KR102331249B1 - Complex composition for differentiation of periodontal ligament cells to cementoblast - Google Patents

Abstract

The present invention relates to a complex composition for inducing periodontal ligament cell differentiation including a transforming growth factor-beta (TGF-β) active receptor-like kinase (ALK) 5 inhibitor, bone morphogenetic protein-7 (BMP-7), and ascorbic acid. According to the present invention, periodontal ligament cells are isolated from human adult-derived periodontal ligament tissues and the isolated periodontal ligament cells are treated with the transforming growth factor-beta (TGF-β) active receptor-like kinase (ALK) 5 inhibitor, bone morphogenetic protein-7 (BMP-7), and ascorbic acid. Accordingly, compared to the existing differentiation method, differentiation of periodontal ligament cells to target cells, cementoblast, can be effective induced, and the crypt cells necessary for the successful regeneration of periodontal tissues are secured, and thus can be used to study cretaceous formation and regeneration and to discover surface expression markers specific therefor.

Description

Complex composition for differentiation of periodontal ligament cells into cementoblasts {Complex composition for differentiation of periodontal ligament cells to cementoblast}

The present invention relates to periodontal ligament cells comprising a transforming growth factor-beta (TGF-β) receptor activin receptor-like kinase (ALK) 5 inhibitor, bone morphogenetic protein-7 (BMP-7) and ascorbic acid. It relates to a composition for inducing differentiation into parent cells.

Periodontal tissue is a tissue that supports and surrounds the teeth, and includes the root cementum, periodontal ligament, alveolar bone, and gingiva. Periodontal tissue regeneration to prevent tooth loss is a complex process, and two hard tissues, cementum and alveolar bone, and two soft tissues, periodontal ligament and gingival tissue, must all be properly made. Among these, cementum is an important tissue that holds the position of teeth in the alveolar bone, so interest in tooth attachment and regeneration of cementum is increasing, and the need for research on the formation and regeneration of cementum is increasing. The understanding of the mechanism of root cementum is still lacking. In particular, the biggest difficulty in the study of cretaceous formation and regeneration is the lack of understanding of the origin and properties of cretaceous cells because there are no specific markers of the cementum. In addition, there are still many controversies about the origin of cretaceous cells, cells that form the cementum.

In general, after the crown is completely formed, the tooth begins to develop together with the crown, erupts into the oral cavity, and then develops toward the root. The structure that causes tooth growth is the neck part of the enamel organ called the cervical loop, which is a two-layered surrounding structure composed of enamel epithelium. The alveolar rings grow from the newly completed crown to form Hertwig's root sheath, form the shape of the tooth root, and induce root dentin formation to connect with the crown dentin. When the formation of root dentin is completed, Hertwig's root sheath is disintegrated, and after the collapse, these cells become epithelial rests of malassez, and thereafter, the epithelial-mesenchymal transition (epithelial-mesenchymal transition) in contact with the newly formed root dentin surface. It differentiates into cementoblasts through the epithelial-mesenchymal transition process. Cretaceous cells are known to form cementum by accumulating cementoid when they come into contact with the root dentin.

In order to culture the cretaceous cells, which play an important role in generating the cementum, epithelial root sheath cells and Malassez epithelial rests of malassez cells should be used, but it is difficult to extract these cells, and cells that can be obtained Since the amount of chemotherapy is very limited, there is a great technical limitation in the study of cretaceous cells and tissue regeneration using cretaceous cells.

Accordingly, the present inventors isolated periodontal ligament cells from adult human periodontal ligament tissue and studied the optimal differentiation method for differentiating them into cretaceous cells. As a result, the TGF-β (transforming growth factor-beta) receptor ALK (activin) When a receptor-like kinase) 5 inhibitor, BMP-7 (bone morphogenetic protein-7) and ascorbic acid are used in combination, it was confirmed that periodontal ligament cells can be effectively differentiated into cetinoblasts, and the present invention has been completed. .

Therefore, an object of the present invention is a periodontal ligament comprising a transforming growth factor-beta (TGF-β) receptor activin receptor-like kinase (ALK) 5 inhibitor, bone morphogenetic protein-7 (BMP-7) and ascorbic acid. To provide a composition, a medium composition, a kit for inducing differentiation of cells into chorionic cells, and a method for inducing differentiation using the same.

In order to achieve the above object, the present invention TGF-β (transforming growth factor-beta) receptor ALK (activin receptor-like kinase) 5 inhibitor, BMP-7 (bone morphogenetic protein-7) and ascorbic acid (Ascorbic acid) It provides a composition for inducing differentiation of periodontal ligament cells into chorionic cells, comprising.

In addition, the present invention is a TGF-β (transforming growth factor-beta) receptor ALK (activin receptor-like kinase) 5 inhibitor, BMP-7, and ascorbic acid (Ascorbic acid) containing periodontal ligament cells to induce differentiation into cetinoblasts. A medium composition for use is provided.

In addition, the present invention is a TGF-β (transforming growth factor-beta) receptor ALK (activin receptor-like kinase) 5 inhibitor, BMP-7, and ascorbic acid (Ascorbic acid) containing periodontal ligament cells to induce differentiation into cetinoblasts. kits are provided for

Periodontal ligament cells comprising the; TGF-β (transforming growth factor-beta) receptor ALK (activin receptor-like kinase) 5 inhibitor, BMP-7 and ascorbic acid (Ascorbic acid) in vitro treatment in periodontal ligament cells; It provides a method for inducing differentiation into cretaceous cells.

According to the present invention, periodontal ligament cells are isolated from human adult periodontal ligament tissue, and thus TGF-β (transforming growth factor-beta) receptor ALK (activin receptor-like kinase) 5 inhibitor, BMP-7 and ascorbic acid (Ascorbic acid) ), it is possible to induce the differentiation of periodontal ligament cells into a target cell, a chorionic cell, compared to the existing differentiation method, and to secure the cementum cells necessary for the successful regeneration of periodontal tissue, thereby conducting a study on the formation and regeneration of the periodontal ligament. It can be used to discover specific surface expression markers.

1 is a diagram showing the results of confirming the shape change of hPDLC after treatment with TGF-β1, SB431542, BMP-7 and/or ascorbic acid by performing the same experiment twice and using a phase-contrast microscope. (TGF: 10 ng/ml TGF-β1, BMP7: 100 ng/ml BMP-7, SB: 10 μM TGF-β receptor inhibitor SB431542, Vit c: 100 μM ascorbic acid)
2 is a view showing the results of confirming the expression of CAP (Cementum attachment protein) and CEMP1 (Cementum protein 1) according to TGF-β1, SB431542, BMP-7 and/or ascorbic acid treatment through RT-qPCR analysis. (TGF: 10 ng/ml TGF-β1, B7: 100 ng/ml BMP-7, VtC: 100 μM ascorbic acid, SB: 10 μM TGF-β receptor inhibitor SB431542, B/C: 100 ng/ml BMP-7 and 100 μM ascorbic acid combined treatment, B/SB: 100 ng/ml BMP-7 and 10 μM TGF-β receptor inhibitor SB431542 combined treatment, C/SB: 100 μM ascorbic acid and 10 μM TGF-β receptor inhibitor SB431542 combined treatment , B/C/SB: 100 ng/ml BMP-7, 100 μM ascorbic acid and 10 μM TGF-β receptor inhibitor SB431542 combined treatment)
3 is a view showing the results of confirming the expression of CAP (Cementum attachment protein) and CEMP1 (Cementum protein 1) according to TGF-β1, SB431542, BMP-7 and/or ascorbic acid treatment through RT-qPCR analysis. (TGF: 10 ng/ml TGF-β1, B7: 100 ng/ml BMP-7, VtC: 100 μM ascorbic acid, SB: 10 μM TGF-β receptor inhibitor SB431542, B/C: 100 ng/ml BMP-7 and 100 μM ascorbic acid combined treatment, B/SB: 100 ng/ml BMP-7 and 10 μM TGF-β receptor inhibitor SB431542 combined treatment, C/SB: 100 μM ascorbic acid and 10 μM TGF-β receptor inhibitor SB431542 combined treatment , B/C/SB: 100 ng/ml BMP-7, 100 μM ascorbic acid and 10 μM TGF-β receptor inhibitor SB431542 combined treatment)
4 is a view showing the results of confirming the expression of SCX, PLAP-1 and OPN according to TGF-β1, SB431542, BMP-7 and / or ascorbic acid treatment through RT-qPCR analysis. (TGF: 10 ng/ml TGF-β1, B7: 100 ng/ml BMP-7, VtC: 100 μM ascorbic acid, SB: 10 μM TGF-β receptor inhibitor SB431542, B/C: 100 ng/ml BMP-7 and 100 μM ascorbic acid combined treatment, B/SB: 100 ng/ml BMP-7 and 10 μM TGF-β receptor inhibitor SB431542 combined treatment, C/SB: 100 μM ascorbic acid and 10 μM TGF-β receptor inhibitor SB431542 combined treatment , B/C/SB: 100 ng/ml BMP-7, 100 μM ascorbic acid and 10 μM TGF-β receptor inhibitor SB431542 combined treatment)
5 is a view showing the results of confirming the expression of OSX and BSP according to TGF-β1, SB431542, BMP-7 and / or ascorbic acid treatment through RT-qPCR analysis. (TGF: 10 ng/ml TGF-β1, B7: 100 ng/ml BMP-7, VtC: 100 μM ascorbic acid, SB: 10 μM TGF-β receptor inhibitor SB431542, B/C: 100 ng/ml BMP-7 and 100 μM ascorbic acid combined treatment, B/SB: 100 ng/ml BMP-7 and 10 μM TGF-β receptor inhibitor SB431542 combined treatment, C/SB: 100 μM ascorbic acid and 10 μM TGF-β receptor inhibitor SB431542 combined treatment , B/C/SB: 100 ng/ml BMP-7, 100 μM ascorbic acid and 10 μM TGF-β receptor inhibitor SB431542 combined treatment)

Hereinafter, the present invention will be described in detail.

The present invention is a TGF-β (transforming growth factor-beta) receptor ALK (activin receptor-like kinase) 5 inhibitor, BMP-7 (bone morphogenetic protein-7), and the differentiation of periodontal ligament cells containing ascorbic acid into cerebroblasts A composition for induction is provided.

The TGF-β receptor ALK5 inhibitor of the present invention may include, without limitation, a substance capable of inhibiting the TGF-β receptor ALK5, preferably A-83-01 or SB431542.

In the present invention, “A-83-01 ((3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide )” and “SB431542 (4-(5-Benzol[1,3]dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)-benzamide)” are TGFβ receptors ( ALK5) and Activin receptors (ALK4/7) (ie TGFβR inhibitors).

Specifically, in the present invention, “SB431542” is an inhibitor of TGF-β receptor Alk5 (Activin Receptor-Like Kinase-5), C ₂₂ H ₁₆ N ₄ O ₃ Means a material having a chemical formula and a molecular weight of 384.4, In the present invention, a derivative of SB431542 may also be included in the scope of the present invention as long as it is a substance that induces differentiation of periodontal ligament cells into chorionic cells through inhibition of TGF-β receptor ALK5, as desired in the present invention.

The TGF-β receptor ALK5 inhibitor of the present invention may be treated to gingival epithelial cells at 0.1 to 30 μM, 1 to 20 μM, and most preferably 5 to 10 μM.

BMP-7 of the present invention is a bone morphogenetic protein, and the BMP-7 may be treated in gingival epithelial cells at 10 to 200 ng/ml, preferably 50 to 150 ng/ml, more preferably 100 ng/ml.

Ascorbic acid of the present invention is a kind of vitamin, and may be used interchangeably with terms such as "Ascorbic acid", "ascorbic acid", "vitamin C" and "vit C". The ascorbic acid may be treated in gingival epithelial cells at 10 to 300 μM, preferably 50 to 200 μM, more preferably 100 μM, but is not limited thereto.

Using the TGF-β receptor ALK5 inhibitor, BMP-7 and ascorbic acid of the present invention, differentiation into leukoblasts with increased expression of markers such as CEMP1 (cementum protein 1) and CAP (cementum attachment protein) in periodontal ligament cells can be effectively induced.

The present invention can effectively induce the differentiation of periodontal ligament cells derived from adult periodontal ligament tissue into cetinoblasts. Therefore, the periodontal ligament cells used as the material of the present invention may be derived from adult periodontal ligament tissues isolated from humans. Accordingly, the present invention overcomes the difficulties of extraction and the limited cell yield of the method of culturing cetaceous cells using epithelial root sheath cells or Malassez residual epithelial cells, and effectively obtains a large amount of desired cetaceous cells from human tissue. can do.

Accordingly, in the present invention, the periodontal ligament cells may be cells derived from animals, preferably mammals, more preferably humans, and may be autologous, allogenic, or xenogenic cells.

In the present invention, the term “cementoblast” can be used interchangeably with “creativity progenitor cell”, and refers to a cretaceous-specific progenitor cell that can be differentiated into cementum.

“Cementum” is one of the hard tissues of teeth, and it is known to be a different tissue from bone in that it accumulates much more slowly than bone or mineralized tissue similar to bone, has no vascular system or innervation, and does not undergo remodeling. In addition, the cementum is not formed in osteoblasts, unlike alveolar bone, and is distinguished from alveolar bone, similar to the development of bone, in that the cretaceous cells form the cementum through cretaceous cells, not bone cells. Therefore, the cementum is one of the general mineralized tissues, but it must be distinguished from the mineralization of bone or alveolar bone.

Cretaceous cells induced to differentiate according to the present invention may be characterized in that the expression of the cettoblast marker is increased, preferably, the expression of markers such as CEMP1 (cementum protein 1) and CAP (cementum attachment protein) is increased. can be done with

The method for isolating periodontal ligament cells of the present invention can be used without limitation, a method widely known in the art for isolating periodontal ligament cells from adult human periodontal tissue, and the periodontal ligament cells of the present invention can be directly isolated or purchased and used in this way. can

The composition of the present invention may include a medium, and the present invention relates to periodontal ligament cells comprising a transforming growth factor-beta (TGF-β) receptor activin receptor-like kinase (ALK) 5 inhibitor, BMP-7 and ascorbic acid. Provided is a medium composition for inducing differentiation into crypt cells.

As used herein, the term medium includes all mediums conventionally used in the art suitable for culturing cells in vitro. Depending on the type of cell, the medium and culture conditions can be selected. The medium used for culture is DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal essential Medium), BME (Basal Medium Eagle), RPMI1640, F-10, F-12, α-MEM (α-Minimal essential Medium), GMEM (Glasgow's Minimal essential Medium), Iscove's Modified Dulbecco's Medium medium, etc., but is not limited thereto.

In addition to the TGF-β receptor ALK5 inhibitor, BMP-7, and ascorbic acid, the composition of the present invention may further include a component capable of inducing differentiation of periodontal ligament cells into cerebroblasts.

In addition, the present invention provides a kit for inducing differentiation of periodontal ligament cells into cerebral blast cells, comprising a transforming growth factor-beta (TGF-β) receptor activin receptor-like kinase (ALK) 5 inhibitor, BMP-7 and ascorbic acid. do.

The kit is not limited to the above, and may include other reagents or instruments. For example, it may contain a culture plate for culturing the target cells or a reagent capable of evaluating the differentiation status into chorionic cells (eg, alizarin red, etc.), and may contain periodontal ligament cells to be cultured. may be

The provided form of the kit according to the present invention is an additive for a differentiation induction medium, or a TGF-β receptor ALK5 inhibitor, such as SB431542 or A-83-01, BMP-7 and ascorbic acid or cytokines, phospholipids, or a basal medium and others. It may be one container that contains all of the reagents in appropriate doses and/or forms, or may be provided by different containers.

The kit according to the present invention may include instructions describing the procedure for carrying out the above-described method according to the present invention.

In addition, the present invention is a periodontal ligament comprising the; TGF-β (transforming growth factor-beta) receptor ALK (activin receptor-like kinase) 5 inhibitor, BMP-7 and ascorbic acid to periodontal ligament cells in vitro treatment; Provided is a method for inducing differentiation of a cell into a cretaceous cell.

Cretaceous cells differentiated in vitro according to the differentiation method of the present invention are used as such after being separated or separated through a separation method using a flow cytometer, etc. can be used as

The term “cell therapy product” means proliferating or selecting living autologous, allogenic, or xenogenic cells in vitro or changing the biological properties of cells in other ways in order to restore the functions of cells and tissues. It refers to drugs used for the purpose of treatment, diagnosis and prevention through a series of methods.

Through the cementum regeneration effect of the cell therapy agent or pharmaceutical composition according to the present invention, it is possible to treat various periodontal diseases, such as periodontitis.

The TGF-β receptor ALK5 inhibitor of the present invention, BMP-7, and ascorbic acid can promote differentiation into cetinoblasts by treating periodontal ligament cells for 5 to 20 days, preferably 7 to 14 days.

The TGF-β receptor ALK5 inhibitor may be treated with 0.1 to 30 μM, preferably from 1 to 20 μM, and more preferably from 5 to 10 μM. In addition, the BMP-7 may be treated at 10 to 200 ng/ml, preferably at 50 to 150 ng/ml, more preferably at 100 ng/ml. The ascorbic acid may be treated at 10 to 300 μM, preferably at 50 to 200 μM, more preferably at 100 μM, but is not limited thereto.

Terms not otherwise defined herein have meanings commonly used in the art to which the present invention pertains.

Hereinafter, the present invention will be described in detail by way of Examples. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples.

Example 1. SB431542, BMP-7 and ascorbic acid Confirmation of Cretaceous Cell Differentiation Induction by Treatment

An experiment was performed to confirm the conditions for inducing cell differentiation from periodontal ligament cells cultured primarily from adult periodontal ligament tissues to cetacean cells. The P -values of the experiments performed in the present invention were calculated through one-way ANOVA using SPSS software. P -values of 0.05 or less were considered statistically significant.

1.1 Cell Isolation, Culture and Confirmation of Morphological Changes

For the primary culture of human periodontal ligament cells (hPDLCs), third molars were obtained from patients aged 19 to 23 years according to the guidelines approved by the IRB of Dankook University and Dental Hospital. Human periodontal ligament tissue was isolated from the root surface, which was treated with 3 mg/mL type I collagenase (Millpore) and 4 mg/mL dispase (sigma) and enzymatically digested at 37° C. for 1 hour. Cell supernatant was cultured in a humidified atmosphere supplemented with 5% CO2 and 37°C conditions using α-MEM (HyClone) medium containing 20% FBS and 1% antibiotics.

Thereafter, hPDLCs ^{were aliquoted in a 6-well plate at a density of 4 x 10 4} cells and cultured in α-MEM medium containing 5% FBS, and then treated with cytokines or inhibitors alone or in combination under the following conditions: 10 ng /ml TGF-β1 (Sino Biological), 100 ng/ml BMP-7 (Prospec), 10 μM TGF-β receptor inhibitor SB431542 (TOCRIS), 100 μM ascorbic acid (sigma). The cytokines or inhibitors were treated in the initial generation of the second subculture of hPDLC, changing the medium every other day for 9 days.

Thereafter, the shape of hPDLC was observed with a phase contrast microscope for 9 days, and the results are shown in FIG. 1 .

As shown in FIG. 1 , there was no significant difference between cells treated with 10 ng/ml TGF-β1 and untreated hPDLC, but in hPDLC treated with SB431542, BMP-7, and ascorbic acid, it was found that minerals were agglomerated. There were clear morphological differences, such as visible parts.

1.2. Check for gene expression changes

The hPDLC isolated and cultured in 1.1 was aliquoted in a 6-well plate at a density of ^{4 x 10 4 cells and cultured in α-MEM medium containing 5% FBS.} Then, cytokines or inhibitors were treated alone or in combination: 10 ng/ml TGF-β1 (Sino Biological), 100 ng/ml BMP7 (Prospec), 10 μM SB431542 (TOCRIS). After 9 days, total RNA was obtained from the cells using Easy-SpinTM Total RNA extraction Kit (iNtRON), cDNA was reverse transcribed therefrom using ReverTra Ace qPCR RT Master MixTM (TOYOBO), and RT-qPCR was performed using iTaqTM Quantification was performed after performing on a StepOn™ system (Applied Biosystems) instrument using a Universal SYBR™ Green Supermix (Bio-Rad) system. Primers for each gene are shown in Table 1 below.

Gene Primer sequence Cementum protein 1 (CEMP1) Forward
Reverse 5'-GATCAGCATCCTGCTCATGTT-3'
5'-AGCCAAATGACCCTTCCATTC-3' Cementum attachment protein (CAP) Forward
Reverse 5'-TCCAGACATTTGCCTTGCTT-3'
5'-TTACAGCAATAGAAAAACAGCATG-3' Scleraxis (SCX) ForwardReverse 5'-AGAAAGTTGAGCAAGGACC-3'
5'-CTGTCTGTACGTCCGTCT-3' Periodontal ligament-associated protein-1 (PLAP-1) Forward
Reverse 5'-TTGACCTCAGTCCCAACCAA-3'
5'-TCGTTAGCTTGTTGTTGTTCAG-3' Bone sialoprotein (BSP) ForwardReverse 5'-TACCGAGCCTATGAAGATGA-3'
5'-CTTCCTGAGTTGAACTTCGA-3' Osterix (OSX) ForwardReverse 5'-GAAGGGAGTGGTGGAGCCAAAC-3'
5'-ATTAGGGCAGTCGCAGGAGGAG-3' Osteopontin (OPN) ForwardReverse 5'-GTGGGAAGGACAGTTATGAA-3'
5'-CTGACTTTGGAAAGTTCCTG-3' GAPDH Forward
Reverse 5'-GTATGACAACAGCCTCAAGAT-3'
5'-CCTTCCACGATACCAAAGTT-3'

GAPDH was used as a standardized control gene. Dissociation curves during PCR were formed in the range of 65°C to 95°C, and qPCR was performed under the following conditions: 1 cycle at 95°C for 1 minute, 40 cycles at 95°C for 15 seconds, and 1 minute at 60°C. CT (threshold cycle) was obtained, and relative comparison was performed for each target gene.

1.2.1 Confirmation of CAP and CEMP1 gene expression

The results of measuring changes in CAP and CEMP1 gene expression by performing two experiments in the method of Example 1.2 are shown in FIGS. 2 and 3 .

As shown in FIGS. 2 and 3 , the combined effect of SB431542, BMP-7 and ascorbic acid at the mRNA level was clearly observed.

More specifically, CAP and CEMP1 are known as markers involved in the differentiation of cementum, and, as shown in FIG. 2, when SB431542, BMP-7 and ascorbic acid were complexly treated in the medium, cementum attachment protein (CAP) and cementum The expression of protein 1 (CEMP1) was increased by about 4.42-fold and 7.5-fold compared to the control group. Furthermore, the highest expression of CAP and CEMP1 was confirmed when SB431542, BMP-7, and ascorbic acid were treated together, compared to the case of a combination of SB431542, BMP-7, or ascorbic acid alone, or when both components were treated together. did.

Similarly in FIG. 3, when SB431542, BMP-7 and ascorbic acid were complexly treated in the medium, the expression of cementum attachment protein (CAP) and cementum protein 1 (CEMP1) was increased by about 3.52 times and 3.47 times compared to the control group. Furthermore, the highest expression of CAP and CEMP1 was confirmed when SB431542, BMP-7, and ascorbic acid were treated together, compared to the case of a combination of SB431542, BMP-7, or ascorbic acid alone, or when both components were treated together. did.

This is a result showing that the combined treatment of SB431542, BMP-7 and ascorbic acid has a more significant effect than any combination containing SB431542, BMP-7 and ascorbic acid on the inhibition of the TGF-β pathway and promotion of cementogenesis. .

1.2.2 Confirmation of SCX, PLAP-1, OPN, OSX and BSP expression

SCX, a ligament progenitor-specific transcription factor, PLAP-1, a peridontal ligament associates protein, which is known not to be expressed in calcification, OPN, a ligament marker in periodontal ligament cells, OSX, which is known to regulate cell differentiation during bone and dentin formation, and Expression of the osteogenic marker BSP was confirmed, and the results are shown in FIGS. 4 and 5 . SCX, PLAP-1 and OPN are negative markers of Cretaceous differentiation, and OSX and BSP are known markers of Cretaceous differentiation.

As shown in Figure 4, SCX and PLAP-1 showed low expression compared to the control group when all of the SB431542, BMP-7 and ascorbic acid components were treated, OPN showed reduced expression compared to the TGF treatment group did. Meanwhile, SCX, PLAP-1 and OPN, which are negative markers of chalky differentiation, were expected to show a decreasing trend even when SB431542, BMP-7 and ascorbic acid were treated alone or in combination of two types, but the observed results did not show a consistent trend. . However, when all of the SB431542, BMP-7 and ascorbic acid components of the present invention were treated, a consistent decrease in expression was observed in all negative markers.

As shown in FIG. 5, OSX and BSP were treated with both SB431542, BMP-7, and ascorbic acid components than in the case of SB431542, BMP-7, or ascorbic acid alone, or a combination of two selected and treated together. The highest expression was confirmed when In addition, OSX and BSP, as markers of chalky differentiation, were expected to show an increase trend even when B431542, BMP-7 and ascorbic acid were treated alone or in combination of two types, but the observed results did not show a consistent trend. However, when all of SB431542, BMP-7, and ascorbic acid of the present invention were treated, a clear increase in expression was observed.

Through the above results, it was confirmed that SB431542, BMP-7, and ascorbic acid can induce very different differentiation patterns depending on either alone or in combination. It was confirmed that it can induce the consistent differentiation of periodontal ligament cells into chorionic cells.

Although the present invention has been described as the preferred embodiment mentioned above, it is possible to make various modifications and variations without departing from the spirit and scope of the invention. It is also intended that the appended claims cover such modifications and variations as fall within the scope of the present invention.

Claims (12)

A composition for inducing differentiation of periodontal ligament cells into ceretoblasts, comprising a transforming growth factor-beta (TGF-β) receptor activin receptor-like kinase (ALK) 5 inhibitor, bone morphogenetic protein-7 (BMP-7) and ascorbic acid .
According to claim 1, wherein the TGF-β receptor ALK5 inhibitor is SB431542, characterized in that, the composition for inducing differentiation of periodontal ligament cells into nephroblasts.
According to claim 1, wherein the TGF-β receptor ALK5 inhibitor is characterized in that it is contained in 0.1 to 30 μM, the composition for inducing differentiation of periodontal ligament cells into nephroblasts.
According to claim 1, wherein the BMP-7 is characterized in that contained in the 10 to 200ng / ml, the composition for inducing differentiation of periodontal ligament cells into chorionic cells.
[Claim 2] The composition for inducing differentiation of periodontal ligament cells into chorionic cells according to claim 1, wherein the ascorbic acid is contained in an amount of 10 to 300 μM.
According to claim 1, wherein the TGF-β receptor ALK5 inhibitor, BMP-7, and ascorbic acid is characterized in that to increase the expression of CEMP1 (cementum protein 1) and CAP (cementum attachment protein), periodontal ligament cells to the ceticular cell composition for inducing differentiation.
According to claim 1, wherein the periodontal ligament cells are characterized in that derived from adult periodontal ligament tissue, the composition for inducing differentiation of periodontal ligament cells into cetinoblasts.
According to claim 1, wherein the periodontal ligament cells are characterized in that derived from human periodontal ligament tissue, the composition for inducing differentiation of periodontal ligament cells into cetinoblasts.
A medium composition for inducing differentiation of periodontal ligament cells into cerebral cells, comprising a transforming growth factor-beta (TGF-β) receptor activin receptor-like kinase (ALK) 5 inhibitor, BMP-7 and ascorbic acid.
A kit for inducing differentiation of periodontal ligament cells into cerebral blast cells, comprising a transforming growth factor-beta (TGF-β) receptor activin receptor-like kinase (ALK) 5 inhibitor, BMP-7 and ascorbic acid.
Periodontal ligament cells including the; TGF-β (transforming growth factor-beta) receptor ALK (activin receptor-like kinase) 5 inhibitor, BMP-7 and ascorbic acid in vitro treatment; of differentiation induction method.
The method of claim 11 , wherein the treatment is performed for 7 to 14 days.

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