WO2021221199A1 - Procédé d'accélération de la différenciation de cellules souches à partir de papille apicale à l'aide d'une stimulation optique - Google Patents

Procédé d'accélération de la différenciation de cellules souches à partir de papille apicale à l'aide d'une stimulation optique Download PDF

Info

Publication number
WO2021221199A1
WO2021221199A1 PCT/KR2020/005637 KR2020005637W WO2021221199A1 WO 2021221199 A1 WO2021221199 A1 WO 2021221199A1 KR 2020005637 W KR2020005637 W KR 2020005637W WO 2021221199 A1 WO2021221199 A1 WO 2021221199A1
Authority
WO
WIPO (PCT)
Prior art keywords
stem cells
apical
infrared
irradiated
group
Prior art date
Application number
PCT/KR2020/005637
Other languages
English (en)
Korean (ko)
Inventor
김홍배
정필훈
정종훈
강문호
Original Assignee
서울대학교산학협력단
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 서울대학교산학협력단 filed Critical 서울대학교산학협력단
Priority to PCT/KR2020/005637 priority Critical patent/WO2021221199A1/fr
Publication of WO2021221199A1 publication Critical patent/WO2021221199A1/fr

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61C—DENTISTRY; APPARATUS OR METHODS FOR ORAL OR DENTAL HYGIENE
    • A61C8/00—Means to be fixed to the jaw-bone for consolidating natural teeth or for fixing dental prostheses thereon; Dental implants; Implanting tools
    • A61C8/0003—Not used, see subgroups
    • A61C8/0004—Consolidating natural teeth
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N5/00—Radiation therapy
    • A61N5/06—Radiation therapy using light
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/42—Apparatus for the treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

  • 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.
  • LEDs light emitting diodes
  • 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.
  • stem cells from apical papilla 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.
  • 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. .
  • SCAP stem cells from apical papilla
  • PBM photobiological control
  • composition for dental implantation comprising apical papillary stem cells irradiated with near-infrared rays.
  • composition for generating a tooth root comprising the near-infrared irradiated apical papillary stem cells.
  • kits for dental implantation including a light source for apical papillary stem cells, and near-infrared irradiation.
  • kits 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.
  • apical papillary stem cells 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.
  • 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.
  • 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.
  • an example of the present invention provides a composition for dental implantation, comprising apical papillary stem cells irradiated with near infrared rays.
  • 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.
  • compositions 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.
  • 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.
  • the apical papillary stem cells may be isolated from the root apex of immature permanent teeth.
  • the near-infrared light may be a pulsed wave having a wavelength of 800 nm to 900 nm.
  • the near-infrared radiation may have the following characteristics:
  • the near-infrared irradiated apical papillary stem cells, ALP, Col1A, RUNX2, OCN, TGF- ⁇ , 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.
  • 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.
  • 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.
  • osteocytes eg, alveolar osteocytes
  • odontoblasts e.g., odontoblasts
  • adipocytes e.g, chondrocytes.
  • kits for dental implantation comprising a light source for apical papillary stem cells, and near-infrared irradiation.
  • 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.
  • kits 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.
  • the apical papillary stem cells may be isolated from the root apex of immature permanent teeth.
  • the near-infrared light may be a pulsed wave having a wavelength of 800 nm to 900 nm.
  • the near-infrared radiation may have the following characteristics:
  • the apical papillary stem cells are ALP, Col1A, RUNX2, OCN, TGF- ⁇ , 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.
  • 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.
  • 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.
  • 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
  • stem cells from apical papilla 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).
  • DPSC dental pulp stem cells
  • 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.
  • a mesenchymal stem cell positive marker for example, STRO-1, CD13, CD90, CD146, CD18, CD34, CD45, and the like.
  • the proportion of cells expressing a mesenchymal stem cell positive marker 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.
  • NIR Near-infrared light
  • 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.
  • the near-infrared light may be a pulsed wave having a wavelength of 800 nm to 900 nm, but is not limited thereto.
  • the near-infrared rays are
  • 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.
  • 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.
  • the present invention is not limited thereto.
  • 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.
  • 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.
  • apical papillary stem cells eg, the above-described composition for tooth transplantation and/or root generation
  • near-infrared in vitro
  • 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.
  • 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.
  • HA/TCP Hydroapatite/Tricalcium phosphate
  • PRF platelet rich fibrin
  • PLGA poly(D) , L-lactide-co-glycolide
  • PLG poly(L-lactic acid)
  • 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.
  • 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.
  • 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.
  • differentiation of apical papillary stem cells includes differentiation into root tissue.
  • the 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.
  • 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- ⁇ 1 (Transforming growth factor-beta1), DSPP (dentin sialophosphoprotein), DMP1 (dental matrix protein 1), CEMP-1 (cementoblastoma-derived protein 1), and CAP (cementum attachment protein)
  • ALP alkaline phosphatase
  • Col1A type 1 collagen A
  • RUNX2 Raster-related transcription factor 2
  • OCN osteocalcin
  • TGF- ⁇ 1 Transforming growth factor-beta1
  • DSPP disforming growth factor-beta1
  • DMP1 dental matrix protein 1
  • CEMP-1 cemento
  • the apical papillary stem cells are selected from the group consisting of ALP, Col1A, RUNX2, OCN, TGF- ⁇ 1, 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.
  • 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.
  • SCAP apical papillary stem cells
  • 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.
  • NIR near-infrared
  • B is an optical sensor of delayed emission (power range: 500pW-0.5mW, S130VC) , Thorlabs, USA)
  • 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.
  • FIG. 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/ ⁇ -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
  • D is a schematic diagram of near-infrared irradiation for nude mice.
  • FIG. 5 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.
  • FIG. 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.
  • 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.
  • FIG. 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.
  • RT-PCR quantitatively real-time polymerase chain reaction
  • FIG. 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
  • 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.
  • Fig. 1A 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.
  • the cells (passage 3, 1.0 ⁇ 10 6 cells) prepared in Reference Example 1.1 were suspended in 100 ⁇ l 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).
  • 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 .
  • near-infrared irradiation was performed once a day, for 10 minutes each time, for a total of 6 weeks.
  • DL delayed luminescence
  • NIR near infrared
  • 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.
  • 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.
  • 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 decay probability P(t) can be calculated by the following equations (2) and (3):
  • Control DL data of non-irradiated cells show no intensity levels (10-19 to 10-16 W/cm 2 ) outside the optical sensor range.
  • 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
  • ALP alkaline phosphatase
  • An energy density of 750 mJ/cm 2 was applied to the cells daily.
  • 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
  • Cells were incubated at 4° C. for 10 minutes.
  • Cells were transferred to 96-well plates in an amount of 50 ⁇ l per well.
  • 50 ⁇ l 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).
  • 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.
  • the dye was removed with deionized water and the stained cells were observed by stereo type microscopy.
  • 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.
  • RT-PCR real-time polymerase chain reaction
  • a real-time polymerase chain reaction was performed using the synthesized cDNA using the CFX96TM Real-Time System (BioRad, CA, USA).
  • the relative expression levels of genes were evaluated using the comparative cycle threshold method.
  • the gene expression of ALP, Col1A, RUNX2, OCN, TGF- ⁇ 1, 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- ⁇ 1, transforming growth factor- ⁇ 1; DSPP, dentin sialophosphoprotein; DMP1, dentin matrix protein 1)
  • 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).
  • 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).
  • CAP cementum attachment protein
  • CEMP-1 cementoblastoma-derived protein 1
  • OCN osteocalcin
  • DMP-1 dentin matrix protein-1
  • SCAP was mixed with the xenogenic scaffold in a total amount of 20.0 ⁇ 10 6 cells.
  • As a scaffold 50 mg of hydroxyapatite/ ⁇ -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.
  • HA/TCP hydroxyapatite/ ⁇ -tricalcium phosphate ceramic particles
  • 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 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%.
  • Table 2 The groups for the animal experiments are summarized in Table 2 below:
  • HA/TCP hydroxyapatite/ ⁇ -tricalcium phosphate
  • SCAP stem cells from apical papilla
  • NIR near-infrared light
  • Implants were harvested 6 weeks after transplantation.
  • PBM near infrared irradiation
  • 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 ⁇ m. Histological sections were stained with hematoxylin and eosin (H&E staining).
  • 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.
  • Example 1 Optimal PBM (Photobiomodulation) status and PBM effect on stem cells from apical papilla (SCAP) ( in vitro )
  • 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.
  • 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.
  • 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).
  • SCAP 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).
  • 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)).
  • 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).
  • SCAP absorbs a lot of near-infrared rays at frequencies of 30 Hz or higher (eg, 30 Hz to 3000 Hz), eg, 300 Hz.
  • ALP alkaline phosphatase
  • 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.
  • 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.
  • RT for 7 genes (ALP, Col1A, RUNX2, OCN, TGF- ⁇ , 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.
  • 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).
  • 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.
  • 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.
  • 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)].
  • FIGS. 16a stained photograph, x80
  • 16b quantification graph
  • 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.

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Epidemiology (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • General Engineering & Computer Science (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • Botany (AREA)
  • Radiology & Medical Imaging (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Dermatology (AREA)
  • Pathology (AREA)
  • Transplantation (AREA)
  • Sustainable Development (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Dentistry (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne une technique d'accélération de la différenciation de cellules souches de papille apicale (SCAP) à l'aide d'une stimulation optique. Plus particulièrement, la présente invention concerne une composition, un kit et un procédé de transplantation de dent ou de formation de racine dentaire, comprenant des SCAP irradiées par un rayonnement proche infrarouge, et un procédé de différenciation de SCAP, comprenant une étape d'irradiation de SCAP avec un rayonnement proche infrarouge.
PCT/KR2020/005637 2020-04-28 2020-04-28 Procédé d'accélération de la différenciation de cellules souches à partir de papille apicale à l'aide d'une stimulation optique WO2021221199A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/KR2020/005637 WO2021221199A1 (fr) 2020-04-28 2020-04-28 Procédé d'accélération de la différenciation de cellules souches à partir de papille apicale à l'aide d'une stimulation optique

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/KR2020/005637 WO2021221199A1 (fr) 2020-04-28 2020-04-28 Procédé d'accélération de la différenciation de cellules souches à partir de papille apicale à l'aide d'une stimulation optique

Publications (1)

Publication Number Publication Date
WO2021221199A1 true WO2021221199A1 (fr) 2021-11-04

Family

ID=78332031

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2020/005637 WO2021221199A1 (fr) 2020-04-28 2020-04-28 Procédé d'accélération de la différenciation de cellules souches à partir de papille apicale à l'aide d'une stimulation optique

Country Status (1)

Country Link
WO (1) WO2021221199A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101212548B1 (ko) * 2007-10-01 2012-12-14 재단법인서울대학교산학협력재단 새로운 치소낭 유래의 치아 줄기세포 및 그의 배양 방법
KR101859683B1 (ko) * 2016-03-17 2018-07-02 서강대학교산학협력단 줄기세포 분화 제어를 위한 바이오몰레트론

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101212548B1 (ko) * 2007-10-01 2012-12-14 재단법인서울대학교산학협력재단 새로운 치소낭 유래의 치아 줄기세포 및 그의 배양 방법
KR101859683B1 (ko) * 2016-03-17 2018-07-02 서강대학교산학협력단 줄기세포 분화 제어를 위한 바이오몰레트론

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
IVANA MARIA ZACCARA, LETÍCIA BOLDRIN MESTIERI, MARIA STELLA MOREIRA, FABIANA SOARES GRECCA, MANOELA DOMINGUES MARTINS, PATRÍCIA MA: "Photobiomodulation therapy improves multilineage differentiation of dental pulp stem cells in three-dimensional culture model", JOURNAL OF BIOMEDICAL OPTICS, vol. 23, no. 9, 10 September 2018 (2018-09-10), pages 1 - 9, XP055862387, DOI: 10.1117/1.JBO.23.9.095001 *
KIM HONG BAE, BAIK KU YOUN, CHOUNG PILL-HOON, CHUNG JONG HOON: "Pulse frequency dependency of photobiomodulation on the bioenergetic functions of human dental pulp stem cells", SCIENTIFIC REPORTS, vol. 7, no. 1, 15927, 1 December 2017 (2017-12-01), pages 1 - 12, XP055862383, DOI: 10.1038/s41598-017-15754-2 *
KIM HONG BAE, BAIK KU YOUN, SEONWOO HOON, JANG KYOUNG-JE, LEE MYUNG CHUL, CHOUNG PILL-HOON, CHUNG JONG HOON: "Effects of pulsing of light on the dentinogenesis of dental pulp stem cells in vitro", SCIENTIFIC REPORTS, vol. 8, no. 1, 2057, 1 December 2018 (2018-12-01), pages 1 - 11, XP055862381, DOI: 10.1038/s41598-018-19395-x *

Similar Documents

Publication Publication Date Title
Zhou et al. Dental follicle cells: roles in development and beyond
Kawaguchi et al. Enhancement of periodontal tissue regeneration by transplantation of bone marrow mesenchymal stem cells
Wang et al. Gingiva-derived mesenchymal stem cell-mediated therapeutic approach for bone tissue regeneration
Huggins et al. Dentin matrix transformation: rapid induction of alkaline phosphatase and cartilage
Nakashima et al. Stimulation of reparative dentin formation by ex vivo gene therapy using dental pulp stem cells electrotransfected with growth/differentiation factor 11 (Gdf11)
Wang et al. Preliminary study on dental pulp stem cell–mediated pulp regeneration in canine immature permanent teeth
Ajay Sharma et al. Advances in regeneration of dental pulp–a literature review
Dahake et al. Response of stem cells from human exfoliated deciduous teeth (SHED) to three bioinductive materials–An in vitro experimental study
Torreggiani et al. Preosteocytes/osteocytes have the potential to dedifferentiate becoming a source of osteoblasts
CN1636054A (zh) 来自人胚胎干细胞的间充质细胞和成骨细胞
Wang et al. Effects of the enamel matrix derivative on the proliferation and odontogenic differentiation of human dental pulp cells
Saczko et al. A simple and established method of tissue culture of human gingival fibroblasts for gingival augmentation.
Tuisku et al. Evidence for a neural influence on tooth germ generation in a polyphyodont species
Kim et al. The dynamic healing profile of human periodontal ligament stem cells: histological and immunohistochemical analysis using an ectopic transplantation model
WO2017200197A1 (fr) Composition pour favoriser la croissance de cellules souches comprenant de la phytosphingosine-1-phosphate ou des dérivés de celle-ci, et composition pour la culture de milieux de cellules souches comprenant celle-ci
Yang et al. Tooth Germ‐Like Construct Transplantation for Whole‐Tooth Regeneration: An In Vivo Study in the Miniature Pig
WO2021221199A1 (fr) Procédé d'accélération de la différenciation de cellules souches à partir de papille apicale à l'aide d'une stimulation optique
CN109847098A (zh) 一种用于修复骨缺损的复合生物支架材料
WO2010036009A2 (fr) Support poreux pour la régénération tissulaire guidée et procédé de préparation de ce support
JP2008511363A (ja) 歯原基を使用する顎骨の増強
Robey et al. Skeletal stem cells: Tissue-specific stem/progenitor cells of cartilage, bone, stroma, and marrow adipocytes
Dong et al. Metformin enhances the osteogenic activity of rat bone marrow mesenchymal stem cells by inhibiting oxidative stress induced by diabetes mellitus: An in vitro and in vivo study
KR102313380B1 (ko) 광자극을 이용한 치근단유두 줄기세포의 분화 촉진 방법
Ishizeki et al. Formation of the sphenomandibular ligament by Meckel's cartilage in the mouse: possible involvement of epidermal growth factor as revealed by studies in vivo and in vitro
Tian et al. The effects of Sema3A overexpression on the proliferation and differentiation of rat gingival mesenchymal stem cells in the LPS-induced inflammatory environment

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20933054

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20933054

Country of ref document: EP

Kind code of ref document: A1