WO2021221199A1 - Method of accelerating differentiation of stem cells from apical papilla by using optical stimulation - Google Patents

Abstract

The present invention relates to a technique for accelerating differentiation of stem cells from apical papilla (SCAP) by using an optical stimulation. More specifically, the present invention provides a composition, kit, and method for tooth transplantation or dental root formation, comprising SCAP irradiated with near-infrared radiation, and a method for differentiating SCAP, comprising a step of irradiating SCAP with near-infrared radiation.

Description

Method for promoting differentiation of apical papillary stem cells using photostimulation

It relates to a technology for promoting differentiation of stem cells from apical papilla (SCAP) using photostimulation, and more specifically, a composition, kit, for tooth transplantation or root generation, comprising apical papilla stem cells irradiated with near-infrared rays, and a method, and a method for differentiation of apical papillary stem cells comprising the step of irradiating near-infrared rays to the apical papillary stem cells is provided.

Photobiomodulation (PBM) therapy is a non-invasive light therapy that uses low-level laser light or light emitting diodes (LEDs) to affect cellular functions such as cell growth, proliferation and differentiation. . In PBM therapy, the red visible light region in the wavelength range of 600 to 700 nm and the near infrared region (NIR) in the wavelength range of 780 to 1100 nm are being used for clinical purposes. PBM therapy is also being tried in the field of tissue engineering using stem cells. PBM therapy contributes to creating an optimal environment for newly transplanted stem cells to survive well. PBM therapy increases blood supply and promotes cell proliferation, synthesis and activation of growth factors.

On the other hand, stem cells from apical papilla (SCAP) are a unique group of mesenchymal stem cells, which are located in the root apex of a developing permanent tooth. Apical papillary stem cells have osteogenic (osteogenic) differentiation, odontogenic (odontogenic) differentiation, chondrogenic (chondrogenic) differentiation, and adipogenic (adipogenic) differentiation ability. Because apical papillary stem cells have a high proliferation rate and mineralization potential, they can be usefully used for bone and dentin regeneration.

Since the apical papillary stem cells are cells derived from the immature apex, they are similar to embryos compared to other dental stem cells. Since apical papillary stem cells are a source of odontoblasts responsible for the formation of root dentin, they are expected to be advantageously used in the regeneration of tooth-related tissues, but many studies have not been conducted on this. .

Accordingly, in the present specification, it is an object of the present specification to provide a technique for differentiation of stem cells from apical papilla (SCAP) into tooth tissue using photobiological control (PBM) and optimal PBM conditions for the same.

One example provides a composition for dental implantation, comprising apical papillary stem cells irradiated with near-infrared rays.

Another example provides a composition for generating a tooth root, comprising the near-infrared irradiated apical papillary stem cells.

Another example provides a kit for dental implantation, including a light source for apical papillary stem cells, and near-infrared irradiation.

Another example provides a kit for generating a tooth root, including a light source for apical papillary stem cells, and near-infrared irradiation.

Another example provides a method for manufacturing a dental implant, comprising irradiating near-infrared rays to the isolated apical papillary stem cells.

Another example provides a differentiation method of apical papillary stem cells, comprising the step of irradiating near-infrared rays to the isolated apical papillary stem cells. The differentiation of the apical papillary stem cells may be differentiation into one or more selected from the group consisting of osteocytes, odontoblasts, adipocytes, and chondrocytes.

Another example provides a method for producing a tooth root, comprising irradiating near-infrared rays to the isolated apical papillary stem cells.

In the present specification, it is proposed that the growth, proliferation, and/or differentiation of apical papillary stem cells is promoted by photostimulation using near-infrared rays, thereby having an advantageous effect on the regeneration of teeth (particularly, root). The effect of the photostimulation may vary depending on the wavelength, frequency, and irradiation conditions of the near-infrared rays. Since the apical papillary stem cells adopted herein are stem cells derived from the apical end of immature permanent teeth, they have more similar properties to embryonic stem cells compared to other odontogenic adult stem cells. For example, whereas pulp-derived stem cells differentiate into crown odontoblasts, apical papillary stem cells differentiate into odontoblasts that generate root in immature permanent teeth. Therefore, in tooth regeneration, apical papillary stem cells are more advantageous than pulp origin stem cells in order to regenerate the tooth root. In the present specification, the differentiation of the apical papillary stem cells into teeth including the root through multi-lineage differentiation may be performed in vitro and/or in vivo after transplantation.

Accordingly, an example of the present invention provides a composition for dental implantation, comprising apical papillary stem cells irradiated with near infrared rays. Another example provides the use of near-infrared irradiated apical papillary stem cells for use in the preparation of a dental implant or a composition for dental implantation.

Another example provides a composition for generating a tooth root, comprising the near-infrared irradiated apical papillary stem cells. Another example provides the use for the production of a composition for generating a root or generating a root of the near-infrared irradiated apical papillary stem cells.

Another example provides a method for manufacturing a dental implant, comprising irradiating near-infrared rays to the isolated apical papillary stem cells.

Another example provides a differentiation method of apical papillary stem cells, comprising the step of irradiating near-infrared rays to the isolated apical papillary stem cells. The differentiation of the apical papillary stem cells may be differentiation into one or more selected from the group consisting of osteocytes, odontoblasts, adipocytes, and chondrocytes. Accordingly, another example provides a method for generating osteocytes, a method for generating dentin blasts, a method for generating adipocytes, and/or a method for generating chondrocytes, comprising irradiating near-infrared rays to the isolated apical papillary stem cells.

Another example provides a method for producing a tooth root, comprising irradiating near-infrared rays to the isolated apical papillary stem cells.

In the composition and method, the apical papillary stem cells may be isolated from the root apex of immature permanent teeth.

In the composition and method, the near-infrared light may be a pulsed wave having a wavelength of 800 nm to 900 nm.

In the composition and method, the near-infrared radiation may have the following characteristics:

(1) a frequency of 30 Hz to 3000 Hz;

(2) an in-wavelength duty cycle of 25 to 35%; or

(3) A combination of (1) and (2) above.

In the composition and method, the near-infrared irradiated apical papillary stem cells, ALP, Col1A, RUNX2, OCN, TGF-β, DSPP, and in the group consisting of DMP1, compared to the apical papillary stem cells not irradiated with near-infrared rays The level of one or more selected coding genes may be increased.

In the composition and method, the near-infrared irradiated apical papillary stem cells are one selected from the group consisting of Col1A, DMP-1, CEMP-1, CAP, and OCN, compared to the apical papillary stem cells that are not irradiated with near-infrared rays. It may be an increased protein level.

In the composition and method, the near-infrared irradiated apical papillary stem cells may be differentiated into one or more selected from the group consisting of osteocytes (eg, alveolar osteocytes), odontoblasts, adipocytes, and chondrocytes.

Another example of the present invention provides a kit for dental implantation, comprising a light source for apical papillary stem cells, and near-infrared irradiation.

Another example provides a dental implantation method, comprising the step of implanting the apical papillary stem cells, and irradiating near-infrared rays to the apical papillary stem cells. The transplanting step and the step of irradiating near-infrared rays may be performed in any order, for example, the step of irradiating near-infrared rays to the transplanted stem cells after performing the transplantation first may be performed.

Another example provides a kit for generating a tooth root, including a light source for apical papillary stem cells, and near-infrared irradiation.

Another example provides a method for generating a tooth root, comprising the step of implanting apical papillary stem cells, and irradiating near-infrared rays to the transplanted apical papillary stem cells.

In the kit and method, the apical papillary stem cells may be isolated from the root apex of immature permanent teeth.

In the kit and method, the near-infrared light may be a pulsed wave having a wavelength of 800 nm to 900 nm.

In the kit and method, the near-infrared radiation may have the following characteristics:

(1) a frequency of 30 Hz to 3000 Hz;

(2) an in-wavelength duty cycle of 25 to 35%; or

(3) A combination of (1) and (2) above.

In the kit and method, the apical papillary stem cells are ALP, Col1A, RUNX2, OCN, TGF-β, DSPP, and DMP1, compared to the apical papillary stem cells that are not irradiated with near-infrared rays by near-infrared irradiation. It may be that the level of one or more coding genes selected from is increased.

In the kit, the apical papillary stem cells are one or more selected from the group consisting of Col1A, DMP-1, CEMP-1, CAP, and OCN, compared to the apical papillary stem cells that are not irradiated with near-infrared rays by near-infrared irradiation. It could be an increase in protein levels.

In the kit, the apical papillary stem cells may be differentiated into one or more selected from the group consisting of osteocytes, odontoblasts, adipocytes, and chondrocytes by near-infrared irradiation.

Hereinafter, the present invention will be described in more detail:

The tooth region described herein may refer to the tooth cross-section structure shown at https://st4.depositphotos.com/10957306/20985/v/1600/depositphotos_209857956-stock-illustration-labeled-tooth-cross-section-anatomy.jpg. can

As used herein, "stem cells from apical papilla (SCAP)" are stem cells isolated from the root apex of immature permanent teeth (eg, molars, etc.), and include root and/or dentin. It is a multi-lineage differentiation stem cell capable of differentiating into various cells necessary for regeneration, for example, osteocytes, otoblasts, adipocytes, chondrocytes, and the like. Immature permanent teeth are permanent teeth that erupt early in life (approximately until adolescence in humans) or are separable from adult wisdom teeth, and are also called early permanent teeth or first molars. The apical papillary stem cells exhibit a higher proliferation rate and mineralization potential for regenerating bone and dentin, compared to dental pulp stem cells (DPSC). In one example, the apical papillary stem cells are immature permanent teeth of a mammal selected from the group consisting of companion animals such as humans, dogs and cats, livestock such as cattle, horses, pigs, sheep, and goats, rodents such as mice and rats, etc. (For example, it may be separated from the apical end of the molar). The apical papillary stem cells may have a high expression rate of one or more selected from the group consisting of a mesenchymal stem cell positive marker, for example, STRO-1, CD13, CD90, CD146, CD18, CD34, CD45, and the like. For example, in the apical papillary stem cells, the proportion of cells expressing a mesenchymal stem cell positive marker (eg, at least one selected from the group consisting of CD13, CD90, CD146, etc.) is about 75% or more, about 77% or more, at least about 80%, at least about 82%, at least about 83%, at least about 85%, at least about 87%, or at least about 90%, and/or expressing a mesenchymal stem cell negative marker (eg, CD34, etc.) The proportion of cells may be about 5% or less, about 3% or less, about 2.5% or less, or about 2% or less.

"Near-infrared light (NIR)" means an electromagnetic wave having a wavelength close to the visible light region while having a longer wavelength than visible light. It has a stronger thermal action than visible light or ultraviolet light, and is useful in various fields thanks to its optical properties showing a reflectance different from that of visible light. For example, in the medical field, it is generally used for disinfection, sterilization, and treatment of joints and muscles. The near-infrared rays usable herein have a wavelength of 700 nm to 1200 nm, for example, 750 nm to 900 nm, 750 nm to 850 nm, 750 nm to 830 nm, 800 nm to 900 nm, 800 nm to 850 nm, or 800 nm to 830 nm. As long as it has an accelerating effect, it is not limited thereto.

In the present specification, by using a pulsed wave as the near-infrared rays, it is possible to obtain an excellent effect of promoting differentiation of apical papillary stem cells compared to the case of using a continuous wave. As used herein, "pulse wave" refers to a waveform that has an extremely short duration of about picoseconds (=10-12 seconds) and is repeated at regular intervals. According to the shape of the pulse, it is divided into a square (square) wave, a rectangular wave, a peak wave, and the like. Because pulsed waves have a large intensity for a very short time, it is possible to compensate for the disadvantages of heat-induced cell damage and deformation that continuous waves can exhibit. Meanwhile, the “continuous wave” refers to a waveform in which amplitude or intensity does not change, unlike the pulse wave, and the waveform does not change with time and exhibits continuous characteristics.

In an embodiment, the near-infrared light may be a pulsed wave having a wavelength of 800 nm to 900 nm, but is not limited thereto.

Considering the absorption rate of apical papillary stem cells, the near-infrared rays are

a frequency of 30 Hz to 3000 Hz, 30 Hz to 1000 Hz, 30 Hz to 500 Hz, 30 Hz to 330 Hz, 30 Hz to 300 Hz, 100 Hz to 3000 Hz, 100 Hz to 1000 Hz, 100 Hz to 500 Hz, 100 Hz to 330 Hz, 100 Hz to 300 Hz, 200 Hz to 3000 Hz, 200 Hz to 1000 Hz, 200 Hz to 500 Hz, 200 Hz to 330 Hz, 200 Hz to 300 Hz, or 270 Hz to 330 Hz, and/or

an in-wavelength duty cycle of 10% to 40%, 10% to 35%, 20% to 40%, 20% to 35%, 25% to 40%, 25% to 35%, or 27% to 33% %.

As used herein, the term “duty cycle” refers to a period representing the ratio of pulse width to pulse period, also referred to as pulse width. That is, as the width of the pulse wave, in a square wave, the ratio of the high part and the low part of one period level is expressed as a percentage.

The total energy density of the near infrared is 50 to 2000 mJ/cm ² , 50 to 1500 mJ/cm ² , 50 to 1000 mJ/cm ² , 50 to 750 mJ/cm ² , 250 to 2000 mJ/cm ² , 250 to 1500 mJ/cm ² , 250 to 1000 mJ/cm ² , 250 to 750 mJ/cm ² , 500 to 2000 mJ/cm ² , 500 to 1500 mJ/cm ² , 500 to 1000 mJ/cm ² , 500 to 750 mJ/ cm ² , 750 to 2000 mJ/cm ² , 750 to 1500 mJ/cm ² , 750 to 1000 mJ/cm ² , or about 750 mJ/cm ² It may be, but is not limited thereto.

The near-infrared rays may be obtained from all commonly available light sources. In the present specification, the light source for near-infrared irradiation can be used without limitation as long as it emits near-infrared rays, for example, at least one selected from the group consisting of lasers, light-emitting diodes (LEDs), etc. However, the present invention is not limited thereto.

In the composition for tooth implantation, the composition for generating a tooth root, the method for producing a tooth implant, and/or the method for producing a tooth root, the near-infrared irradiation may be applied in vitro, and prior to culturing of the apical papillary stem cells , during culturing, and/or after culturing. The near-infrared irradiation time may be appropriately adjusted in consideration of the energy density described above (that is, so that the energy density can be achieved), for example, about 1 to 20 minutes, 1 to 18 minutes, 1 to 15 per one time basis. minutes, 1-12 minutes, 3-20 minutes, 3-18 minutes, 3-15 minutes, 3-12 minutes, 5-20 minutes, 5-18 minutes, 5-15 minutes, 5-12 minutes, 8-20 minutes minutes, 8 to 18 minutes, 8 to 15 minutes, or 8 to 12 minutes. The culture may be performed in a commonly used differentiation medium, for example, at least one medium selected from the group consisting of a commonly used bone formation induction medium, a chondrogenesis induction medium, an adipocyte production induction medium, and the like.

In the tooth transplantation kit, tooth transplantation method, tooth root generation kit, and/or root generation method, apical papillary stem cells (eg, the above-described composition for tooth transplantation and/or root generation) irradiated with near-infrared (in vitro) before transplantation composition for production), and/or near-infrared irradiation may be performed (in vivo) at the transplant site after transplantation of apical papillary stem cells. The near-infrared irradiation time before the transplantation is the same as described above, and the near-infrared irradiation after transplantation may be performed once a day or twice a day at intervals of 12 hours, and the irradiation time per one time is about 1 to 20 minutes, 1 to 18 minutes. , 1 to 15 minutes, 1 to 12 minutes, 3 to 20 minutes, 3 to 18 minutes, 3 to 15 minutes, 3 to 12 minutes, 5 to 20 minutes, 5 to 18 minutes, 5 to 15 minutes, 5 to 12 minutes , 8 to 20 minutes, 8 to 18 minutes, 8 to 15 minutes, or about 8 to 12 minutes.

In the present specification, dental implantation may mean implanting (injecting, inserting, or administering) the apical papillary stem cells or a composition comprising the same as described above in a region requiring restoration and/or regeneration of teeth. The implantation site may be selected from all sites requiring restoration and/or regeneration of teeth, for example, it may be one or more sites selected from the group consisting of pulp or pulp chamber, periodontal (gingival), etc., but is not limited thereto. it is not In the present specification, restoration and/or regeneration of a tooth is interpreted to include generation, restoration, and/or regeneration of a tooth root. The dental implantation may be performed by implanting a mixture of the apical papillary stem cells or a composition comprising the same with an appropriate scaffold. The scaffold may be appropriately selected from all scaffolds available for dental implantation, and for example, the material may be HA/TCP (Hydroxyapatite/Tricalcium phosphate), PRF (platelet rich fibrin), PLGA (poly(D) , L-lactide-co-glycolide), may be selected from the group consisting of PLG (poly(L-lactic acid)), but is not limited thereto.

In the present specification, a dental implant refers to a material or a structure implanted in a tooth, and a near-infrared irradiated apical papillary stem cell or a composition comprising the same as described above, or the apical papillary stem cell or a composition comprising the same and an appropriate scan It may be a mixture mixed with folds.

In the present specification, the tooth implantation target may be selected from mammals including companion animals such as humans, dogs and cats, livestock such as cattle, horses, pigs, sheep and goats, and rodents such as mice and rats.

As used herein, "differentiation of apical papillary stem cells" may mean that the apical papillary stem cells are differentiated into one or more selected from the group consisting of osteocytes, odontoblasts, adipocytes, and chondrocytes. In addition, differentiation of apical papillary stem cells includes differentiation into root tissue.

The osteocytes (osteocytes) are basic cells of bone tissue, and are also called osteoblasts, osteoblasts, bone cells, and the like. Differentiation into osteocytes is carried out by a series of processes called 'osteogenic differentiation'. In one example, the differentiation of the apical papillary stem cells into osteocytes may be performed in vitro and/or in vivo.

The odontoblast is a columnar connective tissue cell that forms the dentin of the tooth and forms the outer surface of the pulp around it. Differentiation into odontoblasts is carried out by a series of processes called 'dentinogenic differentiation'. In one example, the differentiation of the apical papillary stem cells into odontoblasts may be performed in vitro and/or in vivo.

In one example, the apical papillary stem cells by near-infrared irradiation, compared to the apical papillary stem cells not irradiated with near-infrared rays, ALP (alkaline phosphatase), Col1A (type 1 collagen A), RUNX2 (Runt-related transcription factor 2) ), OCN (osteocalcin), TGF-β1 (Transforming growth factor-beta1), DSPP (dentin sialophosphoprotein), DMP1 (dental matrix protein 1), CEMP-1 (cementoblastoma-derived protein 1), and CAP (cementum attachment protein) The level of one or more proteins selected from the group consisting of and/or coding genes thereof may be increased. In one embodiment, the apical papillary stem cells are selected from the group consisting of ALP, Col1A, RUNX2, OCN, TGF-β1, DSPP, and DMP1 by near-infrared irradiation, compared to the apical papillary stem cells not irradiated with near-infrared rays. It may be an increase in the level of one or more coding genes. In another embodiment, the apical papillary stem cells are one or more protein levels selected from the group consisting of Col1A, DMP1, CEMP-1, CAP, and OCN, compared to the apical papillary stem cells that are not irradiated with near infrared rays by near-infrared irradiation. This may be an increase.

The technology for promoting growth, proliferation, and/or differentiation of apical papillary stem cells by photostimulation using near-infrared light provided herein has an excellent tooth (particularly, root) regeneration effect, so it is useful in the field of restoration or regeneration of teeth. can be advantageously applied.

1 shows the results of isolation and culture of apical papillary stem cells (SCAP), (A) shows the appearance of immature third molars obtained from a patient, (B) is a fibroblast in which SCAP is similar to a mesenchymal stem cell population It shows that spindle shapes are displayed.

2 shows the apparatus and process used for delayed emission analysis, (A) shows a near-infrared (NIR) generating LED device, (B) is an optical sensor of delayed emission (power range: 500pW-0.5mW, S130VC) , Thorlabs, USA), and (C) is a schematic diagram of delayed luminescence analysis. Near-infrared irradiation of the cells was carried out for 1 minute, and spontaneous photon re-emission from the cells was measured with an optical sensor for 23 seconds.

3 is a schematic diagram showing the process of irradiating cells with NIR for in vitro experiments (near infrared rays of 830 nm, energy density of 750 mJ/cm 2 applied daily).

Figure 4 schematically shows the in vivo experimental process, (A) shows that SCAP is mixed with a hydroxyapatite/β-tricalcium phosphate (HA/TCP) scaffold, (B) is a mixture of cells and scaffolds It shows the subcutaneous injection into the dorsal surface of immunocompromised nude mouse, (C) shows the LED device for generating near-infrared (NIR) for in vivo experiment, and (D) is a schematic diagram of near-infrared irradiation for nude mice.

5 (A) shows the same sized digital images (2.25mm width × 1.2mm height) of the histological sections of each group after hematoxylin and eosin staining, (B) is trainable weka segmentation of Image J software Selected cellular and connective tissue regions are shown.

6 (A) shows the same sized digital images (2.25mm width × 1.2mm height) of the histological sections of each group after immunohistochemical staining of osteocalcin, (B) is trainable weka segmentation of Image J software. Selected osteocalcin stained regions are shown.

7 shows the flow cytometry results of SCAP.

8 is a micrograph showing multilineage differentiation of SCAP.

9a and 9b show the results of delayed emission analysis when 830 nm near-infrared (NIR) is irradiated for 1 minute;

9a is a graph showing the measured delayed emission intensity (I(t); top) and excitation level (n(t); bottom) at various frequencies,

9b is a graph showing the measured delayed emission intensity (I(t); top) and excitation level (n(t); bottom) at various in-wavelength duty cycles.

10 is a graph showing the degree of osteogenic differentiation of SCAP irradiated with near-infrared rays of various frequencies, showing alkaline phosphatase activity.

11 is a photograph showing the results of Alizarin Red S staining of SCAP irradiated with near-infrared rays of various frequencies (upper) and a graph quantifying the degree of Alizarin Red S staining shown in the photograph (lower).

Calcium nodules were generated up to 2.5 times at 3 Hz, 30 Hz, and 300 Hz at 10 days compared to the control group. On day 21, many groups, including the control group, showed high calcium deposition. This means that the PBM method accelerates the osteogenic differentiation of SCAP at the early stage of differentiation.

12 is a graph showing quantitatively real-time polymerase chain reaction (RT-PCR) results for 7 genes (ALP, Col1A, RUNX2, OCN, TGF-βDSPP, DMP1, and GAPDH) in SCAP irradiated with near-infrared rays.

13a is an electrophoresis picture showing the results of measuring the levels of five proteins (Col1A, DMP-1, CEMP-1, CAP, and OCN) in SCAP irradiated with near-infrared rays by Western blot, and FIG. 13b is a diagram of FIG. 13a. It is a graph that quantifies the results and shows them as relative values to the control group.

14a and 14b show the results of hematoxylin and eosin staining of SCAP implants, 14a is a staining photograph (magnification. × 80), and 14b is a graph quantifying the stained area.

15 is a photograph of SCAP implants stained with hematoxylin and eosin (magnification × 200).

16a and 16b show the results of immunostaining of osteocalcin (OCN), and 16a is a staining photograph (magnification × 80). 16b is a graph quantifying the stained area.

Hereinafter, the present invention will be described in more detail through Examples and Test Examples. However, these Examples and Test Examples are intended to illustrate the present invention, and should not be construed as limiting the present invention.

Reference Example 1. In vitro test

1.1. Isolation and culture of stem cells from apical papilla

An immature third molar was obtained from the patient (Fig. 1A). The patient was selected from among patients (humans) who did not suffer from tooth decay or cavities to the extent of being dislodged. Informed consent was obtained from all patients. All of the tests in this specification were approved by the Clinical Trial Review Committee of Seoul National University Dental Hospital (Seoul, Korea; IRB No. 05004).

SCAP was isolated and cultured according to conventional methods. Specifically, the apical papillary tissue was carefully separated from the root apex of the obtained immature third molar. The isolated tissue was digested in 3 mg/mL collagenase type 1 (Worthington Biochem, Freehold, NJ, USA) and 4 mg/mL dispase (Boehringer, Mannheim, Germany) solution at 37° C. for 1 hour. The obtained solution was passed through a 40 mm strainer (BD Biosciences, Bedford, MA, USA) to obtain a single cell suspension. The obtained cells were treated with 10% FBS (Gibco BRL, Grand Island, NY, USA), 100 mM ascorbic acid 2-phosphate (Sigma-Aldrich, St. Louis, MS, USA), 2 mM glutamine, 100 U/mL penicillin and 100 mg/mL strepto. Eagle's medium (Gibco BRL, Grand Island, NY, USA) supplemented with mycin (Biouids, Rockville, MD, USA) was cultured in an alpha modulation medium at 37° C. and 5% CO ₂ conditions in an incubator. Cultured cells were used for the 3rd or 4th passage.

1.2. Flow Cytometry of Mesenchymal Stem Cell Markers

In order to confirm the stem cell characteristics of the apical papilla-derived cells cultured in Reference Example 1.1, the expression of mesenchymal stem cell surface markers was evaluated by flow cytometry.

Briefly, the cells (passage 3, 1.0×10 ⁶ cells) prepared in Reference Example 1.1 were suspended in 100 μl of 0.5% bovine serum albumin buffer (ICN Biomedicals, Aurora, OH, USA). After addition of antibodies specific for CD34, CD13, CD90 and CD146 (BD Biosciences, Bedford, MA, USA), the cells were incubated at 4° C. for 1 hour. Then, it was incubated with the fluorescently labeled secondary antibody at room temperature for 1 hour. The percentages of CD34 negative, CD13 positive, CD90 positive, and CD146 positive cells were calculated by FACS Calibur ow flow cytometry (Becton & Dickinson Immunocytometry Systems, San Jose, CA, USA), respectively. The obtained data were analyzed with CellQuest Pro software (Becton & Dickinson Immunocytometry Systems, San Jose, CA, USA).

1.3. Multi-lineage differentiation

In order to confirm the multi-lineage differentiation capacity of SCAP, osteogenic, chondrogenic, and adipogenic differentiation induction were performed, respectively. 1.0×10 ⁴ of SCAP (P3) was seeded in 24-well dishes, bone formation induction medium, chondrogenesis induction medium (StemPro, GibcoBRL, Grand Island, NY, USA) and adipogenesis induction medium (Lonza, Switzerland, Basel, Switzerland) respectively. After induction for 21 days, cells were stained with a specific dye. 2% (w/v) Alizarin Red S stain for calcium deposit staining, 1% Alcian Blue dye for glycosaminoglycan precipitation staining, and 0.3% Oil Red O dye for lipid vacuole staining, respectively. (Sigma-Aldrich, St. Louis, MS, USA). The stained cells were observed with an inverted light microscope (Olympus U-SPT, Olympus, Japan).

1.4. Near infrared irradiation

As a device for irradiating near-infrared rays, a device composed of a diffuser and an LED array was used (see FIG. 2A). The wavelength of the LED (PV810-3C6W-EDISAA, KAOS, Korea) was set to 830 nm. LEDs can operate in continuous wave mode and pulsed wave mode. The pulse wave mode was performed in four different frequency modes including 3Hz, 30Hz, 300Hz and 3000Hz. The frequency was tested while changing to an 8-bit microcontroller device (UM_MC95FG308_V3.20_EN, Hynix, Korea). The in-wavelength duty cycle was tested while changing from 10% to 60%. The total energy density was fixed at ^{750 mJ/cm 2 .} In this example, near-infrared irradiation was performed once a day, for 10 minutes each time, for a total of 6 weeks.

1.5. Delayed luminescence spectroscopy

When a cell is irradiated with light, the cell absorbs the photon energy and then spontaneously emits the photon again. By measuring the photon energy re-emitted from an irradiated cell, also called delayed luminescence (DL), it is possible to determine the optimal light conditions for the cell to absorb as much photon energy as possible.

In this example, near infrared (NIR) of 810 nm was used. Near-infrared irradiation was performed for 1 minute, and spontaneous photon re-emission from the cells was measured for 23 seconds ( FIG. 2 ). For DL measurement, SCAP was washed twice with PBS. An optical sensor with a resolution of 37 ms per data (power range: 500 pW to 0.5 mW, S130VC, Thorlabs, USA) was installed _{in a CO 2 incubator.} The SCAP was placed in a darkroom space for 30 min to eliminate natural light. NIR irradiation was performed and delayed luminescence was measured from the cells. The sensing area of the photosensor was similar to the diameter of a 24-well plate. When NIR was irradiated with a continuous wave and four frequencies of 3 Hz, 30 Hz, 300 Hz and 3000 Hz, respectively, delayed emission was measured. After finding the optimal frequency, the optimal in-wavelength irradiance was tested from 10% to 60% at the found optimal frequency. Intra-wavelength irradiance refers to the rate at which light is emitted within one cycle of a pulse at the optimal frequency found. 100% in-wavelength means continuous wave and 10% in-wavelength means that light is emitted only within 10% of the cycle and not during the remaining 90% period.

DL intensity was measured by the following equation (1):

Formula (1)

In Equation 1, I(t) is the re-emitted intensity from the cells obtained from the experimental data, I ₀ is the initial value of the emission intensity, t is the time, and t ₀ is the initial value, respectively. do.

The intensity I(t) of the delayed emission is correlated with the decay probability P(t), and according to the formula I(t)=-dn(t)/dt, the degree of the excitation level n(t) decreases at time t do. The decay probability P(t) can be calculated by the following equations (2) and (3):

All data were subtracted from cell-free conditions. Control DL data of non-irradiated cells show no intensity levels (10-19 to 10-16 W/cm ² ) outside the optical sensor range.

1.6. Alkaline phosphatase activity measurement

SCAP was incubated in culture wells to at least 80% confluence. Thereafter, the culture medium was replaced with a differentiation medium (see Reference Example 1.3). NIR (near infrared; 830 nm) was irradiated daily for 7 days before alkaline phosphatase (ALP) assay. An energy density of 750 mJ/cm ^{2 was applied to the cells daily.} Continuous wave (CW) and NIR of 3 Hz, 30 Hz, and 300 Hz, respectively, were irradiated to the SCAP (see FIG. 3 ). For ALP staining in SCAP, cells were stained with SensoLyte® pNPP (para-nitrophenyl phosphate) alkaline phosphatase assay kit. Lysis buffer (50 mM Tris HCl and 0.1 % Triton X 100, pH 9.5, Sigma-Aldrich) was added to the cells. Cells were incubated at 4° C. for 10 minutes. Cells were transferred to 96-well plates in an amount of 50 μl per well. 50 μl of pNPP substrate was added to each well and the plate was gently shaken for 30 seconds to mix with the cells. The cells were reacted by incubating at 37° C. for 60 minutes. Absorbance was measured with a microplate reader (Synergy HT, Biotek, VT, USA). Absorbance was expressed as a relative value with respect to the control group (NIR untreated group). The absorbance shows the degree of osteogenic differentiation (fold).

1.7. Alizarin Red S staining test

Alizarin Red S staining was performed to evaluate calcium precipitation in the initial mineralization of SCAP. NIR (830 nm) irradiation was performed at 4 frequencies of 3 Hz, 30 Hz, 300 Hz and 3000 Hz, respectively, for 21 days. The in-wavelength irradiation rate of the pulsed wave was fixed at 30%. Cells were seeded at ^{1.0×10 4} cells per well in 96-well plates. On days 10 and 21 after NIR irradiation, cells were washed twice with PBS and fixed in 4% paraformaldehyde for 10 minutes. Cells were washed with deionized water and stained with Alizarin Red S (Sigma-Aldrich, St Louis, Mo, USA) for 30 min at room temperature. After that, the dye was removed with deionized water and the stained cells were observed by stereo type microscopy. To quantify the calcium precipitate, the dye was extracted using 200 mL of 10% glacial acetic acid for 60 minutes at room temperature. The amount was measured using an ELISA plate reader (Tecan, NC, USA at 490 nm) at 490 nm.

1.8. real-time polymerase chain reaction (RT-PCR)

For real-time polymerase chain reaction (RT-PCR) analysis, SCAP was seeded at ^{5.0×10 4 cells per well in 24-well plates.} NIR was irradiated at 4 frequencies: 3Hz, 30Hz, 300Hz and 3000Hz. After 10 days of NIR irradiation for SCAP in a differentiation medium (see Reference Example 1.3), expanded SCAP was harvested and dissolved. Total RNA was extracted from SCAP using RNeasy Mini Kit (Qiagen, MD, USA). The total RNA was converted into cDNA according to the preparation protocol using reverse transcriptase and random primers (cDNA synthesis kit, Toyobo, Japan). A real-time polymerase chain reaction was performed using the synthesized cDNA using the CFX96TM Real-Time System (BioRad, CA, USA). For comparison between the control group and the PBM group, the relative expression levels of genes were evaluated using the comparative cycle threshold method. The gene expression of ALP, Col1A, RUNX2, OCN, TGF-β1, DSPP, DMP1, and GAPDH was evaluated. The relative expression level of mRNA was normalized to the expression level of GAPDH and expressed as a fold change with respect to the control group. The primer sequences used are shown in Table 1 (Primer Sequences for Real-Time Polymerase Chain Reaction).

(The primers listed in Table 1 are SEQ ID NOs: 1 to 16 in order; ALP, alkaline phosphatase; Col1A, type 1 collagen A; RUNX2, Runt-related transcription factor 2; OCN, osteocalcin; TGF-β1, transforming growth factor- β1; DSPP, dentin sialophosphoprotein; DMP1, dentin matrix protein 1)

1.9. Western blot analysis

^{SCAP (1.0×10 6} cells/dish) was seeded in a 60 mm culture dish and cultured for 21 days in a differentiation medium (see Reference Example 1.3). For the PBM group, NIR irradiation was performed daily for 21 days. Illumination was set at a frequency of 300 Hz and an irradiance rate of 30% within the wavelength. Total lysate proteins were isolated and measured with DC Protein Assay Kit (Bio-Rad Laboratories, CA, USA). 30 mg of protein was dissolved in SDS-PAGE loading buffer and transferred to a polyvinylidene difluoride membrane (GE Healthcare, IL, USA). The membrane was coated with primary antibodies against collagen type 1 (Col1A), cementum attachment protein (CAP), cementoblastoma-derived protein 1 (CEMP-1), osteocalcin (OCN), and dentin matrix protein-1 (DMP-1) ( Santa Cruz Biotechnology, TX, USA). After washing the primary antibody, the membrane was incubated with a horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology, MT, USA) for 1 hour. The obtained blot was visualized with an enhanced chemiluminescence kit (GE Healthcare, IL, USA).

Reference Example 2. Effect of PBM on SCAP (in vivo)

2.1. In vivo transplantation of SCAP

Animal testing was performed with approval from Seoul National University Institutional Animal Care and Use Committee (SNU-190426-13). SCAP was mixed with the xenogenic scaffold in a total amount of 20.0×10 ^{6 cells.} As a scaffold, 50 mg of hydroxyapatite/β-tricalcium phosphate ceramic particles (HA/TCP, Dentium, Seoul, South Korea) was used. 1 ml of cell culture medium (see Reference Example 1.1) was added to the cell-scaffold mixture. _{The mixture was incubated overnight in a CO 2} incubator (MC-20A, Science & Technology Inc., Korea) to allow the cells to adhere to the scaffold.

A drop of fibrin (TISSEEL ® Baxter, IL, USA) was mixed with the implant prior to implantation to facilitate handling and maintain bolus shape. A mixture of cells and scaffolds was subcutaneously implanted into the inner dorsal surface of 10 8-week-old immunocompromised nude mice (NIH-bg-nu/nu-xid; Harlan Sprague-Dawley, OrientBio Inc., Seongnam, Korea). A negative control group (Group 1) was defined as a group transplanted with only HA/TCP without SCAP. Two positive controls were defined as follows: one (Group 2) transplanted with the SCAP cell-scaffold mixture without NIR irradiation, and the other positive control (Group 3) after transplantation of the scaffold only without SCAP. The group under NIR irradiation for 5 weeks. The experimental group (group 4) was a group irradiated with NIR for 5 weeks after transplanting the non-NIR-irradiated SCAP cell-scaffold mixture. The irradiated near-infrared rays have a wavelength of 830 nm, a frequency of 300 Hz, and an irradiation rate within the wavelength fixed to 30%. The groups for the animal experiments are summarized in Table 2 below:

(In Table 2,

HA/TCP, hydroxyapatite/β-tricalcium phosphate; SCAP, stem cells from apical papilla; NIR, near-infrared light)

Two pockets were made on the dorsal surface of the mouse, and cell-free HA/TCP was implanted in one pocket, and SCAP and HA/TCP were implanted in the other pocket. For 5 nude mice, NIR of 830 nm was irradiated on the back skin of nude mice for 5 minutes every day. A total energy density of 750 mJ/cm ² was applied to each mouse daily ( FIG. 4 ).

2.2. Histological evaluation and histomorphometry

Implants were harvested 6 weeks after transplantation. For the PBM (near infrared irradiation) group (groups 3 and 4), NIR irradiation was performed daily for 5 weeks. The implants were fixed in 3.7% paraformaldehyde at 4°C for 24 hours. Demineralization was performed with a 12% EDTA solution (pH 7.3) at 4°C for 1 month. The obtained specimen was embedded in paraffin and sectioned to a thickness of 40 μm. Histological sections were stained with hematoxylin and eosin (H&E staining).

A digital image (2.25mm width × 1.2mm height) of the same size of the histological site of each group was obtained and analyzed (FIG. 5). Images were obtained using a Leica microscopy system (Leica SCN400F slide scanner, Leica Microsystem Ltd, Wetzlar, Germany). Histomorphometry was performed using trainable weka segmentation in Image J software (Arganda-Carreras, I., Kaynig, V., Rueden, C., Eliceiri, KW, Schindelin, J., Cardona, A. , and Sebastian Seung, H. (2017);Trainable Weka Segmentation: a machine learning tool for microscopy pixel classification. Bioinformatics 33, 2424-2426, & Malhan, D., Muelke, M., Rosch, S., Schaefer, AB , Merboth, F., Weisweiler, D., Heiss, C., Arganda-Carreras, I., and El Khassawna, T. (2018). An Optimized Approach to Perform Bone Histomorphometry. Front Endocrinol (Lausanne) 9, 666 ). Cells and connective tissues were selected for trainable weka segmentation (FIG. 5B). Selected pixels to total pixels are expressed as a percentage.

2.3. Immunohistochemistry of osteocalcin

Slides with tissue fragments were stained with Discovery XT automated immunohistochemistry stainer (Ventana Medical Systems, Inc., Tucson, AZ, USA). Detection was performed using the Ventana ChromoMap Kit (Ventana Medical Systems, AZ, USA). The fragments were treated with EZ Prep solution (Roche, Basel, Switzerland) to remove paraffin. Slides were incubated with a rabbit polyclonal antibody against osteocalcin (OCN) (1:100; Santa Cruz Biotechnology, TX, USA) at 37° C. for 32 minutes, and incubated with a secondary antibody at 37° C. for 20 minutes. Slides were incubated with DAB+H ₂ O ₂ substrate at 37° C. for 8 minutes. The stained fragments were counterstained with hematoxylin.

In each group, osteocalcin-stained digital images of the same size (2.25mm width × 1.2mm height) were obtained and analyzed ( FIG. 6A ). Tissue morphology analysis was performed using trainable weka segmentation in Image J software. Osteocalcin staining sites were selected by trainable weka segmentation (Fig. 6B). Selected pixels relative to total pixels are expressed as a percentage.

2.4 Statistical Analysis

For in vitro experiments, all experiments were repeated at least 3 times and the data were expressed as mean and standard deviation (SD). Statistical significance was assessed by unpaired Student's t-tests (two-tail, equal SD).

For in vivo histomorphometry experiments, the Mann-Whitney U test was used. All statistical analyzes were performed with SPSS for Windows (version 22.0, SPSS Inc, Chicago, USA). Significance was defined as p <0.05 in in vitro experiments and p < 0.1 in in vivo experiments. P values are indicated on the graph.

Example 1: Optimal PBM (Photobiomodulation) status and PBM effect on stem cells from apical papilla (SCAP) ( in vitro )

1.1. Characteristics of SCAP

As described in Reference Example 1.1, SCAP was isolated and cultured from the root apex of the immature third molar (see FIG. 1A), and the appearance was observed under a microscope and shown in FIG. 1B. As confirmed in FIG. 1B, SCAP exhibited fibroblast spindle shapes similar to the mesenchymal stem cell population.

In addition, flow cytometry was performed according to the method of Reference Example 1.2, and the results obtained are shown in FIG. 7 . As shown in FIG. 7 , it was confirmed that about 91.81% of SCAP expressed CD13, about 99.35% of CD90, about 83.69% of CD146, and about 1.93% of CD34, respectively. Among these markers, CD34 is a negative marker for mesenchymal stem cells. The percentage (%) of cells positive for the markers was measured by quantifying the relative fluorescence intensity of cells bound to the antibody for each marker. The results show that the isolated/cultured SCAP has a high proportion of cells expressing a mesenchymal stem cell positive marker (about 83% or more), and a low proportion of cells expressing a mesenchymal stem cell negative marker (about 2% or less). ), demonstrating that SCAP cells have the characteristics of stem cells (mesenchymal stem cells).

1.2. Multi-lineage differentiation of SCAP

With reference to Reference Example 1.3, the ability of SCAP to differentiate into multiple lines was evaluated. SCAP was cultured in bone formation induction medium, adipocyte formation induction medium, and chondrogenesis induction medium, respectively, and after differentiation induction for 3 weeks, the stained cells were analyzed using an inverted light microscope (Olympus U-SPT, Olympus, Japan). The observed results are shown in FIG. 8 . As shown in FIG. 8 , it was confirmed that small circular Alizarin Red-positive calcium nodules were generated in the SCAP culture (topmost photograph) cultured in the bone formation induction medium. In addition, it was confirmed that SCAP formed Oil Red O-positive lipid clusters after induction of adipogenesis and Alcian Blue-positive nodules after induction of cartilage formation (FIG. 8).

1.3. Delayed luminescence assay

Referring to Reference Examples 1.4 and 1.5, after irradiating the SCAP with near-infrared (NIR) of 830 nm for 1 minute, delayed emission (DL) was measured for 23 seconds from the near-infrared irradiated cells.

First, each of the delayed emission measurements during near-infrared irradiation at various frequencies (CW (continuous wave), 3 Hz, 30 Hz, 300 Hz and 3000 Hz), and the results are shown in FIG. 9A (delayed emission intensity I(t) (top) and Here level n(t) (bottom)). As shown in FIG. 9A , delayed emission (delayed emission intensity and excitation level) was highest at a frequency of 300 Hz, and lowest at a frequency of 3 Hz or continuous wave (the upper and lower graphs of FIG. ≥ 30 Hz > 3 Hz ≒ represents the result of CW). These results show that SCAP absorbs a lot of near-infrared rays at frequencies of 30 Hz or higher (eg, 30 Hz to 3000 Hz), eg, 300 Hz.

Next, by measuring the delayed luminescence at various in-wavelength irradiation rates (duty cycles; 10%, 20%, 30%, 40%, 50%, and 60%) at 300 Hz NIR (830 nm) irradiation, respectively, The results are shown in FIG. 9B (delayed emission intensity I(t) (top) and excitation level n(t) (bottom)). As shown in FIG. 9B, delayed emission (delayed emission intensity and excitation level) appeared highest at 30% in-wavelength irradiation and lowest at 50% in-wavelength irradiation (top and bottom graphs in FIG. 9b are above 30% > 60% ≥ 20% > 10% ≒ 40% ≒ 50%), and SCAP shows that near-infrared absorption is high when the in-wavelength irradiance is about 30%.

These results show that SCAP more effectively absorbs near-infrared rays (eg, 830 nm) when the near-infrared rays are irradiated with a frequency of 300 Hz and an in-wavelength irradiance of 30%.

1.4. Alkaline phosphatase activity

The effect of PBM therapy (near infrared irradiation) on alkaline phosphatase (ALP) activity of SCAP was confirmed. ALP is an indicator of osteogenic differentiation, and in this example, ALP activity was tested by measuring the degree of osteogenic differentiation.

More specifically, with reference to Reference Example 1.6, the degree of osteogenic differentiation of SCAP irradiated with near-infrared rays (830 nm) of various frequencies for 7 days was measured on the 3rd, 5th, and 7th days of NIR irradiation, respectively, and ALP Activity was assayed. The measured osteogenic differentiation degree was calculated as a relative value with respect to the control group (cont; non-near-infrared treatment group), and is shown in FIG. 10 . As shown in FIG. 10 , all groups irradiated with near-infrared rays of 3 Hz, 30 Hz, and 300 Hz for 7 days showed an increase in the degree of osteogenic differentiation at least 1.3 times compared to the control group (p < 0.05), indicating that the increase in ALP activity was Confirmed. Among them, the highest ALP activity was observed in the group irradiated with near-infrared rays of 300 Hz for 7 days. These results show that near-infrared rays having a frequency of around 300 Hz are the optimal frequency to stimulate SCAP to induce osteogenic differentiation.

1.5. Calcium deposition evaluation

In order to evaluate calcium precipitation during matrix mineralization in SCAP irradiated with near-infrared rays, Alizarin Red S staining was performed with reference to Reference Example 1.7. After irradiating the SCAP with near-infrared rays (830 nm) having 4 frequencies of 3 Hz, 30 Hz, 300 Hz and 3000 Hz for 21 days (intra-wavelength irradiation rate is fixed at 30%), the result of staining with Alizarin Red S is shown in FIG. It was. The upper part of FIG. 11 is a photograph showing the state of SCAP Alizarin Red S staining according to the near-infrared frequency (the lower two lines are a 40-fold magnification of the upper two lines), and the lower portion is the obtained Alizarin Red S staining degree It is a graph expressed as a relative value with respect to the control group (near-infrared untreated group) by quantifying . As shown in FIG. 11 , it can be seen that the degree of calcium precipitation on the 10th day in the near-infrared treatment group with frequencies of 3 Hz, 30 Hz, 300 Hz and 3000 Hz was significantly increased compared to the control group. These results can be said to show that near-infrared irradiation promotes the differentiation of apical papillary stem cells at an early stage.

1.6. Marker gene and protein measurement

Referring to Reference Example 1.8, RT for 7 genes (ALP, Col1A, RUNX2, OCN, TGF-β, DSPP, DMP1) related to bone and tooth formation, and GAPDH after 10 days of near-infrared (830 nm) irradiation on SCAP -PCR was performed, and the results are shown in FIG. 12 . The ALP gene showed the highest expression rate when irradiated with near-infrared rays with a frequency of 3 Hz. OCN and RUNX2 genes related to bone formation and DMP1, DSPP and TGFb1 genes related to dentin formation all showed the highest expression rates when irradiated with near-infrared rays at a frequency of 300 Hz. The expression of Col1A was increased compared to the control group in all of the near-infrared irradiation with frequencies of 3 Hz, 30 Hz and 300 Hz.

In addition, referring to Reference Example 1.9, SCAP was irradiated with near-infrared (830 nm) at 300 Hz frequency and 30% wavelength for 21 days, followed by Western blot analysis, Col1A, DMP-1, CEMP-1, CAP, and OCN protein levels were measured, and the results are shown in FIGS. 13A (electrophoresis picture) and FIG. 13B (quantification graph). As shown in FIGS. 13A and 13B , the expression levels of Col1A, DMP-1, CEMP-1, CAP, and OCN proteins in the near-infrared irradiation group (PBM) all significantly increased compared to the control group.

Example 2. PBM (Photobiomodulation) effect on SCAP ( in vivo )

2.1. Histological evaluation and histomorphometry

Referring to Reference Example 2.1, SCAP was implanted in mice. Visually, the implant was hardened, and no inflammatory reaction was observed in the skin of the implantation site in all test groups.

With reference to Immersion Example 2.2, the transplant was recovered at 6 weeks after transplantation, and histological evaluation was performed by staining with hematoxylin and eosin, and the results are shown in FIGS. 14a (stained photograph, x80), FIG. 14b (quantification) one graph), and FIG. 15 (stained photograph, x200). As shown in FIGS. 14A and 14B , newly formed hard tissues were not clearly observed in the group in which the SCAP was not transplanted [Group 1 (HA/TCP transplant only) and Group 3 (HA/TCP transplant + near-infrared irradiation)]. didn't Also, in all PBM groups irradiated with near-infrared rays (group 3 (HA/TCP implantation + near-infrared irradiation) and group 4 (HA/TCP implantation with SCAP + near-infrared irradiation)), cell and connective tissue portions were significantly higher than in group 1. increased. In particular, in the case of group 3 irradiated with near-infrared rays without SCAP, the cell and connective tissue portion increased significantly compared to group 1. In addition, as shown in FIG. 15 , only the scaffolds and cell parts in the group transplanted with SCAP [group 2 (HA/TCP transplantation containing SCAP) and group 4 (HA/TCP transplantation including SCAP + near-infrared irradiation)] New hard tissue was observed at the interface between them.

2.2. Immunohistochemical evaluation using osteocalcin (OCN)

With reference to Reference Example 2.3, immunostaining was performed using osteocalcin (OCN) for groups 1 to 4, and the results are shown in FIGS. 16a (stained photograph, x80) and 16b (quantification graph). As shown in FIG. 16A , the new hard tissue was stained with osteocalcin at the interface between the scaffold and the cell part. Such osteocalcin staining was hardly observed in the group in which SCAP transplantation was not performed (groups 1 and 3), whereas distinct staining was observed in the group in which SCAP transplantation was performed (groups 2 and 4), and in group 4 irradiated with near-infrared rays. In this case, it was found that the degree of osteocalcin staining was increased compared to Group 2, which was not irradiated with near-infrared rays.

Claims (21)

1. A composition for tooth transplantation or root generation, comprising near-infrared irradiated apical papilla stem cells (stem cells from apical papilla; SCAP).
2. According to claim 1, wherein the apical papillary stem cells will be isolated from the apex (root apex) of immature permanent teeth, a tooth transplantation or a composition for generating a root.
3. According to claim 1, wherein the near-infrared is a pulsed wave (pulsed wave) having a wavelength of 800nm to 900nm, tooth implantation or tooth root generation composition.
4. According to any one of claims 1 to 3, wherein the near-infrared radiation will have the following characteristics, the composition for dental implantation or root generation:

   (1) a frequency of 30 Hz to 3000 Hz;

   (2) an in-wavelength duty cycle of 25 to 35%; or

   (3) A combination of (1) and (2) above.
5. The method according to any one of claims 1 to 3, wherein the near-infrared irradiated apical papillary stem cells, ALP, Col1A, RUNX2, OCN, TGF-β1, DSPP compared to the apical papillary stem cells not irradiated with near-infrared rays. , And one or more coding genes selected from the group consisting of DMP1 will increase the level, a tooth transplantation or a composition for generating a root.
6. The method according to any one of claims 1 to 3, wherein the near-infrared irradiated apical papillary stem cells, Col1A, DMP-1, CEMP-1, CAP, and One or more protein levels selected from the group consisting of OCN is increased, a composition for dental implantation or root generation.
7. The method according to any one of claims 1 to 3, wherein the near-infrared irradiated apical papillary stem cells are differentiated into one or more selected from the group consisting of osteocytes, odontoblasts, adipocytes, and chondrocytes. composition for production.
8. apical papillary stem cells, and

   Light source for near-infrared irradiation

   A kit for dental implantation or root generation, comprising a.
9. According to claim 8, wherein the apical papillary stem cells will be isolated from the apical end of immature permanent teeth, tooth transplantation or tooth root generation kit.
10. The kit of claim 8 or 9, wherein the near-infrared radiation is a pulsed wave having a wavelength of 800 nm to 900 nm.
11. The kit for dental implantation or root generation according to claim 8 or 9, wherein the near-infrared radiation has the following characteristics:

    (1) a frequency of 30 Hz to 3000 Hz;

    (2) an in-wavelength duty cycle of 25 to 35%; or

    (3) A combination of (1) and (2) above.
12. The method according to claim 8 or 9, wherein the apical papillary stem cells are ALP, Col1A, RUNX2, OCN, TGF-β1, DSPP, and DMP1 by near-infrared irradiation, compared to the apical papillary stem cells not irradiated with near-infrared rays. The level of one or more encoding genes selected from the group consisting of increases, a tooth implantation or tooth root generation kit.
13. The method according to claim 8 or 9, wherein the apical papillary stem cells are made of Col1A, DMP-1, CEMP-1, CAP, and OCN by near-infrared irradiation, compared to the apical papillary stem cells not irradiated with near-infrared rays. One or more protein levels selected from the group increase, a kit for dental implantation or root generation.
14. The method of claim 8 or 9, wherein the apical papillary stem cells are differentiated into one or more selected from the group consisting of osteocytes, odontoblasts, adipocytes, and chondrocytes by near-infrared irradiation, dental implantation or root generation for kit.
15. A method of manufacturing a dental implant comprising the step of irradiating near-infrared rays to the isolated apical papillary stem cells.
16. A method of differentiation of apical papillary stem cells, comprising the step of irradiating near-infrared rays to the separated apical papillary stem cells.
17. The method of claim 16, wherein the differentiation of the apical papillary stem cells is differentiation into one or more selected from the group consisting of osteocytes, odontoblasts, adipocytes, and chondrocytes.
18. A method for producing a tooth root, comprising the step of irradiating near-infrared rays to the separated apical papillary stem cells.
19. The method according to any one of claims 15 to 18, wherein the apical papillary stem cells are isolated from the root apex of immature permanent teeth.
20. 19. The method according to any one of claims 15 to 18, wherein the near infrared is a pulsed wave having a wavelength of 800 nm to 900 nm.
21. 19. The method according to any one of claims 15 to 18, wherein the near infrared has the following characteristics:

    (1) a frequency of 30 Hz to 3000 Hz;

    (2) an in-wavelength duty cycle of 25 to 35%; or

    (3) A combination of (1) and (2) above.

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