts allows cells that have already been determined to undergo differentiation into cementoblasts, which is distinct from induction of differentiation. 1 to 3 according to the present application and the like, CPNE7 according to the present invention promotes differentiation into cementoblasts.

In one embodiment, the composition for promoting differentiation according to the present invention may be used in an invivo. In this case, a composition comprising the CPNE7 protein according to the present invention, or a gene thereof, or a composition comprising at least one of the two, may be administered topically, for example, or around a pathologically damaged tissue requiring periodontal ligament regeneration. In one embodiment, it is administered to the periodontal ligament or alveolar bone.

In another embodiment, the composition for differentiation according to the present invention may be used in Invitro. In this case, for example, the periodontal ligament fibroblast can be isolated and cultured in Invitro, and a CPNE7 protein according to the present invention, or a composition containing the gene or the above two or more, can be added and used. Methods for isolating periodontal ligament fibroblasts can be found in the literature (Seo BM et al. Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet., 364 (9429): 149-55).

In vitro culture of periodontal ligament fibroblasts can be referred to what is known herein or in the literature (Seo BM et al. Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet., 364 (9429): 149-55) have.

Cell therapy

In another embodiment, the present invention also relates to a cell therapy agent comprising periodontal ligament fibroblasts comprising (expressing) a CPNE7 protein or gene, or a cementoblast differentiated from said fibroblast.

In one embodiment, the fibroblasts differentiated from Invitro by the composition and method according to the present invention may be separated and used as such or may be separated by a separation method using a flow cytometer or the like, and then the cell treatment agent or pharmaceutical composition It can be used as an active ingredient. At this time, BSP (bone sialoprotein), OC (osteocalcin), CAP (Cementum attachment protein), CP-23 (Cementum protein-23) and the like can be used as markers for promoting differentiation and they can be detected and separated by a known method.

In another embodiment, the CNPE7 protein or a gene thereof according to the present invention can be used directly by treating the affected part, and can affect the differentiation of the endogenous fibroblast of the patient, and can exhibit periodontal regenerating effect.

In one embodiment according to the present invention, the cells contained in the cell therapeutic agent overexpress CPNE7. Methods for over-expressing proteins in cells are known. The CPNE7 gene may be cloned into an appropriate eukaryotic expression vector, such as a vector or plasmid derived from a phage, virus or retrovirus, operatively linked to an expression promoter to construct an expression vector and introduce it into cells according to the invention have. Methods of introduction are known and include, but are not limited to, calcium phosphate method, DEAE-dextran mediated transfection, transfusion lipid mediated transfection methods, transfection using electroporation, phage system transfection or viruses, It is not.

The route of administration of a cell therapy agent or a pharmaceutical composition comprising cells according to the present invention can be administered through any conventional route so long as it can reach the target tissue. But are not limited to, parenteral administration, for example, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration.

The therapeutic agent or composition according to the present invention may be formulated in a suitable form together with a commonly used pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include, for example, water, suitable oils, saline, aqueous carriers for parenteral administration such as aqueous glucose and glycols, etc., and may further contain stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. In addition, the composition for cell therapy according to the present invention may contain various additives such as suspending agents, solubilizers, stabilizers, isotonizing agents, preservatives, adsorption inhibitors, surfactants, diluents, excipients, pH adjusters, , Buffers, antioxidants, and the like. Pharmaceutically acceptable carriers and formulations suitable for the present invention, including those exemplified above, are described in detail in Remington ' s Pharmaceutical Sciences, Current Edition.

The composition for cell therapy of the present invention may be formulated into a unit dosage form by using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or may be manufactured by penetrating into a multi-dose container.

The composition may also be administered by any device capable of transferring the cell therapy agent to the target cell. The cell therapeutic composition of the present invention may comprise a therapeutically effective amount of a cell therapy agent for the treatment of diseases. The term " therapeutically effective amount " refers to the amount of active ingredient or pharmaceutical composition that elicits a biological or medical response in a tissue system, animal or human, as contemplated by a researcher, veterinarian, physician or other clinician, ≪ / RTI > inducing a reduction of the symptoms of the disease or disorder. It will be apparent to those skilled in the art that the cell therapy agent contained in the composition of the present invention will vary depending on the desired effect.

The optimal cell therapy agent content can therefore be readily determined by those skilled in the art and will depend upon a variety of factors including the nature of the disease, the severity of the disease, the amount of other ingredients contained in the composition, the type of formulation, and the age, weight, , The time of administration, the route of administration and the fraction of the composition, the duration of the treatment, the co-administered drug, and the like.

In one embodiment according to the present application, the cell therapeutic agent can be administered topically around the pathologically damaged tissue requiring regeneration of periodontal tissue.

It is important that the cell therapeutic agent according to the present invention includes an amount capable of achieving the maximum effect in a minimal amount without side effects considering all of the above factors. For example, the dose of the cell treatment agent or composition of the present invention is 1.0 × 10 ⁷ to 1.0 × 10 ⁸ cells / kg (body weight) based on osteoblasts differentiated from stem cells as the active ingredient, more preferably 1.0 × 10 ⁵ To 1.0 x 10 < ⁸ > cells / kg (body weight). However, the dose may be variously prescribed depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, route of administration, excretion rate and responsiveness of the patient, These factors can be taken into account to appropriately adjust the dosage. The number of administrations may be at least once within the range of one or clinically acceptable side effects, and the administration site may be administered at one site or two or more sites. For animals other than humans, the same dose as that of human beings per kg may be used, or the dose may be adjusted to a dose ratio (for example, average value) of ischemic organs (heart, etc.) The dose can be administered in a converted amount. Examples of the animal to be treated according to the present invention include humans and mammals for other purposes and specifically include humans, monkeys, mice, rats, rabbits, sheep, cows, dogs, horses, pigs and the like do.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example

Experimental Method

1. Cell culture and differentiation

Humidified air containing 5% CO ₂ was incubated at 37 ° C and used in experiments. Cucumber cell line (OCCM-30) was cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% heat-inactivated bovine serum. Differentiation into white hair cells was differentiated for 14 days using differentiation medium containing 50 mg / ml ascorbic acid and 10 mM beta-glycosphate.

2. Isolation and cultivation of human dentigerous cells

In human dentin cells, 10 adult (18-22 year old) wisdom teeth were isolated from dental clinic of Seoul National University. Specifically, all the experiments were approved by the hospital's Institutional Review Board and the patient's consent was obtained. The cranial tissues attached to the root canal of the wisdom tooth were separated, cut into both sides, , Covered with cover slip, and cultured in Dulbecco's modified Eagle's medium.

3. Real-time PCR analysis

The TRIzol reagent was used to isolate the total RNAs of cementoblast and human dentigerous cells. CDNA was synthesized using 2 μg of total RNA, 1 μl of reverse transcriptase and 0.5 μg of oligo (dT). The cDNAs of the synthesized CCR and human dendritic cells were used for real - time PCR using the primers of Table 1. Real-time PCR was performed on an ABI PRISM 7500 sequence detection system (Applied Biosystems) using SYBR GREEN PCR Master Mix (Takara, Japan). Real-time polymerase chain reaction was 94 ° C for 1 min; 95 ° C, 15 sec - 60 ° C, and 34 sec. For 40 cycles. The results were evaluated using the comparative cycle threshold (CT) method.

4. Immunohistochemical staining

The mandibular canthus of 14 days old rat was isolated and fixed with 4% paraformaldehyde. After demineralization for 1 week with 10% ethylenediaminetetra-acetic acid (EDTA, pH 7.4) solution, To a thickness of 5 [mu] m. Immunohistochemical staining was performed with 0.6% H2O2 / methanol for 20 minutes after deparaffinization and functionalization, and then blocked with PBS containing 1% bovine serum albumin for 30 minutes to dilute 1: 100 diluted Cpne7 antibody And reacted at low temperature. Anti-rabbit IgG was reacted with avidin-biotin-peroxidase complex (Vector Laboratory, Burlingame, Calif.) For 30 min at room temperature. DAB kit (Vector Laboratory, Burlingame, Calif.), And the slices were sealed with water, dehydrated and observed with an optical microscope.

5. Verification of the effect of CPNE7 in animal experiments

The animals were anesthetized by inhalation of gelloran in 12 dogs (12-16 kg; 6-8 weeks old), intravenously injected with zoletil (5 mg / kg) and xylazine (0.2-0.5 mg / kg) And then treated with lidocaine (Lidocaine 2% with 1: 80,000 epinephrine). The premolar was randomly selected from the mandible of the dog, and the middle of the selected premolar was cut with high-speed carbide burrs and extracted. A notch was then formed in the coronal portion of the mesial or distal root at an interval of about 5 mm. A total of 36 root teeth were extracted and 12 samples (positive control) with periodontal ligaments, 12 samples with periodontal ligament and cementum removed (negative control), periodontal ligament and cementum were removed and treated with CPNE7 recombinant protein (Experimental group). In the negative control group and the experimental group, periodontal ligament and cementum between each scratches were removed by gracie curette. Then, the root of the experimental group was immersed in 0.8 ml of an aqueous solution containing 2.5 占 퐂 of CPNE7 recombinant protein for 2 minutes, and the roots of the control group were immersed in physiological saline solution for 2 minutes to prevent drying.

The root was re-implanted in each alveolar bone, the tubular portion of the carbide burrs was removed, and the upper portion of the root was completely covered with a tubular flap. At the 4 or 8 weeks postplantation, the dogs were sacrificed by overdosage (90-120 mg / kg) of pentobarbital. The teeth of the dogs were removed, fixed with 10% formalin, and then 5% formic acid was added to remove the calcium, molded and embedded in paraffin to obtain a tissue slice having a thickness of 5 탆. The sections were stained with hematoxylin and eosin and observed using an optical microscope (LEICA DM750, Germany) equipped with a digital camera (LEICA ICC50 camera, Germany), and the newly formed connective tissue , The height of the tooth-bone adhesion, the length of the newly formed cementous tissue in the root, etc. were measured, and all measured values were analyzed using i-SOLUTION Lite® and analysis program (IMT i-Solution Corporation, Solution. Com).

Example 1 CPNE7 Recombinant Protein Causes Promulgation of Cementoblast Differentiation During Cementoblast Differentiation

For this purpose, the expression pattern of the cervical cell marker gene was analyzed as in the experimental method. As a result, FIG. 1A is a graph showing the results of confirming the mRNA level of OC (osteocalcin) gene expressed in the CW cell line according to the time of culturing according to the treatment of CPNE7 recombinant protein. As shown in FIG. 1A, the expression of OC was maintained on day 4 and continued until day 14, and the expression level of CPNE7 recombinant protein-treated group was increased compared with the control group.

FIG. 3B is a graph showing the results of confirming the mRNA level of the Bsp (Bone sialoprotein) gene expressed in the CW cell line according to the time of culturing according to the treatment of the CPNE7 recombinant protein. As shown in FIG. 3B, the expression of Bsp was maintained on day 4 and continued until day 14, and the expression level of CPNE7 recombinant protein was increased at 10 and 14 days of culture in comparison with the control.

FIG. 3C is a graph showing the mRNA level of a Cap (cementum attachment protein) gene expressed in a CMP cell line according to the time of culturing according to the treatment of CPNE7 recombinant protein. As shown in FIG. 3C, the expression of Cap was maintained on day 4 and continued until day 14, and the expression level of CPNE7 recombinant protein-treated group was increased compared with the control group.

Example 2. Effect of Cpne7 overexpression on Cap gene expression in cervical stem cells

The experimental method is as described above, and the results are as follows. FIG. 2 is a graph showing the effect of Cpne7 overexpression on the expression of Cap gene, a cervical cell differentiation marker gene, in cainoblast cells. Cap is expressed in cementoblast and cementum, and it is known that periodontal ligament fibers play a role in adhesion to cementum. As shown in FIG. 2A, Cpne7 overexpression was observed in cainoblast cells, and the expression level of Cpne7 was increased by about 500,000 fold compared to the control. FIG. 2B shows that the level of Cap gene mRNA according to Cpne7 overexpression in cementoblast cells was about 20-fold higher than that of the control group.

Example 3. Effect of Cpne7 Recombinant Protein on the Expression of CAP and CP23 Gene in Human Crypt Cells

The dental follicle is a loose connective tissue that surrounds the dentate tooth, and is also present in the ambush wisdom of a human adult. Dental follicle cells isolated from the cystic tissues of the amblyopic wisdom teeth are mixed with various types of cells, and these cells are differentiated into periodontal ligament cells, cementoblasts and / or osteoblasts to form periodontal ligament, cementum and / (J Dent Res 87 (8): 767-771, 2008; TISSUE ENGINEERING: Part A 18 (5): 459-470, 2012).

The experimental method is as described above, and the results are as follows. FIG. 3 is a graph showing the effect of CPNE7 recombinant protein treatment on CP23 and CAP gene expression in human cortical cells. As shown in FIG. 3A, the expression of CP23 was increased about 10-fold in the CPNE7 recombinant protein-treated group compared to the control group. FIG. 3B shows that the expression of CAP in the experimental group treated with CPNE7 recombinant protein increased about twice as compared with the control group. These results show that CPNE7 can promote the differentiation of dentigerous cells into cementoblast - like cells that are easy to adhere to periodontal ligaments.

Example 4. Analysis of Cpne7 protein expression in molar tissue of mice

Expression of CPNE7 protein was confirmed by immunohistochemistry in mouse molars to confirm expression of CPNE7 in cementum and periodontal ligament.

FIG. 4 shows the results of immunohistochemical staining of Cpne7 protein in the molar tissue of normal mice at 14 days of age. In FIG. 4, Cpne7 was expressed in alveolar bone, periodontal ligament, and cementum, and was strongly expressed in the bundle of periodontal ligament fibers. Especially, the periodontal ligament fiber bundles expressing Cpne7 protein were observed to be inserted into alveolar bone and cementum. 4B to 4C are enlarged photographs of the box of Fig. 4A.

Example 5: Regeneration of periodontal regeneration of CPNE7 recombinant protein in an animal model of teeth

The PDL was preserved after extraction of the selected premolars, and the extracted periodontal ligaments and cementum removed from the extracted teeth were treated with negative control and CPNE7 recombinant protein After 4 weeks and 8 weeks after reapplication, they were evaluated histologically. The positive control group is a sample in which the periodontal ligament is maintained.

Fig. 5 shows the results of histological evaluation at 4 weeks after planting. Figures 5A-B are positive control results. As shown in FIG. 5A, formation of a new periodontal ligament-like tissue is observed along the root surface, and osseointegration is observed in some cases, but the continuity of the periodontal ligament is maintained. In FIG. 5B, the surface of the dentin is observed with the existing cementum, and the surface of the cementum is observed in contact with the new periodontal ligament-like tissue. This new periodontal ligament-like tissue is also in contact with adjacent alveolar bone.

Figures 5C-D are negative control results. As shown in FIG. 5C, a part of the periodontal ligament-like tissue is observed along the root surface, its width is narrower than normal, and osseointegration is observed at other sites. In Fig. 5D, a new periodontal ligament is not observed because the image quality is directly in contact with the alveolar bone at the osseointegration site.

Figures 5E-F are the results of the experimental group treated with CPNE7 recombinant protein. As shown in FIG. 5E, formation of a new periodontal ligament-like tissue up to the vicinity of the tooth alignment along the root surface is observed together with the formation of neovascularization. Some osseointegration sites are also observed. Attachment of connective tissues to the upper part of the alveolar bone is observed. In Fig. 5F, a new periodontal ligament-like tissue including a new blood vessel is observed between the dentin and alveolar bone, and a new neoplastic cementous tissue is observed on the dentin surface.

Figure 6 shows the results of histological evaluation at 8 weeks after planting. Figures 6A-B are positive control results. As shown in FIG. 6A, a new periodontal ligament-like tissue is observed along the root surface, and surface absorption and osseointegration are also observed in some cases. In Fig. 6B, neocortical similar tissues are observed on the dentin surface, and fibers of the new periodontal ligament-like tissues are more compact than those of the 4th week group. Neovascularization is rarely observed.

Figures 6C-D are negative control results. As shown in FIG. 6C, osseointegration is remarkably observed along the root surface at the level of the control region, and neovascularization and surface absorption are observed in some cases. In Figure 6D, the dentin at the osseointegration site is in direct contact with the alveolar bone with some surface absorption.

Figures 6E-F are the results of the experimental group treated with CPNE7 recombinant protein. As shown in Fig. 6E, a new periodontal ligament-like tissue is observed along the root surface to the tooth-adjusting region. In Fig. 6F, neocortical similar tissues are observed on the surface of the dentin, fibers in the new periodontal ligament-like tissues are embedded vertically in the neocortical cortex, and more dense fibers can be observed in the new periodontal ligamentous tissues have.

Taken together, these results indicate that CPNE7 not only promotes the formation of periodontal ligaments in periodontal ligament fibroblasts, but also promotes differentiation and cement formation in cementoblasts, indicating that periodontal ligaments formed are important for adhesion to the cementum.

Example 6. Analysis of the effect of CPNE7 recombinant protein on the formation of cementum in the alveolar crest region and the binding (attachment) of the fibers in periodontal ligament fibers and gingiva

The alveolar crest is the highest part of the alveolar bone where the cortical plate and alveolar bone meet. It is located 1.5-2 mm lower than the cemento-enamel junction of the normal teeth. The gingival is located on the upper part of the tooth adjustment and the gingival fibers are attached. Regeneration of the periodontal ligament, including the site of the tooth, prevents the gingival epithelial cell from moving to the apical region and prevents the gingival fibers from penetrating the damaged area. Therefore, we evaluated the effect of CPNE7 on the formation of new cementum and the binding (adherence) of fibers in the periodontal ligament fibers and gingiva at the dentition site.

Fig. 7 shows the results showing specific findings in the dentate area treated with CPNE7 recombinant protein. 7A to 7B show that the experimental group treated with the CPNE7 recombinant protein at the 4th week showed no neurons in the other group (positive control and negative control) (X40). And that the fibers in the gingiva are strongly bonded to the side of the new cementous tissue. 7 (C), (D) and (E) show that the experimental group treated with CPNE7 recombinant protein showed no neuronal cortical similarity above the control level (x40). And that the fibers in the gingiva are strongly bonded to the side of the new cementous tissue.

These results indicate that the connective tissue grafted to the exposed root surface can be used as an adhesive protein to attach the implanted connective tissue to the root surface during the healing process after the procedure.

Example 7. Evaluation of Histopathological Evaluation of Dental Grafting at 8 Weeks

At 8 weeks after the implantation, I-solution program (IMT Isolution corporation, Korea) was used to calculate the rate of formation of new periodontal ligament-like tissues, bone resorption, and new cementum in the positive control, negative control and / And histomorphometry analysis was performed. The alveolar crest is the highest part of the alveolar bone where the cortical plate and alveolar bone meet. It is located 1.5-2 mm lower than the cemento-enamel junction of the normal teeth.

FIG. 8 shows the results of histological analysis of the amount of cementum-like tissue (PDLT), osseointegration (RR), and periodontal tissue in the periodontal ligament-like tissue (CTL) 8 weeks after dental implantation. In FIG. 8, the rate of generation of PDLT in the experimental group treated with CPNE7 recombinant protein was similar to that of the positive control group and increased more than twice as compared with the negative control group.

The ratio of osteosynthesis in the control group and in the group treated with CPNE7 recombinant protein was statistically significantly lower than that of the negative control group (RR) Respectively.

The CPNE7 recombinant protein-treated cementum-like tissue (CTL) of the periodontal ligament was similar to that of the positive control group, and the cementum-like cortical tissue (CTL) Respectively. The ratio of cementum - like tissue to total root was similar to the result of cemento - similar tissues in periodontal tissue.

Taken together, these results indicate that CPNE7 plays an important role in promoting regeneration, attachment and cement formation in periodontal ligaments.

[Table 1] Nucleotide Sequences of Real-Time PCR Primers

While the present invention has been described in connection with what is presently considered to be the preferred embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, .

All technical terms used in the present invention are used in the sense that they are generally understood by those of ordinary skill in the relevant field of the present invention unless otherwise defined. The contents of all publications referred to herein are incorporated herein by reference.

Claims (12)

A composition for regenerating periodontal ligament comprising a CPNE7 protein or gene, Wherein said regeneration of periodontal ligament comprises formation of cementum through promotion of differentiation of periodontal ligament fibroblasts into cementoblasts and attachment of said regenerated periodontal ligament to cementum. The method according to claim 1, Wherein said composition is used for the treatment of periodontal disease or degenerated foreign teeth, or for prevention of tooth-osseointegration by said degenerated traumatic injury. 3. The method of claim 2, Wherein said periodontal disease is gingivitis or periodontitis. The method according to claim 1, Wherein said cementum formation is by promoting cementum attachment protein (CAP) expression in said cementoblasts by said CPNE7 and in the white cells derived from said cementoblast.  The method according to claim 1, Wherein the composition is administered to a periodontal ligament or alveolar bone requiring periodontal ligament regeneration.  A composition for the differentiation of periodontal ligament fibroblasts containing CPNE7 protein or gene into cervical cells. A method for promoting differentiation of periodontal ligament fibroblasts into cervical cells comprising the step of treating the CPNE7 protein or gene with periodontal ligament fibroblasts. A cell therapy agent for regenerating periodontal ligament, comprising periodontal ligament fibroblast overexpressing CPNE7 or cementoblast or chalk cell differentiated from said fibroblast as an active ingredient. A method for regenerating periodontal ligament of a subject comprising administering to a subject in need of regeneration of periodontal ligament a therapeutically effective amount of a cell therapeutic agent according to claim 8. A method of regenerating periodontal ligament of a subject comprising administering to a subject in need of regenerating periodontal ligament a therapeutically effective amount of a CPNE7 protein or gene, or a composition comprising said protein or gene. 11. The method of claim 10, Wherein said periodontal ligament regeneration comprises formation of cementum through promotion of differentiation of periodontal ligament fibroblasts into cementoblasts and attachment of said regenerated periodontal ligament to cementum. 11. The method of claim 10, Wherein said gene or protein, or said composition, is administered to a periodontal ligament or alveolar bone requiring periodontal ligament regeneration.

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