KR102041910B1 - Composition for differentiation of periodontal ligament cells to Cementoblast - Google Patents

Abstract

The present invention relates to a composition, a medium, a kit, and a method for inducing differentiation of periodontal ligament cells into blastocysts comprising a transforming growth factor-beta (TLG-beta) receptor activin receptor-like kinase (ALK) 5 inhibitor. It is about. According to the present invention, by separating the periodontal ligament cells from human adult periodontal ligament cells and treating them with TGF-β receptor ALK5 inhibitors, the periodontal ligament cells can be effectively induced into differentiation into the target cells, the chalk cells, By obtaining the chalk cells necessary for regeneration, it can be used to study chalk formation and regeneration and to discover surface expression markers specific thereto.

Description

Composition for inducing differentiation of periodontal ligament cells into chalk cells [Composition for differentiation of periodontal ligament cells to Cementoblast}

The present invention relates to a composition, a medium, a kit, and a method for inducing differentiation of periodontal ligament cells into blastocysts comprising a transforming growth factor-beta (TLG-beta) receptor activin receptor-like kinase (ALK) 5 inhibitor. It is about.

The periodontal tissue is a tissue that supports and wraps the teeth and includes the alveolar chalky, the periodontal ligament, the alveolar bone, and the gingiva. Regeneration of the periodontal tissue to prevent loss of teeth is a complex process, and two hard tissues, chalky and alveolar bone, and two soft tissues, periodontal ligament and gingival tissue, must all be made properly. Among them, chalk is an important tissue that holds the position of teeth in the alveolar bone. Therefore, interest in tooth attachment and regeneration of the chalk is increasing and the necessity of research on chalk formation and regeneration is increasing. There is still a lack of understanding of the mechanism of root chalk chalk. In particular, the greatest difficulty in the study of chalk formation and regeneration is that there is a lack of understanding of the origin and properties of the chalk cells because there is no specific marker of chalk. There is also much controversy about the origin of the chalky cells, which are the cells that form chalky cells.

In general, teeth begin to develop with the crown and erupt into the oral cavity after the crown is fully formed, and then develop toward the root. The structure that leads to tooth development is the neck of the enamel organ called the cervical loop, which is a two-layered peripheral structure composed of enamel epithelium. The alveolar ring grows from the newly completed crown to form Hertwig's root sheath, forms the roots of the tooth and induces root dentin formation to connect with the crown dentin. When the formation of the root dentin is completed, Hertwig's root sheath collapses, and after collapse, the cells become Malassez epithelial rests of malassez, which then come into contact with the newly formed root dentin planes. Differentiate into cementoblasts through epithelial-mesenchymal transition. When chalkacet cells reach the root dentin, it is known that they accumulate chalkoids and form chalkiness.

In order to cultivate the chalk cells that play an important role in producing the chalk, it is necessary to use Hertwig's root sheath cells and Malassez epipital rests of malassez cells, but these cells are difficult to obtain and obtain. Since the amount of is very limited, there are great technical limitations in the study of chalk cells and tissue regeneration using the chalk cells.

Therefore, the present inventors have investigated the optimal differentiation method for separating periodontal ligament cells from human adult periodontal ligament tissues and differentiating them into chalk cells, and as a result, TGF-β (transforming growth factor-beta) receptor ALK (activin When the receptor-like kinase 5 inhibitor is used, it has been confirmed that the periodontal ligament cells can be effectively differentiated into chalky cells, thereby completing the present invention.

Accordingly, an object of the present invention is to provide a composition, a medium composition, a kit, and a composition for inducing differentiation of periodontal ligament cells into blastocysts comprising a TGF-β (activating receptor-like kinase) 5 inhibitor It is to provide a method of inducing differentiation.

In order to achieve the above object, the present invention provides a composition for inducing differentiation of periodontal ligament cells into blastocysts comprising a TGF-β (activating receptor-like kinase) 5 inhibitor.

In another aspect, the present invention provides a medium composition for inducing differentiation of periodontal ligament cells into chalk cells, including a TGF-β receptor ALK5 inhibitor.

In another aspect, the present invention provides a kit for inducing differentiation of periodontal ligament cells into chalk cells, including a TGF-β receptor ALK5 inhibitor.

In another aspect, the present invention provides a method for inducing differentiation of periodontal ligament cells into chalk cells, comprising the step of treating the periodontal ligament cells with a TGF-β receptor ALK5 inhibitor.

According to the present invention, by separating the periodontal ligament cells from human adult periodontal ligament cells and treating them with TGF-β receptor ALK5 inhibitors, the periodontal ligament cells can be effectively induced into differentiation into the target cells, the chalk cells, By obtaining the chalk cells necessary for regeneration, it can be used to study chalk formation and regeneration and to discover surface expression markers specific thereto.

1 is a diagram showing the results of morphological changes of hPDLC after SB431542, TGF-β1 and / or BMP7 treatment (a, untreated; b, 10 μM SB431542; c, 10 ng / ml TGF-B1; d , 100 ng / ml BMP7; e, BMP7 and SB431542; f, TGF-B1 and SB431542).
Figure 2 is a diagram showing the results confirmed by RT-qPCR analysis of the expression of the chalk cell- and ligament-specific markers according to SB431542, TGF-β1 and / or BMP7 treatment.
Figure 3 is a view showing the results confirming the phosphorylation blocking effect of Smad 1, Smad3 by SB431542, TGF-β1 and / or BMP7 treatment.
Figure 4 is a diagram showing the results confirmed by Western blot changes in Smad1 / 3 phosphorylation according to the treatment according to the K02288 concentration.
5 is a diagram showing the results of RT-qPCR analysis of the expression of chalk cells, ligament cells and mineralization-related genes in PDLC after SB431542, K02288 and TGFβ-1 treatment.
6 is a diagram showing the results of mineral formation after SB431542 or TGF-β1 treatment in PDLC cultured in growth medium and osteogenic medium. Figure 6 A is a diagram showing the results confirmed by staining the mineral accumulation results with alizarin A after culturing for 3 weeks in each medium. FIG. 6B is a diagram showing the results of quantifying the absorbance at 405 nm after dissolving the mineral node. FIG.
Figure 7 is a schematic diagram showing the mechanism of induction of differentiation of SB431542 by SB431542 by blocking Smad2 / 3 phosphorylation.

Hereinafter, the present invention will be described in detail.

The present invention provides a composition for inducing differentiation of periodontal ligament cells into chalk cells, comprising a TGF-β receptor ALK5 inhibitor.

The TGF-β receptor ALK5 inhibitor of the present invention inhibits Smad3 phosphorylation in the periodontal ligament cells to block signaling pathways, thereby enhancing expression of markers such as CEMP1 (cementum protein 1) and CAP (cementum attachment protein). Differentiation into leukocytes can be effectively induced.

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 compounds known as inhibitors (ie TGFβR inhibitors) of the Activin receptor (ALK4 / 7).

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

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

 Treatment with TGF-β receptor ALK5 inhibitors may increase the specific chalk cell specific markers, indicating that the periodontal ligament cells effectively differentiated into the chalk cell. When treating the TGF-β receptor ALK5 inhibitor of the present invention, preferably SB431542, to periodontal ligament cells, the chalk cell specific markers can be increased by about 2.32 and 1.59 compared to the untreated group.

The TGF-β receptor ALK5 inhibitors of the present invention can block the Smad2 / 3 pathway involved in downstream signaling induced by TGF-β1 treatment, and more specifically specifically block the expression of chalk formation genes by effectively blocking Smad3 phosphorylation. Can be promoted.

The present invention can effectively induce differentiation of periodontal ligament cells derived from adult periodontal ligament tissue into chalk cells. Thus, the periodontal ligament cells of the present invention may be derived from adult periodontal ligament tissue isolated from humans. Accordingly, the present invention overcomes the problems of extraction and limited cell yield problems of the method of culturing chalky cells using epithelial myocytes or Malassez epithelial residual cells, and effectively obtains a large amount of desired chalky cells from human tissue. can do.

Thus, in the present invention, the periodontal ligament cells may be cells derived from an animal, preferably a mammal, more preferably a human, and may be autologous, allogenic, xenogenic cells.

In the present invention, the term “cementoblast” may be used interchangeably with “chalk progenitor cells” and means chalky specific precursor cells that can differentiate into chalky.

"Cementum" is one of the hard tissues of the tooth and is known to be different from bone in that it accumulates much more slowly than bone-like mineralized tissue or bone and does not have a vascular or nerve distribution and does not undergo a remodeling process. In addition, unlike the alveolar bone, chalky cells are not formed in osteoblasts, and they are distinguished from alveolar bone similar to that of bone in that chalky cells form chalky cells through chalky cells rather than bone cells. Therefore, chalkiness should be distinguished from one of the common mineralized tissues, but not from mineralization of bone or alveolar bone.

The differentiation-induced chalkhead cells according to the present invention may be characterized by an increased expression of a chalk cell marker, and preferably, expression of markers such as CEMP1 (cementum protein 1) and CAP (cementum attachment protein) is increased. You can do

The method of isolating periodontal ligament cells of the present invention can be used without limitation, a method widely known in the art for separating periodontal ligament cells from adult human periodontal tissue, the periodontal ligament cells of the present invention can be used to directly isolate or purchase Can be.

The composition of the present invention may include a medium, and the present invention provides a medium composition for inducing differentiation of periodontal ligament cells into blastocysts comprising a TGF-β receptor ALK5 inhibitor.

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

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

The present invention also provides a kit for inducing differentiation of periodontal ligament cells into chalk cells, comprising a TGF-β receptor ALK5 inhibitor.

The kit is not limited to the above, and may include other reagents or instruments. For example, a culture plate for culturing the target cells or a reagent (for example, alizarin red, etc.) capable of evaluating the differentiation state to the chalk cells may be included, and the periodontal ligament cells to be cultured may be provided. It may be.

Provided forms of the kits according to the present invention may be formulated with an additive for differentiation induction medium, or a TGF-β receptor ALK5 inhibitor such as SB431542 or A-83-01, or cytokines, phospholipids, or basal medium and other reagents, It may be one container contained in a form or / or may be provided by each of the different containers.

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

The present invention also provides a method of inducing differentiation of periodontal ligament cells into chalk cells, comprising the step of treating a periodontal ligament cell with a TGF-β receptor ALK5 inhibitor.

The chalk cells differentiated in vitro according to the differentiation method of the present invention are used as such or after being separated by a separation method using a flow cytometer or the like, and then, an active ingredient of a cell therapeutic agent for tooth regeneration, or a pharmaceutical composition. Can be used as

The term "cell therapy" refers to the proliferation or selection of autologous, allogenic and xenogenic cells in vitro or to alter the biological characteristics of the cells in order to restore the function of cells and tissues. Medicines used for the purpose of treatment, diagnosis and prevention through a series of methods.

Through the chalky regeneration effect of the cell therapy 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 may be treated with periodontal ligament cells, preferably 5 to 20 days, preferably 7 to 14 days, to promote differentiation into chalk cells.

Terms not defined otherwise in this specification are intended to have a meaning 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 to illustrate the present invention is not limited to the contents of the present invention.

Example  1.by SB431542 treatment Chalk cells  Identify the effect of differentiation induction

Experiments were performed to identify conditions that can induce cell differentiation from periodontal ligament cells cultured from adult periodontal ligament tissues to chalk cells. The P -values of the experiments performed in the present invention were calculated through one-way analysis of variance using SPSS software. P -values below 0.05 were considered statistically significant.

1.1 Cell isolation, culture and morphological changes

For primary culture of human periodontal ligament cells (hPDLCs), third molars were obtained from patients aged 19-23 years according to the IRB's approved guidelines at Dankook University Dental Hospital. Human periodontal ligament tissue was isolated from the root surface and treated with 3 mg / mL type I collagenase (Millpore) and 4 mg / mL dispase (sigma) enzymatically digested at 37 ° C. for 1 hour. Cell supernatants were incubated at 37 ° C. and humidified atmosphere supplemented with 5% CO ₂ using α-MEM (HyClone) medium containing 20% FBS and 1% antibiotic.

HPDLC was then aliquoted into 6-well plates at 4 × 10 ⁴ cell density and cultured in α-MEM medium containing 5% FBS, after which cytokines or inhibitors were treated alone or in combination under the following conditions: 10 ng / ml TGF-β1 (Sino Biological), 100 ng / ml BMP7 (Prospec), 10 μM TGF-β receptor inhibitor SB431542 (TOCRIS). The cytokine or inhibitor was treated with medium exchange every other day for 9 days in the initial generation of the second subculture of hPDLC.

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

As shown in FIG. 1, no morphologically distinct differences were found between untreated cytokine or inhibitor-treated hPDLC and treated hPDLC.

1.2 Confirmation of Gene Expression Change

HPDLC isolated and cultured at 1.1 were aliquoted into 6-well plates at 4 × 10 ⁴ cell density and incubated in α-MEM medium containing 5% FBS. Cytokines or inhibitors were then 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 cells using Easy-Spin ^™ Total RNA extraction Kit (iNtRON), and cDNA was reverse transcribed from ReverTra Ace qPCR RT Master Mix ^™ (TOYOBO). Quantification was performed on a StepOn ^™ system (Applied Biosystems) instrument using the iTaq ™ 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'-TTACAGCAATAGAAAAACAGCATGA-3 ' Scleraxis (SCX) ForwardReverse 5'-AGAAAGTTGAGCAAGGACC-3 '
5'-CTGTCTGTACGTCCGTCT-3 ' Osteocalcin (OCN) ForwardReverse 5'-TGAGTCCTGAGCAGCAG-3 '
5'-TCTCTTCACTACCTCGCT-3 ' Bone sialoprotein (BSP) ForwardReverse 5'-TACCGAGCCTATGAAGATGA-3 '
5'-CTTCCTGAGTTGAACTTCGA-3 ' GAPDH Forward
Reverse 5'-GTATGACAACAGCCTCAAGAT-3 '
5'-CCTTCCACGATACCAAAGTT-3 '

 GAPDH was used as a standardized control gene. Dissociation curves were formed during the PCR 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. A threshold cycle was obtained and relative comparisons were made for each target gene. The result of measuring the change of gene expression is shown in FIG.

As shown in FIG. 2, there was a marked change in the mRNA level between the treated and untreated groups. After 9 days of treatment with BMP7 , the expression of cementum protein 1 ( CEMP1 ) and cementum attachment protein ( CAP ) increased by 1.33 and 1.17 fold, respectively, compared to the control. On the other hand, hPDLC treated with 10 ng / ml of TGF-β1 for 9 days did not change the expression of the two chalk cell specific markers, CEMP1 and CAP. On the other hand, scleraxis , a ligament progenitor-specific transcription factor, was 12.84 times higher in expression than the control group.

In addition, when the cells were treated with 10 μM of SB431542, it was confirmed that the expression of CEMP1 and CAP increased 2.32 and 1.59 times compared to the control. This is a result showing that inhibition of the TGF-β pathway and BMP7 treatment promotes cementogenesis.

Processing of TGF-β1 is induced increased expression of scleraxis, treatment of SB431542 and BMP7 induced increased expression of the austenite to Riggs 2.15 and 2.44 compared to control group. On the other hand, co-treatment with BMP7 and TGF-β1 inhibitors did not show a clear synergistic effect on differentiation into chalk cells.

1.3 Smad3  Confirmation of phosphorylation inhibitory effect

HPDLC isolated and cultured in Example 1.1 were dispensed into 6-well plates at 1.5 x 10 ⁵ cell density and incubated for 4 hours in α-MEM medium containing 5% FBS. The cytokine or inhibitor was then treated alone or together for 1 hour; 10 ng / ml TGF-β1 (Sino Biological), 100 ng / ml BMP7 (Prospec), 10 μM SB431542 (TOCRIS). Harvest the cells and use 20 using elution buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% NP40, 2 mM EDTA pH 8.0, 2 mM EGTA pH 8.0) containing protease / phosphatase inhibitors. Eluted on ice for minutes. Proteins contained in clarified lysate were separated on SDS-PAGE and transferred to PVDF membrane and incubated overnight at 4 ° C. with the corresponding antibody. Antibodies used were: anti-Smad-1, anti-p-Smad-1 / 5/9, anti-Smad-3, and anti-p-Smad-3 (Cell Signaling Technology); anti-actin (Santa Cruz Biotechnology). After continuous incubation with HRP-conjugated antibodies, protein bands were detected using ECL (GE Healthcare) and Automatic X-ray firm processor (JPI Healthcare). Western blot results are shown in FIG. 3.

As shown in FIG. 3, phosphorylation of Smad1 was induced by BMP7 treatment, and phosphorylation of Smad3 was induced by TGF-β1. Since Smad1 / 5 is known to be inducible by the BMPs / BMPR2 / ALK1 / 2/3/6 signaling pathway, no increase in the phosphorylation signal of Smad 1/5 was observed by TGF-β1 treatment.

SB431542 treatment completely blocked the phosphorylation of Smad3 induced by TGF-β1. Therefore, it was confirmed that SB431542 promotes the expression of the chalky forming gene by blocking the Smad2 / 3 pathway.

However, Smad1 phosphorylation by BMP7 treatment did not further increase under conditions of inhibition of the TGF-β pathway by SB431542 treatment. In addition, when TGF-β1 and BMP7 were simultaneously treated, phosphorylation of Smad1 and Smad3 was also confirmed at the same level as that of the individual treatment. These results show that signaling pathways by TGF-β1 and BMP7 do not show synergistic effects on chalkiness.

Example  2. Of SB431542  Check mechanism of action

To determine whether the BMP-activating signaling pathway is involved in differentiation for chalk formation, selective type I BMP receptor inhibitors such as ALK2 / 6, K02288 were treated with hPDLC. K02288 is known to reduce BMP4-induced Smad 1/5/8 phosphorylation in vitro. Inhibition of the Smad1 / 5 pathway does not have a marked effect on chalky differentiation. In consideration of this, qPCR and Western blot using K02288 were performed in the same manner as in Example 1 to confirm whether K02288 may be involved in chalky formation differentiation, and 0.5 μM K02288 (TOCRIS), respectively. And 0.01 to 1 μM K02288 (TOCRIS). Western blot results for Smad1 and Smad3 phosphorylation are shown in FIG. 4, and the results of CEMP1, CAP, BSP, and OCN gene expression are shown in FIG. 5.

As shown in FIG. 4, K02288 suppressed Smad1 phosphorylation in a dose-dependent manner, and the phosphorylation signal was completely disappeared by K02288 treatment of 500 nM or more, but phosphorylation of Smad3 did not change in a dose-dependent manner.

In addition to the expression of the chalk cell-associated genes, as shown in FIG. 5, the inhibition of the TGF-β pathway by SB431542 treatment caused the expression of osteogenic markers such as osteocalcin (OCN) and bone sialoprotein (BSP). Increased, indicating the interaction between the TGF-β and BMP pathways. In addition, 500 nM of K02288 was treated with SB431542 for 9 days in hPDLC. As a result, CEMP1, CAP, OCN, and BSP, which are related to chalk cells and osteoblasts, were maintained at the same level as SB431542 alone. K02288 had no significant effect on the expression of chalk cell markers, but promoted the expression of mineralization markers.

In addition, treatment of TGF-β1 with these two inhibitors resulted in a 1.3-2.3-fold decrease in gene expression increase compared to the expression level of cells treated with inhibitors only. These results show that, unlike the general case, TGF-β1 promotes fibroblast differentiation more than hPDLC differentiation of hard tissues.

Example  3. SB431542 AT  by hPDLC  Differentiation into hard tissue

TGF-β or SB431542 obtained and cultured in Example 1 after 9 days of treatment with hPDLC, osteogenic medium [100 nM dexamethasone (Sigma), 100 μM ascorbic acid and 5 mM β-glycerophosphate containing 5% FBS α-MEM medium] was added for 2 weeks with 10 ng / ml TGF-β or 10 μM SB431542. As a general growth medium, α-MEM medium containing 20% FBS was used.

The cells were washed with PBS and fixed with 4% formaldehyde for 15 minutes. Cells were stained by treatment with 2% alizarin red S (pH 4.2, Sigma) for 5 minutes and washed four times with distilled water. For quantification of alizarin red staining, cells stained for 30 minutes in 10% acetic acid and the absorbance was measured at 405nm with a microplate reader, the results are shown in FIG.

As shown in FIG. 6, after 21 days of incubation in osteogenic medium, hPDLC differentiated into hard tissue and minerals accumulated on the cell surface. The addition of SB431542 during the culturing step in osteogenic medium resulted in a 2.01 fold increase in mineralization levels compared to the untreated control. On the other hand, the treatment of TGF-β1 was found to reduce the mineralization level rather than the untreated control. These results show that SB431542 induces periodontal ligament cells to differentiate into chalky cells, whereas TGF-β1 inhibits chalky differentiation and increases the differentiation of periodontal ligament soft tissues.

Taken together, SB431542 blocks Smad2 / 3 pathway and induces cell differentiation from periodontal ligament cells primary cultured from adult periodontal ligament tissues to chalk cells, and treatment of SB431542 prevents the expression of chalk cell markers. It can be seen that it can increase (FIG. 7).

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

Claims (10)

SB431542, a transforming growth factor-beta (TLF-) receptor activin receptor-like kinase 5 inhibitor, is included as a single active ingredient, and SB431542 increases expression of cement protein 1 (CEMP1) and cementum attachment protein (CAP). A composition for inducing differentiation of human periodontal ligament cells into chalk cells.
delete According to claim 1, wherein the TGF-β receptor ALK5 inhibitor is characterized in that it comprises 0.1 to 30 μM, composition for inducing differentiation of human periodontal ligament cells into chalk cells.
delete The composition of claim 1, wherein the TGF-β receptor ALK5 inhibitor inhibits Smad3 phosphorylation.
The composition of claim 1, wherein the human periodontal ligament cells are derived from adult periodontal ligament tissues.
SB431542, a transforming growth factor-beta (TLF-) receptor activin receptor-like kinase 5 inhibitor, is included as a single active ingredient, and SB431542 increases expression of cement protein 1 (CEMP1) and cementum attachment protein (CAP). A medium composition for inducing differentiation of human periodontal ligament cells into chalk cells.
SB431542, a transforming growth factor-beta (TLF-) receptor activin receptor-like kinase 5 inhibitor, is included as a single active ingredient, and SB431542 increases expression of cement protein 1 (CEMP1) and cementum attachment protein (CAP). A kit for inducing differentiation of human periodontal ligament cells into chalk cells.
Treating the human periodontal ligament cells with a single active ingredient, SB431542, a TGF-β receptor ALK5 inhibitor, wherein SB431542 is characterized by increasing expression of CEMP1 (cementum protein 1) and CAP (cementum attachment protein) to induce differentiation of human periodontal ligament cells into chalk cells in vitro.
The method of claim 9, wherein the treatment is performed for 7 to 14 days.

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