WO2015050315A2 - Method for promoting osteogenesis using magnetic nanoparticles and eletromagnetic field - Google Patents

Method for promoting osteogenesis using magnetic nanoparticles and eletromagnetic field Download PDF

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WO2015050315A2
WO2015050315A2 PCT/KR2014/006404 KR2014006404W WO2015050315A2 WO 2015050315 A2 WO2015050315 A2 WO 2015050315A2 KR 2014006404 W KR2014006404 W KR 2014006404W WO 2015050315 A2 WO2015050315 A2 WO 2015050315A2
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bone
electromagnetic field
magnetic nanoparticles
cells
differentiation
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Korean (ko)
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ÖdæÖqa²¶ð
서영권
Ô¢nÒ¸ÐÖµn
박희정
²¾ØÖæµÔ®½
김유미
Ô¢nÖµn²¼¾
박정극
²¾ØÖcaÒpð
김상헌
²¾ØÔ®¯Öqv
김민옥
ÖæuÒ¸ÐÒºh
윤희훈
ÖµnÒqd
정현
Ö¸©ÖseÒq½
이종호
²¾ØÖhqÖ¢Ð
김수찬
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Ô±r²§ÝÔðØÒnr²§°€Öb¢ÒnrÒqjÔµvÔ»y
동국대학교 산학협력단
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    • 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
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • 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
    • A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
    • A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
    • 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
    • A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
    • A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
    • A61L27/446—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix with other specific inorganic fillers other than those covered by A61L27/443 or A61L27/46
    • 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
    • A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
    • A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
    • A61L27/46—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix with phosphorus-containing inorganic fillers
    • 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
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • 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
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/12—Nanosized materials, e.g. nanofibres, nanoparticles, nanowires, nanotubes; Nanostructured surfaces
    • 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
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants

Definitions

  • the present invention relates to methods of using magnetic nanoparticles and electromagnetic fields to induce bone regeneration, in particular healing or regeneration of damaged alveolar bone and bone tissue.
  • electromagnetic fields have been used for neurodifferentiation, bone differentiation, and pain treatment using low intensity of less than 20 G (Gaussian) in the low frequency region of 7.5 Hz to 100 Hz.
  • the inventors of the present invention intend to improve the therapeutic efficacy of bone injury sites by promoting bone cell activation and differentiation of mesenchymal stem cells into bone cells by using an electromagnetic field.
  • the present invention has been completed by developing a technique for maximizing the effect of electromagnetic treatment of the electromagnetic field by briefly irradiating the electromagnetic field of strength and high intensity within 1 hour.
  • An object of the present invention is to apply a hydrogel or implant containing magnetic nanoparticles to a bone injury site, and then by irradiating an electromagnetic field of a specific frequency and intensity to improve the treatment efficiency of the bone injury site or promote bone fusion to promote bone regeneration To provide a way.
  • the method of promoting bone regeneration using the magnetic nanoparticles and the electromagnetic field according to the present invention may induce rapid bone treatment as a method of physical therapy after surgery, and introduce the magnetic nanoparticles or the bone formation promoting substance into the hydrogel or the scaffold.
  • by implanting the bone damage site can maximize the efficiency of bone treatment.
  • 1 is a diagram showing the microscopic results of osteoblasts irradiated with electromagnetic fields of various frequencies for 3 days.
  • FIG. 2 mRNA of collagen, bone sialo protein (bonesialoprotein), osteonectin, osteocalcin, osteopontin, non-mentin, type 2 bone morphogenetic protein (BMP-2) after irradiating electromagnetic fields of various frequencies to bone cells for 3 days It is a figure which shows the result of analyzing expression.
  • Figure 3 is a diagram showing the microscopic results of mesenchymal stem cells irradiated with electromagnetic fields of various frequencies for 3 days.
  • Figure 4 shows the results of analyzing the mRNA expression of collagen, osteonectin, osteocalcin, osteopontin, non-mentin, type 2 bone morphogenetic protein (BMP-2) after irradiating electromagnetic fields of various frequencies to mesenchymal stem cells for 3 days Is a diagram showing.
  • FIG. 5 shows the results of analyzing protein expression of collagen, osteonectin, type 2 osteoblastic protein (BMP-2), p-ERK, and p-CREB after irradiating electromagnetic fields of various frequencies to mesenchymal stem cells for 14 days. The figure shown.
  • FIG. 6 is a diagram showing the results of immunostaining osteopontin after irradiation of mesenchymal stem cells with electromagnetic fields of various frequencies for 14 days.
  • FIG. 7 is a diagram showing microscopic results of mesenchymal stem cells irradiated with high-intensity electromagnetic fields of various frequencies for 3 days.
  • FIG. 8 shows the results of analyzing mRNA expression of collagen, type 2 osteoblastic protein (BMP-2), osteonectin, osteocalcin, and osteopontin after irradiating high intensity electromagnetic fields of various frequencies to mesenchymal stem cells for 3 days. The figure shown.
  • BMP-2 type 2 osteoblastic protein
  • osteonectin osteocalcin
  • osteopontin osteopontin
  • Figure 9 shows the shape of the cells of the magnetic nanoparticle injection group (MP, 5 ⁇ g / ml), electromagnetic field irradiation group (EMF, 45Hz, 8 hours ⁇ 2 times / day), and the experimental group irradiated with the electromagnetic field after the magnetic nanoparticles Figure shows the results.
  • MP magnetic nanoparticle injection group
  • EMF electromagnetic field irradiation group
  • FIG. 11 is a group of magnetic nanoparticles injected (MP, 50 ⁇ g / ml), electromagnetic field irradiation group (EMF, 45Hz, 8 hours ⁇ 2 times / day), and the experimental group after the injection of magnetic nanoparticles to investigate the electromagnetic field Osteocalcin and Osteo Figure shows the immunostaining results of Onnectin.
  • FIG. 12 is a magnetic nanoparticle injection group (MP, 5 ⁇ g / ml), electromagnetic field irradiation group (EMF, 45Hz, 8 hours ⁇ 2 times / day), and magnetic nanoparticles into the bone cell line (Saos-2)
  • MP magnetic nanoparticle injection group
  • EMF electromagnetic field irradiation group
  • Saos-2 magnetic nanoparticles into the bone cell line
  • FIG. 13 is a magnetic nanoparticle injection group (MP, 5 ⁇ g / ml), electromagnetic field irradiation group (EMF, 45Hz, 8 hours ⁇ 2 times / day), and magnetic nanoparticles into the bone cell line (Saos-2)
  • MP magnetic nanoparticle injection group
  • EMF electromagnetic field irradiation group
  • Saos-2 magnetic nanoparticles into the bone cell line
  • a three-dimensional hydrogel (MP, 20 ⁇ g / ml) containing magnetic nanoparticles in bone cell line (Saos-2), electromagnetic field irradiation group on the three-dimensional hydrogel (EMF, 60Hz, 8 hours ⁇ 2 times / day) And morphological results of cells on day 3 of the experimental group irradiated with electromagnetic fields on magnetic nanoparticle-containing three-dimensional hydrogel.
  • 15 is a three-dimensional hydrogel containing magnetic nanoparticles (MP, 20 ⁇ g / ml) in the bone cell line (Saos-2), electromagnetic field irradiation group (EMF, 60Hz, 8 hours ⁇ 2 times / day) in three-dimensional hydrogel , And the results of analyzing the mRNA of the third day of the experimental group irradiated with the electromagnetic field on the magnetic nanoparticle-containing three-dimensional hydrogel.
  • MP magnetic nanoparticles
  • EMF electromagnetic field irradiation group
  • the present invention is a.
  • the cells are preferably bone cells or mesenchymal stem cells. It is not limited to this.
  • the hydrogel was prepared using a form that can be injected using a buffer solution and collagen.
  • the buffer solution may be a buffer solution of pH 8.0 containing 8.4% sodium bicarbonate or NaOH, but may be used in combination with other injection solutions.
  • the buffer solution may be added to the magnetic nanoparticles, bone morphogenic protein (BMP) and / or nanohydroxyapatite.
  • BMP bone morphogenic protein
  • the magnetic nanoparticles are mixed at a concentration of 20 ug / ml, hydroxyapatite is mixed at a concentration of 0.015 g / ml.
  • the magnetic nanoparticles may have a particle diameter of 10 to 200 nm and nanohydroxyapatite of 1 to 500 nm.
  • the magnetic nanoparticles of step 1) is preferably selected from the group consisting of FeO 2 , FeO 3 , and FeO 4 , but is not limited thereto.
  • the magnetic nanoparticles are preferably attached to the terminal of the PEG, but is not limited thereto.
  • the magnetic nanoparticles preferably use SiO 2 as a surface modification derivative, but are not limited thereto.
  • the modified derivative is a surface modification induction in order to control the polarity of the magnetic nanoparticles and induce inflow into cells and minimize toxicity, and further induce surface modification to bind desired functional groups or molecules.
  • the size of the magnetic nanoparticles is preferably less than 200 nm, but is not limited thereto.
  • the electromagnetic field of step 2) is continuous or pulsed, but is not limited thereto.
  • the frequency of the electromagnetic field of step 2) is preferably 45 Hz to 75 Hz, but is not limited thereto.
  • the frequency of the electric field is greater than 75 Hz, bone differentiation-related osteonectin, osteocalcin and osteopontin protein expression is slightly reduced, thereby reducing the bone differentiation effect.
  • the strength of the electromagnetic field of step 2) is preferably 10 G to 1.5 T, but is not limited thereto.
  • the pH of the hydrogel of step 1) is preferably 3 to 5, more preferably 4 to 5, most preferably 4, but is not limited thereto.
  • the magnetic nanoparticles of step 1) are preferably loaded on a hydrogel, alone or in combination with bone formation inducing substances, or coated or contained on an implant surface, but are not limited thereto.
  • the bone formation inducing substance may be one or more components selected from the group consisting of BMP (Bone morphogenetic protein) family, hydroxyapatite, Tricalcium phosphate (TCP) and dicalcium phosphate (DCP), but is not limited thereto.
  • BMP Bis morphogenetic protein
  • TCP Tricalcium phosphate
  • DCP dicalcium phosphate
  • the implant is a natural polymer consisting of collagen, Haronic acid, alginate and chitosan, poly (lactic acid) (PLA), poly (lactic-co-glycolic acid) (PLGA), poly (glycolic acid) (PGA), polycaprolactone (PCL) ) And one or more components selected from the group consisting of synthetic polymers composed of poly (methyl methacrylate) and PMMA, and synthetic polymers composed of titanium, titanium alloys, and nickel-cobalt alloys.
  • PLA poly (lactic acid)
  • PLGA poly (lactic-co-glycolic acid)
  • PGA poly (glycolic acid)
  • PCL polycaprolactone
  • Bone marrow mesenchymal stem cells were seeded into 100 mm dishes for cell culture, followed by 1 ⁇ 10 5 cells, followed by an 8 hour / day electromagnetic field (10 gauss) at frequencies of 7.5, 30, 45, 50, 60, 75 and 100 Hz, respectively. After 3 days of irradiation, cells were analyzed for morphological changes, mRNA expression and protein expression. And bone marrow mesenchymal stem cells cultured on a cover slide (diameter 12 mm) is fixed with 10% formalin for 30 minutes and washed three times with pH 7.2 PBS. Osteopontin antibody was treated and incubated for 24 hours at room temperature and then developed with EnVision Plus reagent.
  • the protein expression of collagen, osteonectin, type 2 osteoblastic protein (BMP-2) increases as the electromagnetic field frequency of 30 Hz or more increases, and this result is a result of activating pERK and p-CREB. there was. However, in the 100 Hz electromagnetic field it was confirmed that the reduction of the frequency of the electromagnetic field is effective at the frequency of 45 ⁇ 75 Hz (Fig. 5).
  • Bone marrow mesenchymal stem cells were inoculated with 1 ⁇ 10 5 cells in a 100 mm dish for cell culture, and then irradiated with electromagnetic fields three times a day at frequencies of 30, 45, 50, 60, 75 and 100 Hz, respectively. 20 minutes were performed. At this time, the intensity was investigated at 1.12, 0.89, 0.68, 0.63, 0.57 and 0.4 T, respectively, and after 3 days, the morphological changes and mRNA expression were analyzed.
  • Bone marrow mesenchymal stem cells were inoculated with 1 ⁇ 10 5 cells in a 100 mm dish for cell culture, and then replaced with a medium for bone differentiation.
  • control comparison group
  • MP magnetic particle
  • EMF electromagnettic field
  • MP + EMF electromagnetic field irradiation group
  • Magnetic nanoparticle injection group MP, 50 ⁇ g / ml
  • electromagnetic field irradiation group EMF, 45Hz, 8 hours ⁇ 2 times / day
  • the osteocalcin and austerity As a result of the immunostaining of Onnectin, protein secretion was promoted in most experimental groups as compared to the control group, especially in the group irradiated with magnetic nanoparticles and the electromagnetic field (Fig. 11).
  • the bone cell line (Saos-2) was inoculated with 1 ⁇ 10 5 cells in a 100 mm dish for cell culture, and the experiment was carried out as follows.
  • control comparison group
  • MP magnetic particle
  • EMF electromagnettic field
  • MP + EMF electromagnetic field irradiation group
  • magnetic nanoparticle injection group (MP, 5 ⁇ g / ml), electromagnetic field irradiation group (EMF, 45Hz, 8 hours ⁇ 2 times / day), and magnetic nanoparticles were injected into the bone cell line (Saos-2).
  • EMF electromagnetic field irradiation group
  • FIG. 12 As a morphological picture of the cells on day 3 of the experimental group irradiated with, no cytotoxicity such as cell death was observed in all experimental groups (FIG. 12), As a result of mRNA analysis on day 3, the expression of osteocalcin, collagen, and bone sialo protein was increased in the group administered with the magnetic nanoparticles and the electromagnetic field, which was confirmed by the activation of calcium channel and Cbfa1 (FIG. 13). .
  • Collagen hydrogel was prepared by mixing 1% collagen, 5 times concentrated medium (5 times concentrated DMEM cell culture medium), and buffer solution (pH 8.0) at a ratio of 7: 2: 1, and bone cell line (Saos-2). 3) artificial bone tissue was prepared by mixing 1.0 ⁇ 10 6 cells with 1 ml of collagen hydrogel, inoculating 1 ml into a 100 mm culture dish, and inducing gelation in a 37 ° C. incubator for 30 minutes. Then, about 15 ml of medium was added, and the experiment was conducted under the following conditions.
  • control hydrogel
  • MP magnetic particle
  • EMF electromagnettic field
  • MP + EMF electromagnetic field irradiation group after the manufacture of hydrogel ( 60 Hz, 8 hours ⁇ 2 times / day)
  • MP + EMF electromagnetic field irradiation group after preparation of the hydrogel containing the magnetic nanoparticles, the electromagnetic field irradiation group was subjected to bone-related mRNA analysis for three days after each experiment.

Description

자성나노입자와 전자기장을 이용한 골 재생 촉진 방법Method of promoting bone regeneration using magnetic nanoparticles and electromagnetic field
본 발명은 골 재생, 특히 손상된 치조골 및 골 조직을 치유 또는 재생을 유도하기 위하여 자성나노입자와 전자기장을 이용하는 방법에 관한 것이다.The present invention relates to methods of using magnetic nanoparticles and electromagnetic fields to induce bone regeneration, in particular healing or regeneration of damaged alveolar bone and bone tissue.
최근에 손상된 조직의 치료를 향상시키기 위하여 초음파, 전자기장 및 LED 등 다양한 외부적 자극 치료법이 이용되고 있다. 특히 전자기장은 7.5 Hz부터 100 Hz의 저주파 영역에서 20 G(가우스) 미만의 저강도를 이용하여 신경분화, 골 분화, 그리고 통증 치료 등에 이용되어 왔다.Recently, various external stimulation therapies such as ultrasound, electromagnetic fields, and LEDs have been used to improve the treatment of damaged tissues. In particular, electromagnetic fields have been used for neurodifferentiation, bone differentiation, and pain treatment using low intensity of less than 20 G (Gaussian) in the low frequency region of 7.5 Hz to 100 Hz.
Fregni 등(NeuroImage, 2013)은 다양한 전기 및 전자기장자극이 척추 손상으로 인한 만성신경통의 통증을 완화시킴을 보고하였고, Ahmadian S 등(Biotechnol. Appl. Biochem. 2006, 43, 71-75)은 쥐의 피부에 25 Hz 및 2 mT 하루에 2.5시간을 조사하였을때 피부의 콜라겐이 증가됨을 보고하였다. 또한 Bae 등(Cytotherapy. 2013 15(8):961-970)은 50 Hz 및 5 mT의 전자기장을 하루에 60 분씩 12일간 중간엽 줄기세포에 조사한 결과 신경세포로 분화가 촉진됨을 보고하였다. Fregni et al. (NeuroImage, 2013) reported that various electrical and electromagnetic field stimuli alleviate the pain of chronic neuralgia due to spinal injury, and Ahmadian S et al. (Biotechnol. Appl. Biochem. 2006, 43, 71-75) When the skin was irradiated at 25 Hz and 2 mT for 2.5 hours a day, it was reported that the collagen of the skin was increased. Bae et al. (Cytotherapy. 2013 15 (8): 961-970) reported that mesenchymal stem cells were stimulated for differentiation into neurons by irradiation of 50 Hz and 5 mT electromagnetic fields for 60 minutes per day for 12 days.
최근에 전자기장을 이용한 골 재생 관련 연구들도 보고되고 있다. Nascimento 등(Gerodontlogy, 2012, 29:e1249-1251)은 동물에 임플란트 식립 후 1.5 MHz 및 0.8 mT의 전자기장을 2주간 20분씩 조사한 결과 골 융합이 촉진된 것을 보고하였고, Sun 등은 15 Hz 및 1.8 mT의 전자기장에서 골수 유래 중간엽 줄기세포를 배양하여, 알카라이니포스파테이즈(ALP)와 골형성단백질(BMP-2) 등의 발현이 촉진되어 골세포로의 분화가 촉진됨을 보고하였으며, Ceccarelli 등(BioResearch Open Access, 2013, 2(4): 283-294)은 75Hz 및 2 mT의 전자기장으로 다양한 중간엽 줄기세포의 골 분화를 촉진시켰다. 이러한 전자기장을 이용한 골 분화 촉진 연구는 7.5 ~ 75 Hz 및 0.1 ~ 5 mT의 전자기장을 이용하였다. Recently, studies on bone regeneration using electromagnetic fields have been reported. Nascimento et al. (Gerodontlogy, 2012, 29: e1249-1251) reported that bone fusion was promoted after 20 minutes of 1.5 MHz and 0.8 mT electromagnetic fields for 2 weeks after implant placement in animals, and Sun et al. 15 Hz and 1.8 mT. Cultivation of bone marrow-derived mesenchymal stem cells in the electromagnetic field of the cells promotes the expression of alkaliniphosphate (ALP) and bone morphogenetic protein (BMP-2), thereby promoting differentiation into bone cells. (BioResearch Open Access, 2013, 2 (4): 283-294) promoted bone differentiation of various mesenchymal stem cells with electromagnetic fields at 75 Hz and 2 mT. In order to promote bone differentiation using the electromagnetic fields, electromagnetic fields of 7.5 to 75 Hz and 0.1 to 5 mT were used.
이에, 본 발명자들은 전자기장을 이용하여 골세포 활성화와 중간엽줄기세포의 골세포로의 분화를 촉진하여 골 손상 부위의 치료 효능을 향상시키고자, 자성나노입자를 골 결손부위에 주입 또는 식립 후 저강도 및 고강도의 전자기장을 1시간 이내로 짧게 조사하여 전자기장의 골 치료 효과를 극대화하는 기술을 개발함으로써 본 발명을 완성하였다. Therefore, the inventors of the present invention intend to improve the therapeutic efficacy of bone injury sites by promoting bone cell activation and differentiation of mesenchymal stem cells into bone cells by using an electromagnetic field. The present invention has been completed by developing a technique for maximizing the effect of electromagnetic treatment of the electromagnetic field by briefly irradiating the electromagnetic field of strength and high intensity within 1 hour.
본 발명의 목적은 자성나노입자를 함유한 하이드로젤 또는 임플란트를 골손상부위에 적용한 뒤, 특정 주파수와 강도의 전자기장을 조사함으로서 골손상부위의 치료효율을 향상시키거나 골 융합을 촉진시켜 골 재생을 촉진하는 방법을 제공하는 것이다.An object of the present invention is to apply a hydrogel or implant containing magnetic nanoparticles to a bone injury site, and then by irradiating an electromagnetic field of a specific frequency and intensity to improve the treatment efficiency of the bone injury site or promote bone fusion to promote bone regeneration To provide a way.
상기 목적을 해결하기 위하여, 본 발명은In order to solve the above object, the present invention
1) 세포 또는 조직에 자성나노입자를 함유한 하이드로젤을 주입하는 단계; 및1) injecting a hydrogel containing magnetic nanoparticles into cells or tissues; And
2) 상기 세포 또는 조직에 전자기장을 조사하는 단계를 포함하는 골 분화 및 골 재생을 촉진하는 방법을 제공한다. 2) providing a method for promoting bone differentiation and bone regeneration comprising irradiating the electromagnetic field to the cells or tissues.
본 발명에 따른 자성나노입자와 전자기장을 이용한 골 재생 촉진 방법은 수술 후 물리치료의 한 방법으로 빠른 골 치료를 유도할 수 있으며, 자성나노입자 또는 골 형성 촉진 물질을 하이드로젤 또는 스캐폴드에 도입시켜, 골 손상 부위에 이식함으로써 골 치료 효율을 극대화 시킬 수 있다. The method of promoting bone regeneration using the magnetic nanoparticles and the electromagnetic field according to the present invention may induce rapid bone treatment as a method of physical therapy after surgery, and introduce the magnetic nanoparticles or the bone formation promoting substance into the hydrogel or the scaffold. In addition, by implanting the bone damage site can maximize the efficiency of bone treatment.
도 1은 다양한 주파수의 전자기장을 3일간 조사한 골세포의 현미경 결과를 나타낸 도이다. 1 is a diagram showing the microscopic results of osteoblasts irradiated with electromagnetic fields of various frequencies for 3 days.
도 2는 골세포에 다양한 주파수의 전자기장을 3일간 조사한 뒤 콜라젠, 본시알로프로테인(bonesialoprotein), 오스테오넥틴, 오스테오칼신, 오스테오폰틴, 비멘틴, 제 2형 골형성단백질(BMP-2)의 mRNA 발현을 분석한 결과를 나타낸 도이다.Figure 2 mRNA of collagen, bone sialo protein (bonesialoprotein), osteonectin, osteocalcin, osteopontin, non-mentin, type 2 bone morphogenetic protein (BMP-2) after irradiating electromagnetic fields of various frequencies to bone cells for 3 days It is a figure which shows the result of analyzing expression.
도 3은 다양한 주파수의 전자기장을 3일간 조사한 중간엽줄기세포의 현미경 결과를 나타낸 도이다. Figure 3 is a diagram showing the microscopic results of mesenchymal stem cells irradiated with electromagnetic fields of various frequencies for 3 days.
도 4는 중간엽줄기세포에 다양한 주파수의 전자기장을 3일간 조사한 뒤 콜라젠, 오스테오넥틴, 오스테오칼신, 오스테오폰틴, 비멘틴, 제2형 골형성단백질(BMP-2)의 mRNA 발현을 분석한 결과를 나타낸 도이다. Figure 4 shows the results of analyzing the mRNA expression of collagen, osteonectin, osteocalcin, osteopontin, non-mentin, type 2 bone morphogenetic protein (BMP-2) after irradiating electromagnetic fields of various frequencies to mesenchymal stem cells for 3 days Is a diagram showing.
도 5는 중간엽줄기세포에 다양한 주파수의 전자기장을 14일간 조사한 뒤 콜라젠, 오스테오넥틴, 제 2형 골형성단백질(BMP-2), p-ERK, p-CREB의 단백질 발현을 분석한 결과를 나타낸 도이다. FIG. 5 shows the results of analyzing protein expression of collagen, osteonectin, type 2 osteoblastic protein (BMP-2), p-ERK, and p-CREB after irradiating electromagnetic fields of various frequencies to mesenchymal stem cells for 14 days. The figure shown.
도 6는 중간엽줄기세포에 다양한 주파수의 전자기장을 14일간 조사한 뒤 오스테오폰틴을 면역염색한 결과를 나타낸 도이다.6 is a diagram showing the results of immunostaining osteopontin after irradiation of mesenchymal stem cells with electromagnetic fields of various frequencies for 14 days.
도 7은 다양한 주파수의 고강도 전자기장을 3일간 조사한 중간엽줄기세포의 현미경 결과를 나타낸 도이다. FIG. 7 is a diagram showing microscopic results of mesenchymal stem cells irradiated with high-intensity electromagnetic fields of various frequencies for 3 days.
도 8은 중간엽줄기세포에에 다양한 주파수의 고강도 전자기장을 3일간 조사한 뒤 콜라젠, 제2형 골형성단백질(BMP-2), 오스테오넥틴, 오스테오칼신, 오스테오폰틴의 mRNA 발현을 분석한 결과를 나타낸 도이다.FIG. 8 shows the results of analyzing mRNA expression of collagen, type 2 osteoblastic protein (BMP-2), osteonectin, osteocalcin, and osteopontin after irradiating high intensity electromagnetic fields of various frequencies to mesenchymal stem cells for 3 days. The figure shown.
도 9는 자성나노입자 투입군(MP, 5 ㎍/ml), 전자기장 조사군(EMF, 45Hz, 8시간 × 2회/일), 그리고 자성나노입자를 투입한 뒤 전자기장을 조사한 실험군의 세포의 형태학적 결과를 나타낸 도이다.Figure 9 shows the shape of the cells of the magnetic nanoparticle injection group (MP, 5 ㎍ / ml), electromagnetic field irradiation group (EMF, 45Hz, 8 hours × 2 times / day), and the experimental group irradiated with the electromagnetic field after the magnetic nanoparticles Figure shows the results.
도 10은 자성나노입자 투입군(MP, 50 ㎍/ml), 전자기장 조사군(EMF, 45Hz, 8시간 × 2회/일), 그리고 자성나노입자를 투입한 뒤 전자기장을 조사한 실험군의 mRNA를 분석한 결과를 나타낸 도이다.10 is analyzed for the mRNA of the magnetic nanoparticle injection group (MP, 50 ㎍ / ml), the electromagnetic field irradiation group (EMF, 45Hz, 8 hours × 2 times / day), and the experimental group irradiated with the electromagnetic field after the magnetic nanoparticles Figure 1 shows the results.
도 11은 자성나노입자 투입군(MP, 50 ㎍/ml), 전자기장 조사군(EMF, 45Hz, 8시간 × 2회/일), 그리고 자성나노입자를 투입한 뒤 전자기장을 조사한 실험군의 오스테오칼신과 오스테오넥틴의 면역염색 결과를 나타낸 도이다. 11 is a group of magnetic nanoparticles injected (MP, 50 ㎍ / ml), electromagnetic field irradiation group (EMF, 45Hz, 8 hours × 2 times / day), and the experimental group after the injection of magnetic nanoparticles to investigate the electromagnetic field Osteocalcin and Osteo Figure shows the immunostaining results of Onnectin.
도 12는 골세포주(Saos-2)에 자성나노입자 투입군(MP, 5 ㎍/ml), 전자기장 조사군(EMF, 45Hz, 8시간 × 2회/일), 그리고 자성나노입자를 투입과 전자기장을 조사한 실험군의 3일째 세포의 형태학적 결과를 나타낸 도이다.12 is a magnetic nanoparticle injection group (MP, 5 ㎍ / ml), electromagnetic field irradiation group (EMF, 45Hz, 8 hours × 2 times / day), and magnetic nanoparticles into the bone cell line (Saos-2) Figure 3 shows the morphological results of cells on day 3 of the experimental group.
도 13은 골세포주(Saos-2)에 자성나노입자 투입군(MP, 5 ㎍/ml), 전자기장 조사군(EMF, 45Hz, 8시간 × 2회/일), 그리고 자성나노입자를 투입과 전자기장을 조사한 실험군의 3일째 mRNA를 분석한 결과를 나타낸 도이다. 13 is a magnetic nanoparticle injection group (MP, 5 ㎍ / ml), electromagnetic field irradiation group (EMF, 45Hz, 8 hours × 2 times / day), and magnetic nanoparticles into the bone cell line (Saos-2) Figure 3 shows the results of mRNA analysis of the experimental group examined.
도 14는 골세포주(Saos-2)에 자성나노입자 함유 3차원 하이드로젤(MP, 20 ㎍/ml), 3차원 하이이드로젤에 전자기장 조사군(EMF, 60Hz, 8시간 × 2회/일), 그리고 자성나노입자 함유 3차원 하이드로젤에 전자기장을 조사한 실험군의 3일째 세포의 형태학적 결과를 나타낸 도이다.14 is a three-dimensional hydrogel (MP, 20 ㎍ / ml) containing magnetic nanoparticles in bone cell line (Saos-2), electromagnetic field irradiation group on the three-dimensional hydrogel (EMF, 60Hz, 8 hours × 2 times / day) And morphological results of cells on day 3 of the experimental group irradiated with electromagnetic fields on magnetic nanoparticle-containing three-dimensional hydrogel.
도 15는 골세포주(Saos-2)에 자성나노입자 함유 3차원 하이드로젤(MP, 20 ㎍/ml), 3차원 하이이드로젤에 전자기장 조사군(EMF, 60Hz, 8시간 × 2회/일), 그리고 자성나노입자 함유 3차원 하이드로젤에 전자기장을 조사한 실험군의 3일째 mRNA를 분석한 결과를 나타낸 도이다. 15 is a three-dimensional hydrogel containing magnetic nanoparticles (MP, 20 ㎍ / ml) in the bone cell line (Saos-2), electromagnetic field irradiation group (EMF, 60Hz, 8 hours × 2 times / day) in three-dimensional hydrogel , And the results of analyzing the mRNA of the third day of the experimental group irradiated with the electromagnetic field on the magnetic nanoparticle-containing three-dimensional hydrogel.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 The present invention
1) 세포 또는 조직에 자성나노입자를 함유한 하이드로젤을 주입하는 단계; 및1) injecting a hydrogel containing magnetic nanoparticles into cells or tissues; And
2) 상기 단계 1)에서 제조된 하이드로젤에 전자기장을 조사하는 단계를 포함하는 골 분화 및 골 재생을 촉진하는 방법을 제공한다.2) it provides a method for promoting bone differentiation and bone regeneration comprising the step of irradiating the electromagnetic field to the hydrogel prepared in step 1).
상기 세포는 골세포 또는 중간엽줄기세포인 것이 바람직하나. 이에 한정하지 않는다.The cells are preferably bone cells or mesenchymal stem cells. It is not limited to this.
상기 하이드로젤은 완충용액과 콜라겐을 이용하여 주입이 가능한 형태로 이용하여 제조하였다. 완충용액은 8.4% 탄산수소나트륨 또는 NaOH를 함유한 pH 8.0의 버퍼 용액이 사용되어질 수 있으나 다른 주사액과 혼용되어 사용되어 질 수도 있다. 또한, 완충용액은 자성나노입자와 골형성단백질(BMP) 및/또는 나노하이드록시아파타이트를 첨가할 수도 있다. The hydrogel was prepared using a form that can be injected using a buffer solution and collagen. The buffer solution may be a buffer solution of pH 8.0 containing 8.4% sodium bicarbonate or NaOH, but may be used in combination with other injection solutions. In addition, the buffer solution may be added to the magnetic nanoparticles, bone morphogenic protein (BMP) and / or nanohydroxyapatite.
이때, 자성나노입자는 20 ug/ml의 농도로 혼합되어지며, 하이드록시아파타이트는 0.015 g/ml의 농도로 혼합되어진다. 골재생을 촉진하기 위해 자성나노입자는 10 내지 200 nm, 나노하이드록시아파타이트는 1 내지 500 nm의 입경을 가질 수 있다.At this time, the magnetic nanoparticles are mixed at a concentration of 20 ug / ml, hydroxyapatite is mixed at a concentration of 0.015 g / ml. To promote bone regeneration, the magnetic nanoparticles may have a particle diameter of 10 to 200 nm and nanohydroxyapatite of 1 to 500 nm.
상기 단계 1)의 자성나노입자는 FeO2, FeO3, 및 FeO4로 이루어진 군으로부터 선택되는 것이 바람직하나, 이에 한정되지 않는다.The magnetic nanoparticles of step 1) is preferably selected from the group consisting of FeO 2 , FeO 3 , and FeO 4 , but is not limited thereto.
상기 자성나노 입자는 PEG의 말단에 부착되는 것이 바람직하나, 이에 한정하지 않는다. The magnetic nanoparticles are preferably attached to the terminal of the PEG, but is not limited thereto.
상기 자성나노입자는 SiO2를 표면 개질 유도체로 이용하는 것이 바람직하나, 이에 한정되지 않는다.The magnetic nanoparticles preferably use SiO 2 as a surface modification derivative, but are not limited thereto.
상기 개질 유도체란 자성나노입자의 극성을 조절 및 세포안으로 유입을 유도하고 독성을 최소화으로 하기 위해 표면개질 유도를 시행하는 것으로, 추가적으로 원하는 작용기나 분자들을 결합시키기 위해서 표면 개질을 유도한다. The modified derivative is a surface modification induction in order to control the polarity of the magnetic nanoparticles and induce inflow into cells and minimize toxicity, and further induce surface modification to bind desired functional groups or molecules.
상기 자성나노입자의 크기는 200 nm 미만인 것이 바람직하나, 이에 한정되지 않는다.The size of the magnetic nanoparticles is preferably less than 200 nm, but is not limited thereto.
상기 단계 2)의 전자기장은 연속 또는 펄스 형태인 것이 바람직하나, 이에 한정되지 않는다.Preferably, the electromagnetic field of step 2) is continuous or pulsed, but is not limited thereto.
상기 단계 2)의 전자기장의 주파수는 45 Hz 내지 75 Hz인 것이 바람직하나, 이에 한정되지 않는다. 전기장의 주파수가 75 Hz 보다 클 경우 골 분화 관련오스테오넥틴과, 오스테오칼신, 오스테오폰틴의 단백질의 발현이 다소 감소하므로 골 분화 효과가 적어진다.The frequency of the electromagnetic field of step 2) is preferably 45 Hz to 75 Hz, but is not limited thereto. When the frequency of the electric field is greater than 75 Hz, bone differentiation-related osteonectin, osteocalcin and osteopontin protein expression is slightly reduced, thereby reducing the bone differentiation effect.
상기 단계 2)의 전자기장의 강도는 10 G 내지 1.5 T인 것이 바람직하나, 이에 한정되지 않는다.The strength of the electromagnetic field of step 2) is preferably 10 G to 1.5 T, but is not limited thereto.
상기 단계 1)의 하이드로젤의 pH는 3 내지 5인 것이 바람직하고, 4 내지 5인 것이 보다 바람직하며, 4인 것이 가장 바람직하나, 이에 한정되지 않는다.The pH of the hydrogel of step 1) is preferably 3 to 5, more preferably 4 to 5, most preferably 4, but is not limited thereto.
상기 단계 1)의 자성나노입자는 단독 또는 골 형성 유도 물질들과 함께 하이드로젤에 담지되어 골 결손 부위에 충진되거나, 임플란트 표면에 코팅 또는 함유되어지는 것이 바람직하나, 이에 한정되지 않는다.The magnetic nanoparticles of step 1) are preferably loaded on a hydrogel, alone or in combination with bone formation inducing substances, or coated or contained on an implant surface, but are not limited thereto.
상기 골 형성 유도 물질은 BMP(Bone morphogenetic protein) 패밀리, 하이드록시아파타이트, TCP(Tricalcium phosphate) 및 DCP(dicalcium phosphate)로 구성되는 군으로부터 선택되는 하나 이상의 성분일 수 있으나, 이에 한정되지 않는다.The bone formation inducing substance may be one or more components selected from the group consisting of BMP (Bone morphogenetic protein) family, hydroxyapatite, Tricalcium phosphate (TCP) and dicalcium phosphate (DCP), but is not limited thereto.
상기 임플란트는 콜라겐, 하루론산, 알지네이트 및 키토산으로 구성된 천연고분자, PLA(poly(lactic acid)), PLGA(poly(lactic-co-glycolic acid)), PGA(poly(glycolic acid)), PCL(polycaprolactone) 및 PMMA(poly(methyl methacrylate))로 구성된 합성고분자, 및 티타늄, 티타늄 합금 및 니켈-코발트 합금으로 구성된 합성고분자로 구성된 군으로부터 선택되는 하나 이상의 성분을 포함할 수 있으나, 이에 한정되지는 않는다.The implant is a natural polymer consisting of collagen, Haronic acid, alginate and chitosan, poly (lactic acid) (PLA), poly (lactic-co-glycolic acid) (PLGA), poly (glycolic acid) (PGA), polycaprolactone (PCL) ) And one or more components selected from the group consisting of synthetic polymers composed of poly (methyl methacrylate) and PMMA, and synthetic polymers composed of titanium, titanium alloys, and nickel-cobalt alloys.
상기 다른 목적에 따라, 본 발명은 According to the above another object, the present invention
1) 본 발명의 나노자성나노입자 함유 하이드로젤을 임플란트 표면에 코팅하는 단계; 1) coating the nano-magnetic nanoparticle-containing hydrogel of the present invention on the implant surface;
2) 상기 단계 1)에서 제조한 임플란트를 골 결손 부위에 고정 또는 충진하는 단계; 및2) fixing or filling the implant prepared in step 1) at the bone defect site; And
3) 상기 2)에서 고정 또는 충전된 결손부위에 전자기장을 조사하는 단계를 포함하는골 융합을 촉진하는 방법을 제공한다.3) It provides a method for promoting bone fusion comprising the step of irradiating the electromagnetic field to the defects fixed or filled in the 2).
또한, 본 발명은In addition, the present invention
1) 본 발명의 자성나노입자를 함유한 골 재생 스캐폴드를 제조하는 단계;1) preparing a bone regeneration scaffold containing the magnetic nanoparticles of the present invention;
2) 상기 단계 1)에서 제조한 스캐폴드를 골 결손 부위에 고정 또는 충진하는 단계; 및2) fixing or filling the scaffold prepared in step 1) to the bone defect site; And
3) 상기 2)에서 고정 또는 충전된 결손부위에 전자기장을 조사하는 단계를 포함하는 골 융합을 촉진하는 방법을 제공한다.3) It provides a method for promoting bone fusion comprising the step of irradiating the electromagnetic field to the fixed or filled defects in 2).
이하, 본 발명의 이해를 돕기 위하여 실시예를 들어 상세하게 설명하기로 한다. 다만 하기의 실시예는 본 발명의 내용을 예시하는 것일 뿐 본 발명의 범위가 하기 실시예에 한정되는 것은 아니다. 본 발명의 실시예는 당업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해 제공되는 것이다.Hereinafter, examples will be described in detail to help understand the present invention. However, the following examples are merely to illustrate the content of the present invention is not limited to the scope of the present invention. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.
<실시예 1> 저강도 전자기장을 이용한 골세포 활성 분석Example 1 Analysis of Bone Cell Activity Using Low Intensity Electromagnetic Field
일차 배양된 골세포를 세포배양용 100 mm 디쉬에 1 × 105 세포를 접종한 뒤 각각 30, 45, 50, 60, 75 및 100 Hz의 주파수로 전자기장(10 가우스)을 8 시간/일, 3일간 조사한 뒤 세포의 형태학적 변화와 mRNA 발현을 분석하였다.Primary cultured osteoblasts were inoculated into 1 × 10 5 cells in a 100 mm dish for cell culture and subjected to electromagnetic fields (10 gauss) at frequencies of 30, 45, 50, 60, 75 and 100 Hz, respectively, for 8 hours / day, 3 After daily irradiation, the morphological changes and mRNA expression of the cells were analyzed.
그 결과, 세포 독성으로 인한 세포의 형태학적인 변화는 관찰되지 않았고(도 1), 전자기장의 주파수가 증가할수록 콜라젠, 본시알로프로테인(bonesialoprotein), 오스테오넥틴, 오스테오칼신, 오스테오폰틴, 비멘틴 및 제 2형 골형성단백질(BMP-2)의 mRNA 발현이 증가되었다(도 2).As a result, no morphological changes of the cells due to cytotoxicity were observed (FIG. 1), and collagen, bonesialoprotein, osteonectin, osteocalcin, osteopontin, nonmentin and zeogen were increased with increasing frequency of the electromagnetic field. MRNA expression of type 2 osteoblastic protein (BMP-2) was increased (FIG. 2).
<실시예 2> 저강도 전자기장을 이용한 중간엽줄기세포의 골 분화 분석Example 2 Bone Differentiation Analysis of Mesenchymal Stem Cells Using Low-Intensity Electromagnetic Fields
골수 중간엽줄기세포를 세포배양용 100 mm 디쉬에 1 × 105 세포를 접종한 뒤 각각 7.5, 30, 45, 50, 60, 75 및 100 Hz의 주파수로 전자기장(10 가우스)을 8시간/일, 3일간 조사한 뒤 세포의 형태학적 변화와 mRNA발현 및 단백질 발현을 분석하였다. 그리고 커버 슬라이드(지름 12mm)에 배양된 골수 중간엽줄기세포를 10% 포르말린으로 30분 동안 고정하고 pH 7.2 PBS로 3회 세척한다. 오스테오폰틴 항체(Osteopontin antibody) 처리하고 실온에서 24시간 동안 인큐베이션(incubation) 한 뒤 EnVision Plus reagent으로 디벨롭(develop)하였다. Bone marrow mesenchymal stem cells were seeded into 100 mm dishes for cell culture, followed by 1 × 10 5 cells, followed by an 8 hour / day electromagnetic field (10 gauss) at frequencies of 7.5, 30, 45, 50, 60, 75 and 100 Hz, respectively. After 3 days of irradiation, cells were analyzed for morphological changes, mRNA expression and protein expression. And bone marrow mesenchymal stem cells cultured on a cover slide (diameter 12 mm) is fixed with 10% formalin for 30 minutes and washed three times with pH 7.2 PBS. Osteopontin antibody was treated and incubated for 24 hours at room temperature and then developed with EnVision Plus reagent.
그 결과, 세포 독성으로 인한 세포의 형태학적인 변화는 관찰되지 않았고(도 2), 45 ~ 75 Hz의 전자기장 주파수가 증가할수록 콜라젠, 오스테오넥틴, 비멘틴, 제 2형 골형성단백질(BMP-2)의 mRNA발현이 증가됨을 관찰할 수 있었다. 그러나 75 Hz 이상의 전자기장에서는 오스테오넥틴과, 오스테오칼신, 오스테오폰틴의 mRNA 발현이 다소 감소하였다(도 4).As a result, no morphological changes of cells due to cytotoxicity were observed (Fig. 2), and collagen, osteonectin, non-mentin, type 2 bone morphogenetic proteins (BMP-2) as the electromagnetic field frequency increased from 45 to 75 Hz. MRNA expression was increased. However, the mRNA expression of osteonectin, osteocalcin and osteopontin was slightly decreased in the electromagnetic field of 75 Hz or more (FIG. 4).
또한, 30 Hz 이상 전자기장 주파수가 증가할수록 콜라젠, 오스테오넥틴, 제 2형 골형성단백질(BMP-2)의 단백질 발현이 증가되고, 이러한 결과는 pERK와 p-CREB의 활성화시킴으로서 나타나는 결과임을 확인할 수 있었다. 그러나 100 Hz 전자기장에서는 이러한 발현이 줄어드는 것은 전자기장의 주파수는 45 ~75 Hz의 주파수에서 효과적임을 확인하였다(도 5).In addition, the protein expression of collagen, osteonectin, type 2 osteoblastic protein (BMP-2) increases as the electromagnetic field frequency of 30 Hz or more increases, and this result is a result of activating pERK and p-CREB. there was. However, in the 100 Hz electromagnetic field it was confirmed that the reduction of the frequency of the electromagnetic field is effective at the frequency of 45 ~ 75 Hz (Fig. 5).
또한, 오스테오폰틴을 면역염색한 결과 50 Hz이상부터 단백질분비가 증가되어 있는 것을 확인하였다(도 6).In addition, as a result of immunostaining osteopontin it was confirmed that the protein secretion is increased from 50 Hz or more (Fig. 6).
<실시예 3> 고강도 전자기장을 이용한 골 분화 분석Example 3 Bone Differentiation Analysis Using High Intensity Electromagnetic Field
골수 중간엽줄기세포를 세포배양용 100 mm 디쉬에 1 × 105 세포를 접종한 뒤 각각 30, 45, 50, 60, 75 및 100 Hz의 주파수로 전자기장을 하루에 3회 조사하였으며, 1회 조사 시 20분간 시행하였다. 이때 강도는 각각 1.12, 0.89, 0.68, 0.63, 0.57 및 0.4 T의 강도로 조사하였으며, 3일간 조사한 뒤 세포의 형태학적 변화와 mRNA 발현을 분석하였다. Bone marrow mesenchymal stem cells were inoculated with 1 × 10 5 cells in a 100 mm dish for cell culture, and then irradiated with electromagnetic fields three times a day at frequencies of 30, 45, 50, 60, 75 and 100 Hz, respectively. 20 minutes were performed. At this time, the intensity was investigated at 1.12, 0.89, 0.68, 0.63, 0.57 and 0.4 T, respectively, and after 3 days, the morphological changes and mRNA expression were analyzed.
그 결과, 다양한 주파수의 고강도(0.4 ~1.3 T) 전자기장을 3일간 조사하였을 때 액포 형성과 같은 세포사의 형태학적 변화는 관찰되지 않아 중간엽줄기세포에 손상을 주지 않은 것이 관찰되었으며(도 7), 중간엽줄기세포의 골분화 분석에서는 콜라젠, 제 2형 골형성단백질(BMP-2), 오스테오넥틴, 오스테오칼신, 오스테오폰틴의 mRNA 발현이 45 ~ 60 Hz에서 가장 증가되어 있음을 확인하였다(도 8).As a result, morphological changes such as vacuole formation were not observed when high-intensity (0.4-1.3 T) electromagnetic fields of various frequencies were observed for 3 days, which did not damage mesenchymal stem cells (Fig. 7). Bone differentiation analysis of mesenchymal stem cells confirmed that mRNA expression of collagen, type 2 osteoblastic protein (BMP-2), osteonectin, osteocalcin and osteopontin was most increased at 45-60 Hz (Fig. 8).
<실시예 4> 자성나노입자와 저강도 전자기장을 이용한 중간엽줄기세포의 골 분화 분석Example 4 Bone Differentiation Analysis of Mesenchymal Stem Cells Using Magnetic Nanoparticles and Low-Intensity Electromagnetic Fields
골수 중간엽줄기세포를 세포배양용 100 mm 디쉬에 1 × 105 세포을 접종한 뒤 골분화용 배지로 교체한 뒤 다음과 같이 실험을 진행하였다. Bone marrow mesenchymal stem cells were inoculated with 1 × 10 5 cells in a 100 mm dish for cell culture, and then replaced with a medium for bone differentiation.
구체적으로, control: 비교군, MP(magnetic particle): 자성나노입자 투입군(배지 1 ml당 5 ㎍ 자성나노입자 투여, 지름 약 40 nm), EMF(electromagnetic field): 전자기장 조사군 (45 Hz, 8시간 × 2회/일) 및 MP+EMF: 자성 나노입자 투입 후 전자기장 조사군을 3일간 각 조건별 실험 후 골 관련 mRNA분석과 면역화학염색을 실시하였다.Specifically, control: comparison group, MP (magnetic particle): magnetic nanoparticle injection group (5 μg magnetic nanoparticles per 1 ml medium, about 40 nm in diameter), EMF (electromagnetic field): electromagnetic field irradiation group (45 Hz, 8 hours × 2 times / day) and MP + EMF: After the magnetic nanoparticles were injected, the field irradiation group was subjected to bone-related mRNA analysis and immunochemical staining for 3 days.
그 결과, 자성나노입자 투입군(MP, 50 ㎍/ml), 전자기장 조사군(EMF, 45Hz, 8시간 × 2회/일), 그리고 자성나노입자를 투입한 뒤 전자기장을 3일간 조사한 뒤 세포의 형태학적 변화를 관찰한 결과 자성나노입자와 전자기장에 의한 세포 독성은 관찰되지 않았으며(도 9), 3일간 실험 후 mRNA를 분석한 결과, 자성나노입자와 전자기장을 함께 조사한 경우, 골 분화 관련 마커인 오스테오넥틴, 오스테오칼신, 오스테오폰틴, 및 본시알로 프로테인의 발현이 현저히 증가하였고, 이는 칼슘체널의 증가와 Cbfa의 발현 증가로 유발된 것으로 관찰되었다(도 10).As a result, after injecting magnetic nanoparticles (MP, 50 ㎍ / ml), electromagnetic field irradiation group (EMF, 45Hz, 8 hours × 2 times / day), and magnetic nanoparticles were injected for 3 days, When morphological changes were observed, cytotoxicity by magnetic nanoparticles and electromagnetic field was not observed (FIG. 9). After analyzing the mRNA after 3 days of experiments, when the magnetic nanoparticles and the electromagnetic field were irradiated together, bone differentiation-related markers The expression of phosphorus osteonectin, osteocalcin, osteopontin, and bone sialo protein was markedly increased, which was observed to be caused by an increase in calcium channel and increased expression of Cbfa (FIG. 10).
또한, 자성나노입자 투입군(MP, 50 ㎍/ml), 전자기장 조사군(EMF, 45Hz, 8시간 × 2회/일), 및 자성나노입자를 투입한 뒤 전자기장을 조사한 실험군의 10일 후 오스테오칼신과 오스테오넥틴의 면역염색 결과로서, 비교군(control)군에 비해 대부분의 실험군에서 단백질 분비가 촉진되었으며, 특히 자성나오입자와 전자기장을 함께 조사한 군에서 단백질발현이 현저히 증가하였다(도 11).Also, Magnetic nanoparticle injection group (MP, 50 ㎍ / ml), electromagnetic field irradiation group (EMF, 45Hz, 8 hours × 2 times / day), and after 10 days of the experimental group irradiated with the magnetic nanoparticles, the osteocalcin and austerity As a result of the immunostaining of Onnectin, protein secretion was promoted in most experimental groups as compared to the control group, especially in the group irradiated with magnetic nanoparticles and the electromagnetic field (Fig. 11).
<실시예 5> 자성나노입자와 저강도 전자기장을 이용한 골세포주의 활성 분석Example 5 Activity Analysis of Bone Cell Lines Using Magnetic Nanoparticles and Low-Intensity Electromagnetic Fields
골세포주(Saos-2)를 세포배양용 100 mm 디쉬에 1 × 105 세포를 접종한 뒤 다음과 같이 실험을 진행하였다.The bone cell line (Saos-2) was inoculated with 1 × 10 5 cells in a 100 mm dish for cell culture, and the experiment was carried out as follows.
구체적으로, control: 비교군, MP(magnetic particle): 자성나노입자 투입군(배지 1 ml당 5 ㎍ 자성나노입자 투여), EMF(electromagnetic field): 전자기장 조사군 (45 Hz, 8시간 × 2회/일) 및 MP+EMF: 자성 나노입자 투입 후 전자기장 조사군을 3일간 각 조건별 실험 후 골 관련 mRNA 분석을 실시하였다.Specifically, control: comparison group, MP (magnetic particle): magnetic nanoparticle injection group (5 ㎍ magnetic nanoparticles per 1 ml medium), EMF (electromagnetic field): electromagnetic field irradiation group (45 Hz, 8 hours × 2 times / Day) and MP + EMF: After the injection of magnetic nanoparticles, the electromagnetic field irradiation group was subjected to bone-related mRNA analysis for three days.
그 결과, 골세포주(Saos-2)에 자성나노입자 투입군(MP, 5 ㎍/ml), 전자기장 조사군(EMF, 45Hz, 8시간 × 2회/일), 및 자성나노입자를 투입과 전자기장을 조사한 실험군의 3일째 세포의 형태학적 사진으로, 모든 실험군에서 세포사와 같은 세포독성은 관찰되지 않았으며(도 12), 3일째 mRNA를 분석한 결과로서, 자성나노입자를 투여하고 전자기장을 조사한 군에서 오스테오칼신, 콜라겐, 그리고 본시알로 프로테인의 발현이 증가되었고, 이는 칼슘채널과 Cbfa1의 활성화에 의한 것으로 확인되었다(도 13).As a result, magnetic nanoparticle injection group (MP, 5 ㎍ / ml), electromagnetic field irradiation group (EMF, 45Hz, 8 hours × 2 times / day), and magnetic nanoparticles were injected into the bone cell line (Saos-2). As a morphological picture of the cells on day 3 of the experimental group irradiated with, no cytotoxicity such as cell death was observed in all experimental groups (FIG. 12), As a result of mRNA analysis on day 3, the expression of osteocalcin, collagen, and bone sialo protein was increased in the group administered with the magnetic nanoparticles and the electromagnetic field, which was confirmed by the activation of calcium channel and Cbfa1 (FIG. 13). .
<실시예 6> 자성나노입자 함유 하이드로젤과 전기장을 이용한 골세포주 활성 분석Example 6 Analysis of Bone Cell Line Activity Using Hydrogen Containing Magnetic Nanoparticles and Electric Field
콜라겐 하이드로젤은 1% 콜라겐, 5배 농축배지(DMEM 세포배양배지를 5배 농축), 그리고 완충용액(pH 8.0)을 7:2:1의 비율로 혼합하여 제조하였고, 골세포주(Saos-2)를 1.0 × 106 세포를 콜라겐 하이드로젤 1 ml과 혼합한 뒤 100 mm 배양 디쉬에 1 ml을 접종한 뒤 30분간 37℃ 인큐베이터에서 젤화를 유도하여 3차원 인공 골 조직을 제조하였다. 그런 다음 약 15 ml의 배지를 첨가하고, 다음과 같은 조건으로 실험을 진행하였다. Collagen hydrogel was prepared by mixing 1% collagen, 5 times concentrated medium (5 times concentrated DMEM cell culture medium), and buffer solution (pH 8.0) at a ratio of 7: 2: 1, and bone cell line (Saos-2). 3) artificial bone tissue was prepared by mixing 1.0 × 10 6 cells with 1 ml of collagen hydrogel, inoculating 1 ml into a 100 mm culture dish, and inducing gelation in a 37 ° C. incubator for 30 minutes. Then, about 15 ml of medium was added, and the experiment was conducted under the following conditions.
구체적으로, control: 하이드로젤, MP(magnetic particle): 하이드로젤 제조시 자성나노입자 혼합 (하이드로젤 1 ml당 20 ㎍ 자성나노입자 혼합), EMF(electromagnetic field): 하이드로젤 제조후 전자기장 조사군 (60 Hz, 8시간 × 2회/일) 및 MP+EMF: 자성 나노입자 함유 하이드로젤 제조 후 전자기장 조사군을 3일간 각 조건별 실험 후 골 관련 mRNA 분석을 실시하였다.Specifically, control: hydrogel, MP (magnetic particle): mixed magnetic nanoparticles in the manufacture of hydrogel (20 ㎍ magnetic nanoparticles per ml of hydrogel), EMF (electromagnetic field): electromagnetic field irradiation group after the manufacture of hydrogel ( 60 Hz, 8 hours × 2 times / day) and MP + EMF: After preparation of the hydrogel containing the magnetic nanoparticles, the electromagnetic field irradiation group was subjected to bone-related mRNA analysis for three days after each experiment.
그 결과, 골세포주(Saos-2)에 자성나노입자 함유 3차원 하이드로젤(MP, 20 ㎍/ml), 3차원 하이이드로젤에 전자기장 조사군(EMF, 60Hz, 8시간 × 2회/일), 그리고 자성나노입자 함유 3차원 하이드로젤에 전자기장을 조사한 실험군의 3일째 세포의 형태학적 사진으로 모든 실험군의 세포가 잘 살아 있었으며(도 14), 3일째 mRNA를 분석한 결과로서, 자성나노입자 함유하이드로젤에 전자기장을 조사한 실험군에서 본시알로 프로테인과 골형성단백질(BMP-2)의 발현이 증가되고 있음을 확인였다(도 15).As a result, three-dimensional hydrogel (MP, 20 ㎍ / ml) containing magnetic nanoparticles in bone cell line (Saos-2), electromagnetic field irradiation group in three-dimensional hydrogel (EMF, 60Hz, 8 hours × 2 times / day) , And the morphological picture of the cells on the 3rd day of the experimental group irradiated with electromagnetic fields on the magnetic nanoparticle-containing three-dimensional hydrogel, the cells of all experimental groups were well alive (FIG. 14), As a result of analyzing the mRNA on the 3rd day, it was confirmed that the expression of bone sialo protein and bone morphogenetic protein (BMP-2) was increased in the experimental group irradiated with the magnetic nanoparticle-containing hydrogel in the electromagnetic field (FIG. 15).

Claims (11)

1) 세포 또는 조직에 자성나노입자를 함유한 하이드로젤을 주입하는 단계; 및1) injecting a hydrogel containing magnetic nanoparticles into cells or tissues; And
2) 상기 단계 1)에서 제조된 하이드로젤에 전자기장을 조사하는 단계를 포함하는 골 분화 및 골 재생을 촉진하는 방법. 2) A method for promoting bone differentiation and bone regeneration comprising irradiating an electromagnetic field to the hydrogel prepared in step 1).
제 1항에 있어서, 상기 세포는 골세포 또는 중간엽 줄기세포인 것을 특징으로 하는 골 분화 및 골 재생을 촉진하는 방법. The method of claim 1, wherein the cells are bone cells or mesenchymal stem cells.
제 1항에 있어서, 상기 자성나노입자는 FeO2, FeO3, 및 FeO4로 이루어진 군으로부터 선택되는 것을 특징으로 하는 골 분화 및 골 재생을 촉진하는 방법. The method of claim 1, wherein the magnetic nanoparticles are selected from the group consisting of FeO 2 , FeO 3 , and FeO 4 .
제 1항에 있어서, 상기 자성나노입자의 크기는 200 nm 미만인 것을 특징으로 하는 골 분화 및 골 재생을 촉진하는 방법. The method of claim 1, wherein the size of the magnetic nanoparticles is less than 200 nm.
제 1항에 있어서, 상기 전자기장은 연속 또는 펄스 형태인 것을 특징으로 하는 골 분화 및 골 재생을 촉진하는 방법. 10. The method of claim 1, wherein said electromagnetic field is in the form of a continuous or pulsed form.
제 1항에 있어서, 상기 전자기장의 주파수는 45 Hz 내지 75 Hz인 것을 특징으로 하는 골 분화 및 골 재생을 촉진하는 방법. The method of claim 1, wherein the frequency of the electromagnetic field is between 45 Hz and 75 Hz.
제 1항에 있어서, 상기 전자기장의 강도는 10 G 내지 1.5 T인 것을 특징으로 하는 골 분화 및 골 재생을 촉진하는 방법. 2. The method of claim 1, wherein the strength of the electromagnetic field is between 10 G and 1.5 T.
제 1항에 있어서, 상기 하이드로젤의 pH는 3 내지 5인 것을 특징으로 하는 골 분화 및 골 재생을 촉진하는 방법. The method of claim 1, wherein the pH of the hydrogel is 3 to 5 characterized in that to promote bone differentiation and bone regeneration.
제 1항에 있어서, 상기 자성나노입자는 단독 또는 골 형성 유도 물질들과 함께 하이드로젤에 담지되어 골 결손 부위에 충진되거나, 임플란트 표면에 코팅 또는 함유되어지는 것을 특징으로 하는 골 분화 및 골 재생을 촉진하는 방법. The method according to claim 1, wherein the magnetic nanoparticles are alone or in combination with bone formation inducing materials to be filled in the bone defect site, or coated or contained on the implant surface, bone differentiation and bone regeneration characterized in that How to promote.
제 9항에 있어서, 상기 골 형성 유도 물질은 BMP(Bone morphogenetic protein), 하이드록시아파타이트, TCP(Tricalcium phosphate) 및 DCP(dicalcium pyrophosphate)로 구성되는 군으로부터 선택되는 하나 이상의 성분인 것을 특징으로 하는 골 분화 및 골 재생을 촉진하는 방법. The bone forming agent of claim 9, wherein the bone formation inducing substance is at least one component selected from the group consisting of bone morphogenetic protein (BMP), hydroxyapatite, tricalcium phosphate (TCP) and dicalcium pyrophosphate (DCP). How to promote differentiation and bone regeneration.
제 9항에 있어서, 상기 임플란트는 콜라겐, 하루론산, 알지네이트 및 키토산으로 구성된 천연고분자, PLA(poly(lactic acid)), PLGA((poly(lactic-co-glycolic acid))), PGA(poly(glycolic acid)), PCL(polycaprolactone) 및 PMMA(poly(methyl methacrylate))로 구성된 합성고분자, 및 티타늄, 티타늄 합금 및 니켈-코발트 합금으로 구성된 합성고분자로 구성된 군으로부터 선택되는 하나 이상의 성분을 포함하는 것을 특징으로 골 분화 및 골 재생을 촉진하는 방법.The method of claim 9, wherein the implant is a natural polymer composed of collagen, haronic acid, alginate and chitosan, PLA (poly (lactic acid)), PLGA ((poly (lactic-co-glycolic acid))), PGA (poly ( glycolic acid)), PCL (polycaprolactone) and PMMA (poly (methyl methacrylate)) synthetic polymers, and synthetic polymers composed of titanium, titanium alloys and nickel-cobalt alloys Characterized by a method for promoting bone differentiation and bone regeneration.
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