WO2017155328A1 - 생체 내 분해속도 및 물성 조절 가능한 생체적합성 돼지연골 유래 세포외기질 막 제조방법 및 상기 돼지연골 유래 세포외기질을 유효성분으로 함유하는 유착방지용 조성물 - Google Patents
생체 내 분해속도 및 물성 조절 가능한 생체적합성 돼지연골 유래 세포외기질 막 제조방법 및 상기 돼지연골 유래 세포외기질을 유효성분으로 함유하는 유착방지용 조성물 Download PDFInfo
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- WO2017155328A1 WO2017155328A1 PCT/KR2017/002563 KR2017002563W WO2017155328A1 WO 2017155328 A1 WO2017155328 A1 WO 2017155328A1 KR 2017002563 W KR2017002563 W KR 2017002563W WO 2017155328 A1 WO2017155328 A1 WO 2017155328A1
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- A—HUMAN NECESSITIES
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- 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
- A61L27/3604—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 characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
- A61L27/3633—Extracellular matrix [ECM]
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- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
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- A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
- A61L2/0005—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts
- A61L2/0011—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts using physical methods
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- A61L2/0035—Gamma radiation
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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- A61L27/3604—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 characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
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- A61L27/3683—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 subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
- A61L27/3687—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 subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
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- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
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- 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
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
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Definitions
- the present invention relates to a biocompatible porcine cartilage-derived extracellular matrix membrane manufacturing method capable of controlling degradation rate and physical properties in vivo, and an anti-adhesion composition containing the porcine cartilage-derived extracellular matrix as an active ingredient.
- Natural materials are derived from natural materials, animals and the human body and have very good biocompatibility and physiological functions.
- An example is an extracellular matrix (ECM), which is a biomaterial extracted from a complex human body and an animal, and is characterized by controlling or regulating the function of a cell or inducing tissue regeneration. Therefore, the support made of natural materials has been evaluated as an ideal cell therapeutic or tissue engineering support because it can provide excellent bio-functionality and biodegradability as well as less inflammatory response after implantation into a living body.
- ECM extracellular matrix
- allogenic or xenogenic tissues or organs have been harvested, and then, acellularized and attracted attention have been used for various types of scaffolds or membranes.
- cartilage-derived extracellular matrix is avascular and nerve-free tissue, there is a tissue-specific distinction compared to the extracellular matrix of other tissues.
- cartilage tissue is rich in chondroimodulin, thrombospondin, and endostatin that inhibit angiogenesis, as well as lubricin and beaglely, which prevent cell adhesion. Biglycan, decorin, fibromodulin and the like.
- the extracellular matrix of cartilage tissue is difficult to control the degradation period, and due to its weak physical properties, there is a limit to application to the human body. The development of a method to make it necessary.
- Adhesion means that the tissues of the organs that should be separated are connected and fused to the fibrous tissue by surgery or inflammation. Complications are small bowel obstruction, acquired female infertility, ectopic pregnancy, chronic abdominal pain, and reoperation. . These adhesions occur in 50-90% of the patients after surgery, and the frequency of reattachment is 34.6%. Adhesion is similar to the healing process that occurs after tissue damage, so it is important to prevent adhesion only without interrupting the healing process. Adhesions continue to occur during the healing of the surgical site, even though the wound healing process is complete, resulting in binding to other adjacent tissues, and ultimately invading the blood vessels into one complete tissue or organ. . In order to prevent such adhesion, drugs that promote fibrin breakdown are used, but anti-adhesion materials are often used to create a physical barrier between tissues and to space the tissues.
- biodegradable materials that is, cellulose or hyaluronic acid, as a physical barrier, and other natural materials such as collagen are used.
- biodegradable materials have the property of absorbing water and are characterized by hydrolysis and are used mainly after abdominal surgery because of their rapid decomposition rate.
- These products usually have the form of a gel, which has a disadvantage of having a wide administration site and difficult fixation at an injured site.
- Interceed the most commonly used anti-adhesion agent, produces only 60% of its anti-adhesion effect (www.ethicon.com).
- the final stage of adhesion is vascularization and by this stage the adherent tissue becomes a single tissue, irreversibly completing the adhesion. Therefore, it is very important to prevent the formation of blood vessels across damaged tissues.
- existing anti-adhesion agents serve as simple barriers and do not have a pharmacological mechanism that prevents vascular invasion that can ultimately occur due to lack of physiological function.
- An object of the present invention is to provide a biocompatible porcine cartilage-derived extracellular matrix membrane manufacturing method capable of controlling degradation rate and physical properties in vivo and an anti-adhesion composition containing the porcine cartilage-derived extracellular matrix as an active ingredient.
- the present invention comprises the steps of separating the pig cartilage; Freeze-drying and grinding the separated pork cartilage; Decellularizing the ground pig cartilage powder; Mixing the decellularized porcine cartilage powder with an acidic solution and pepsin, and then neutralizing it with a basic solution to prepare an aqueous porcine cartilage powder solution; Preparing a porcine cartilage-derived extracellular matrix membrane by mixing the aqueous solution of pig cartilage with a crosslinking agent; And it provides a biocompatible porcine cartilage-derived extracellular matrix membrane manufacturing method that can control the degradation rate and physical properties in vivo comprising the step of irradiating the pig cartilage-derived extracellular matrix membrane.
- the present invention provides a composition for preventing adhesion, comprising a pig cartilage-derived extracellular matrix prepared by the above method as an active ingredient.
- the present invention relates to a biocompatible porcine cartilage-derived extracellular matrix membrane manufacturing method capable of controlling degradation rate and physical properties in vivo, and to an anti-adhesion composition containing the porcine cartilage-derived extracellular matrix as an active ingredient, having high biocompatibility as a natural material.
- the extracellular matrix of cartilage tissues is difficult to control the degradation period and the physical properties are limited. Therefore, the method has been developed to improve the physical properties through physical or chemical treatment and radiation treatment.
- the porcine cartilage-derived extracellular matrix was treated with physicochemical methods to produce biomaterials of various formulations, and the physicochemical treatment was performed to confirm the characteristics of maintaining cartilage specific functions.
- it can also be used as an anti-adhesion agent having excellent stability in the body and anti-adhesion effect using porcine cartilage-derived extracellular matrix material.
- Figure 1 shows a pig cartilage-derived extracellular matrix membrane manufacturing process of the present invention.
- Figure 2 is a comparison result before and after crosslinking of the cartilage extracellular matrix membrane. Extracellular matrix anti-adhesion film, before (left) crosslinking, after (middle) crosslinking, (right) after irradiation.
- Figure 3 is a component analysis of the cartilage extracellular matrix source material.
- Figure 6 is a result of in vivo disassembly experiment according to the irradiation of the cartilage extracellular matrix membrane.
- Figure 7 shows the results of vascular endothelial cell adhesion to the cartilage extracellular matrix membrane.
- Figure 8 is a result of confirming the effect in vivo using the mouse cecum adhesion model.
- (a) Mouse cecal adhesion model experimental procedure and extracellular matrix membrane transplantation process are shown.
- (b) One week after extracellular matrix membrane transplantation, comparison with control group.
- (c) 1 week after extracellular matrix membrane transplantation.
- the present invention comprises the steps of separating the pig cartilage; Freeze-drying and grinding the separated pork cartilage; Decellularizing the ground pig cartilage powder; Mixing the decellularized porcine cartilage powder with an acidic solution and pepsin, and then neutralizing it with a basic solution to prepare an aqueous porcine cartilage powder solution; Preparing a porcine cartilage-derived extracellular matrix membrane by mixing the aqueous solution of pig cartilage with a crosslinking agent; And it provides a biocompatible porcine cartilage-derived extracellular matrix membrane manufacturing method that can control the degradation rate and physical properties in vivo comprising the step of irradiating the pig cartilage-derived extracellular matrix membrane.
- the crosslinking agent may be glutaraldehyde, but is not limited thereto.
- the step of irradiating the radiation may be irradiated with 5 to 100KGy gamma rays, but is not limited thereto.
- the step of decellularizing is characterized in that it is performed by a physical decellularization method, a chemical decellularization method or a combination of physical and chemical methods.
- the physical decellularization methods include freeze-thaw, sonication, or physical agitation.
- the chemical decellularization method is characterized in that the porcine cartilage derived powder is treated with a stock solution, anionic surfactant, nonionic surfactant, cationic surfactant, DNase, RNase or trypsin.
- the decellularizing step is preferably performed at a temperature range of about 0 ⁇ 50 °C.
- the stock solution is Tris HCl (pH 8.0) solution
- the anionic surfactant is sodium dodecyl sulfate (SDS), sodium deoxycholate, or Triton X-200
- the nonionic surfactant is Triton X-100
- the cationic surfactant is CHAPS, sulfobeta-10 (Sulfobetaine-10, SB- 10), sulfobetaine-16 (SB-16), or tri-n-butyl phosphate.
- the present invention provides a composition for preventing adhesion, comprising a pig cartilage-derived extracellular matrix prepared according to the above method as an active ingredient.
- porcine cartilage-derived extracellular matrix can prevent the formation of adhesion by inhibiting fibrosis and inflammation of the surgical site.
- the composition may be provided in the form of ointment, powder, gel, film, slab, wrap or sponge to prevent adhesion formation at the surgical site.
- porcine cartilage-derived extracellular matrix powders For the preparation of porcine cartilage-derived extracellular matrix powders, see EN 12442, "Animal tissues and their derivatives utilized in the manufacture of medical devices, part 1; Analysis and management of risk, part 2; controls on sourcing, collection and handling". The pig knee cartilage of the facility that meets the criteria was purchased and used.
- Pig cartilage powder manufacturing process is as follows.
- Cartilage was cut from pork cartilage to make cartilage pieces (about 20 X 30 mm), washed three times with physiological saline for 10 minutes, frozen at -80 ° C and lyophilized for 3 days.
- the dried cartilage pieces were lyophilized to a size of about 10 ⁇ m using a freeze mill (JAI, JFC-300, JAPAN) and stored at -80 ° C.
- the pig cartilage powder prepared above was treated with 500 ml per 10 g of a stock solution (Hypotonic buffer) and stirred at 200 rpm for 4 hours at 4 ° C. In order to precipitate and separate the cartilage powder, it was treated for 30 minutes at 10,000 rpm using a centrifuge (US-21SMT, Vision, Korea).
- cartilage powder was added to 0.1% SDS (Sodium dodecyl sulfate, Bio-rad, USA) solution and stirred at 200 ° C. for 2 hours at 200 rpm. After the SDS treatment, the cartilage powder was washed 5 times using tertiary distilled water, and the exchange of the wash water was performed under the centrifugal conditions as described above.
- SDS Sodium dodecyl sulfate, Bio-rad, USA
- the decellularized cartilage powder was lyophilized for 3 days after cooling in an -80 ° C cryogenic freezer.
- the dried cartilage powder was crushed in the same manner as described above to finally obtain a cartilage powder powder of about 10 ⁇ m size and stored at -80 °C until needed.
- Soluble cartilage powder was placed in a dialysis membrane (MWCO 1000, Spectrolab, USA) and stirred in distilled water at 200 rpm for 24 hours at 200 rpm. Thereafter, the received cartilage powder was placed in a container, cooled in a -80 ° C cryogenic freezer, and lyophilized for 3 days. Soluble cartilage powder was pulverized into a powder having a size of about 10 ⁇ m in the same manner as described above and finally stored in a -80 ° C. freezer until needed.
- aqueous cartilage powder prepared above was treated with 100 ml of tertiary distilled water, followed by stirring at 200 rpm for 1 hour at room temperature.
- the aqueous solution of cartilage powder was placed in a centrifuge container and treated at 3000 rpm for 10 minutes.
- the supernatant was dispensed 35 ml into a 100 X 100 mm square silicone mold using a pipet and dried for 48 hours on a clean bench.
- the extracellular matrix membrane prepared above was packaged with silver foil and subjected to gamma irradiation with a dose of 5KGy ⁇ 100KGy.
- Membrane used as anti-adhesion agent was important to the physical properties that can sufficiently protect the surgical site because the tensile strength test according to the product characteristics based on the KFDA guidelines (Fig. 4a).
- the cut extracellular matrix membrane was plotted by indicating the degree of tensile strength as the ultimate force value through a tensile strength meter. As shown in the results, the tensile strength was improved as the thickness of the extracellular matrix membrane was increased, and the physical strength was also significantly increased through chemical crosslinking. In addition, even in the measurement of the tensile strength of the irradiated sample, it was confirmed that the physical strength can be adjusted according to the radiation dose (FIG. 4A).
- the anti-adhesion membrane must be fixed to the surgical site to effectively prevent anti-adhesion. Therefore, even when sutured during surgery, strength that can be broken without breaking is required. Since the seal strength is not specified in the KFDA, it was established by establishing a protocol that can measure the seal strength on its own. Suture strength measurement was performed by cutting the film as shown below and then suture the film using an operating room and attaching the operating room and the extracellular matrix membrane to the tensile strength meter to measure the tensile strength. As a result of the measurement, the sealing strength of the extracellular matrix membrane was significantly increased after crosslinking (FIG. 4B).
- the adhesion prevention film with controlled degradation time for each organ should be used for long-term specific adhesion prevention effect.
- the degree of degradation can be controlled according to the radiation dose of the cartilage extracellular matrix membrane.
- cartilage extracellular matrix membranes treated with different radiation doses were cut to 1 ⁇ 1 cm and treated with PBS treated with collagenase to observe the course of 2 weeks (Fig. 5 left).
- the samples treated with collagenase for 2 weeks were centrifuged to analyze the amount of hydroxyproline in the supernatant to determine the degree of degradation of collagen, which is a major component of the extracellular matrix membrane (FIG. 5).
- the degree of degradation of the extracellular matrix membrane was controlled when the collagenase was treated according to the radiation dose.
- cartilage extracellular matrix membrane prepared in Figure 1 was confirmed the difference in adhesion of vascular endothelial cells acting in the adhesion mechanism as follows.
- the cartilage extracellular matrix membrane and cover glass prepared in FIG. 1 were attached to a separate 24 well dish having a diameter of 5 mm, dried and fixed, and sterilized with IOS. And 2x10 4 vascular endothelial cells were transplanted into membrane coated dish, cover glass and uncoated 24 well plate and attached for 24 hours. After that, the cells attached to each surface were stained with calein and observed through a fluorescence microscope. As a result, it was confirmed that cell adhesion was inhibited in the dish coated with the extracellular matrix membrane as compared to the covel glass and the well plate (FIG. 7).
- An adhesion model was constructed using 8-week-old C57BL6 mice. After cutting the skin layer and the muscle layer of the mouse abdomen, the muscle layer and the skin layer were sutured to make a model in which adhesion occurs between the damaged tissue and confirmed the adhesion effect. Adhesion tissue was generated at 1 week and an adhesion model was formed to strongly adhere muscles and skin (FIG. 8A).
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Claims (5)
- 돼지연골을 분리하는 단계;상기 분리된 돼지연골을 동결건조하여 분쇄하는 단계;상기 분쇄된 돼지연골분말을 탈세포화하는 단계;상기 탈세포화된 돼지연골분말을 산성 용액 및 펩신(pepsin)과 혼합하여 처리 후, 염기성 용액으로 중화시켜 돼지연골분말 수용액을 제조하는 단계;상기 돼지연골분말 수용액을 가교제와 혼합하여 돼지연골 유래 세포외기질 막을 제조하는 단계; 및상기 돼지연골 유래 세포외기질 막에 방사선을 조사하는 단계를 포함하는 생체 내 분해속도 및 물성 조절 가능한 생체적합성 돼지연골 유래 세포외기질 막 제조방법.
- 제1항에 있어서, 상기 가교제는 글루타르알데히드(Glutaraldehyde)인 것을 특징으로 하는 생체 내 분해속도 및 물성 조절 가능한 생체적합성 돼지연골 유래 세포외기질 막 제조방법.
- 제1항에 있어서, 상기 방사선을 조사하는 단계는 5 내지 100KGy 감마선을 조사하는 것을 특징으로 하는 생체 내 분해속도 및 물성 조절 가능한 생체적합성 돼지연골 유래 세포외기질 막 제조방법.
- 제1항 내지 제3항 중 어느 한 항에 따라 제조된 돼지연골 유래 세포외기질을 유효성분으로 함유하는 유착방지용 조성물.
- 제4항에 있어서, 상기 조성물은 연고, 분말, 겔, 필름, 슬랩, 랩 및 스펀지로 이루어진 군에서 선택된 어느 하나의 형태인 것을 특징으로 하는 유착방지용 조성물.
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EP17763589.3A EP3427765A4 (en) | 2016-03-11 | 2017-03-09 | METHOD FOR PRODUCING EXTRACELLULAR MATRIX MEMBRANE OBTAINED FROM BIOCOMPATIBLE PIG CARTILAGE CAPABLE OF CONTROLLING IN VIVO DECOMPOSITION RATE AND PHYSICAL PROPERTIES, AND COMPOSITION FOR PREVENTING ADHESION CONTAINING EXTRACELLULAR CARRINOUS MATRIX |
CN201780016682.7A CN108834404A (zh) | 2016-03-11 | 2017-03-09 | 可调节体内降解速率以及物理性质的生物相容性猪软骨衍生细胞外基质膜制造方法及包含猪软骨衍生细胞外基质作为有效成分的防粘连用组合物 |
JP2018567556A JP6961629B2 (ja) | 2016-03-11 | 2017-03-09 | 生体内分解率及び物性の調節が可能な生体適合性豚軟骨来由細胞外基質膜の製造方法 |
US16/083,564 US20190070336A1 (en) | 2016-03-11 | 2017-03-09 | Method for producing extracellular matrix membrane derived from biocompatible porcine cartilage capable of regulating in vivo decomposition rate and physical properties, and composition for preventing adhesion containing extracellular matrix derived from porcine cartilage as active ingredient |
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KR10-2016-0029579 | 2016-03-11 | ||
KR1020160029579A KR101772316B1 (ko) | 2016-03-11 | 2016-03-11 | 생체 내 분해속도 및 물성 조절 가능한 생체적합성 돼지연골 유래 세포외기질 막 제조방법 및 상기 돼지연골 유래 세포외기질을 유효성분으로 함유하는 유착방지용 조성물 |
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PCT/KR2017/002563 WO2017155328A1 (ko) | 2016-03-11 | 2017-03-09 | 생체 내 분해속도 및 물성 조절 가능한 생체적합성 돼지연골 유래 세포외기질 막 제조방법 및 상기 돼지연골 유래 세포외기질을 유효성분으로 함유하는 유착방지용 조성물 |
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US (1) | US20190070336A1 (ko) |
EP (1) | EP3427765A4 (ko) |
JP (1) | JP6961629B2 (ko) |
KR (1) | KR101772316B1 (ko) |
CN (1) | CN108834404A (ko) |
WO (1) | WO2017155328A1 (ko) |
Cited By (1)
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US10939362B2 (en) | 2018-01-11 | 2021-03-02 | Mediatek Inc. | Apparatuses and methods for system information (SI) request through a contention-based random access procedure |
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KR102038434B1 (ko) * | 2018-01-16 | 2019-10-30 | 한국원자력연구원 | 동물의 연골 유래 세포외기질 막의 분해도 조절 방법 및 이를 이용하여 분해도가 조절된 동물의 연골 유래 세포외기질 막 |
KR102333417B1 (ko) | 2019-08-27 | 2021-12-02 | 주식회사 도프 | 동물지방 유래 세포외기질 및 동물지방 유래 세포외기질 보존액 |
KR102416861B1 (ko) * | 2020-02-28 | 2022-07-07 | 주식회사 엘앤씨바이오 | 연골 성분을 함유하는 연골 재생용 조성물 및 그 제조방법 |
CN114340686B (zh) * | 2020-02-28 | 2023-06-06 | 爱恩斯生物科技(昆山)有限公司 | 含有软骨成分的软骨再生用组合物及其制备方法 |
CN111840642B (zh) * | 2020-07-15 | 2022-04-01 | 四川大学 | 一种软骨脱细胞基质复合支架的制备方法及其应用 |
EP4232050A4 (en) * | 2020-10-26 | 2024-05-08 | Biopryme Biologics, Inc. | PREPARATION AND USE OF POWDER DERIVED FROM TISSUE MATRIX |
KR102549899B1 (ko) * | 2021-02-19 | 2023-07-03 | 한국원자력연구원 | 동물의 연골 유래 세포외기질 막의 접착력 조절 방법 및 이를 이용하여 접착력이 조절된 동물의 연골 유래 세포외기질 막 |
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- 2017-03-09 JP JP2018567556A patent/JP6961629B2/ja active Active
- 2017-03-09 CN CN201780016682.7A patent/CN108834404A/zh active Pending
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EP3427765A1 (en) | 2019-01-16 |
US20190070336A1 (en) | 2019-03-07 |
JP6961629B2 (ja) | 2021-11-05 |
EP3427765A4 (en) | 2020-01-15 |
KR101772316B1 (ko) | 2017-08-29 |
JP2019508213A (ja) | 2019-03-28 |
CN108834404A (zh) | 2018-11-16 |
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