WO2013047936A1 - 섬유상 탈회골 기질의 제조방법 - Google Patents

섬유상 탈회골 기질의 제조방법 Download PDF

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WO2013047936A1
WO2013047936A1 PCT/KR2011/007517 KR2011007517W WO2013047936A1 WO 2013047936 A1 WO2013047936 A1 WO 2013047936A1 KR 2011007517 W KR2011007517 W KR 2011007517W WO 2013047936 A1 WO2013047936 A1 WO 2013047936A1
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Prior art keywords
bone
demineralized
demineralized bone
hours
fibrous
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PCT/KR2011/007517
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English (en)
French (fr)
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송석범
정구원
소정원
서한솔
유현승
김귀남
김병석
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(주)시지바이오
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Priority to EP11873106.6A priority Critical patent/EP2762175B1/en
Priority to US14/347,601 priority patent/US9029077B2/en
Priority to CN201180073750.6A priority patent/CN103998070B/zh
Priority to ES11873106.6T priority patent/ES2617785T3/es
Publication of WO2013047936A1 publication Critical patent/WO2013047936A1/ko

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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
    • A61L27/36—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
    • A61L27/3683—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
    • 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
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/28—Bones
    • 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
    • A61L27/3604—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
    • A61L27/3608—Bone, e.g. demineralised bone matrix [DBM], bone powder
    • 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
    • A61L27/3641—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 site of application in the body
    • A61L27/3645—Connective tissue
    • A61L27/365—Bones
    • 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
    • A61L27/3683—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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/28—Bones
    • A61F2002/2835—Bone graft implants for filling a bony defect or an endoprosthesis cavity, e.g. by synthetic material or biological material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/28—Bones
    • A61F2002/2835—Bone graft implants for filling a bony defect or an endoprosthesis cavity, e.g. by synthetic material or biological material
    • A61F2002/2839—Bone plugs or bone graft dowels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/30—Joints
    • A61F2/46—Special tools or methods for implanting or extracting artificial joints, accessories, bone grafts or substitutes, or particular adaptations therefor
    • A61F2/4644—Preparation of bone graft, bone plugs or bone dowels, e.g. grinding or milling bone material
    • A61F2002/4645—Devices for grinding or milling bone material
    • 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
    • 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
    • A61L2430/40—Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking

Definitions

  • the present invention relates to an improved method for producing fibrous demineralized bone matrices, and more particularly, to an improved method for producing fibrous demineralized bone matrices comprising processes of partial deliming, slicing, complete deliming, and grinding.
  • Demineralized bone matrix consists mostly of highly cross-linked collagen, and remaining non-collagen proteins include TGF- ⁇ , PDGF, osteopontin, osteonectin, and bone morphogenetic protein (BMP). .
  • BMP bone morphogenetic protein
  • the demineralized bone matrix is used in the form of a bone graft composition for the purpose of repairing bone defects and the like.
  • the demineralized bone matrix is prepared in the form of particles by performing a demineralization process on the bone separated in vitro and then grinding it to an appropriate size.
  • the present inventors are demineralized bone matrix having a particle size of 0.05 to 250 ⁇ m; Demineralized bone matrix having a particle size of 250 to 2000 ⁇ m; And a composition for bone regeneration in which a hydration material is mixed at a specific ratio, the composition having excellent injectability and formability (handling) (Korean Patent Registration No. 10-1041784).
  • Cortical bones are composed of collagen fiber bundles parallel to the long axis of the bone, and the fibrous demineralized bone matrix obtained therefrom is used in implants for bone repair and other orthopedic applications. It is known to exhibit useful properties. Fibrous demineralized bone matrix is prepared by pulverizing cortical bone separated in vitro using a special grinder (eg US Pat. No. 5,607,269) until the particles in fibrous form are obtained and then demineralized.
  • a special grinder eg US Pat. No. 5,607,269
  • this manufacturing method has to use only natural cortical shafts as bone raw materials due to the mechanical limitation of the grinding machine, and also has a problem in that the yield of fibrous demineralized bone is low. In order to remedy this problem, U.S. Pat.Nos.
  • 7,323,193 and 7,939,108 cut the bones separated into in vitro to a suitable size and demineralize in acidic solution for 6 hours and then demineralize in acidic solution for 2 days. It has been disclosed a method of preparing fibrous demineralized bone matrix by grinding. However, the improved manufacturing method according to the above-mentioned US Pat. Nos. 7,323,193 and 7,939,108 has a problem of performing the deliming process for a long time of 2 days, that is, 48 hours.
  • the present inventors have conducted various studies to develop a method for producing a fibrous demineralized bone matrix having useful properties, in particular, a method for improving a long time deliming process.
  • a method for improving a long time deliming process As a result, when the bone obtained by partially demineralized is manufactured in the form of a thin sheet through slicing, and then demineralized, it is surprising that the demineralization process can be remarkably shortened to about 6 hours, and the demineralized bone matrix fibers It has been found that while in form it is obtained in high yield.
  • an object of the present invention is to provide a method for producing a fibrous demineralized bone substrate by sequentially performing the processes of partial deliming, slicing, complete deliming, and grinding.
  • step (a) primary demineralization of the bone separated in vitro for 1 to 5 hours in an acidic solution; (b) slicing the bone obtained from step (a) to a thickness of 0.1 to 3 mm to form a bone in sheet form; (c) secondary demineralization of the bone in sheet form obtained in step (b) in an acidic solution for 2 to 6 hours; And (d) crushing the demineralized bone obtained in step (c).
  • the acidic solution of step (a) and step (c) may be 0.1 to 3 N HCl aqueous solution independently of each other, preferably steps (a) and step
  • the acidic solution of (c) may be about 0.6 N HCl aqueous solution.
  • the grinding may be performed to obtain a fibrous demineralized bone matrix having a length in the range of 1000 to 5000 ⁇ m.
  • the deliming process can be significantly shortened by about 6 hours.
  • the manufacturing method according to the present invention can be suitably applied to mass production on an industrial scale.
  • FIG. 1 shows bone A obtained after slicing partially demineralized bone and bone B obtained after pulverizing demineralized bone.
  • FIG. 2 is a photograph of the fibrous demineralized bone matrix (A) and the particulate demineralized bone matrix (B) measured by an optical microscope.
  • the present invention comprises the steps of (a) primary demineralization of the bone separated in vitro for 1 to 5 hours in an acidic solution; (b) slicing the bone obtained from step (a) to a thickness of 0.1 to 3 mm to form a bone in sheet form; (c) secondary demineralization of the bone in sheet form obtained in step (b) in an acidic solution for 2 to 6 hours; And (d) pulverizing the demineralized bone obtained in step (c).
  • the manufacturing method of the present invention sequentially performs the processes of partial deliming, slicing, complete deliming, and pulverization, and in particular, through partial deliming and slicing, the subsequent deliming process can be significantly shortened to about 6 hours, Demineralized bone matrices in the form of fibers can be prepared in high yields.
  • the production method of the present invention includes a partial deliming process, ie, primary deliming of the bone separated in vitro for 1 to 5 hours in an acidic solution (ie, step (a)).
  • the extracorporeal bone may be bone of a mammal, including a human.
  • the bone separated into the body is preferably used after removing the soft tissue, fat, bone marrow, etc. attached to the bone in a conventional manner.
  • the removal can usually be carried out using 60 to 90% by weight of an aqueous solution of ethanol, it may be further used a surfactant as needed.
  • an aqueous HCl solution typically an aqueous HCl solution may be used, and an aqueous 0.1 to 3 N HCl solution, preferably about 0.6 N HCl, may be used.
  • the first deliming time may be 1 to 5 hours, preferably about 3 hours.
  • the manufacturing method of the present invention is a step of slicing the partially demineralized bone in the form of a sheet, that is, slicing the bone obtained from the partial demineralization process to a thickness of 0.1 to 3 mm _ to form a sheet-shaped bone [that is, Step (b)].
  • the slicing may be used without limitation as long as the device can cut thin bones, for example, a bone slicer such as Bone Slicer (YOU IL MC / CO. KR) may be used.
  • the thickness of the sheet-shaped bone obtained through the slicing may be 0.1 to 3 mm, preferably 0.2 to 1.0 mm, more preferably 0.3 to 0.6 mm, most preferably about 0.5 mm.
  • a broken particle shape other than a sheet form is obtained.
  • the thickness is less than 0.1 mm, a sheet form is obtained, but even when demineralized, a particulate form that is not fibrous is obtained.
  • the production method of the present invention is a step of completely demineralizing the bone in the form of a partially demineralized sheet, that is, a step of demineralizing the bone in the form of the sheet obtained in the acid solution for 2 to 6 hours [ie, step (c)] Include.
  • the acidic solution typically an aqueous HCl solution may be used, and an aqueous 0.1 to 3 N HCl solution, preferably about 0.6 N HCl, may be used.
  • the secondary deliming i.e. complete deliming, can be carried out within a significantly shortened time compared to conventional deliming times, i.e. 2 to 6 hours, preferably about 3 hours.
  • the present invention includes the step of crushing the fully demineralized bone (ie step (d)).
  • the grinding process may be performed using a conventional mill.
  • the milling may be carried out to obtain a fibrous demineralized bone matrix having a length in the range of 1000 to 5000 ⁇ m, more preferably in the range of 2,000 to 4,000 ⁇ m, which is suitable for the type of mill. Can be obtained by setting.
  • Bone donated from the human body using surgical instruments to remove the soft tissue attached to the bone, using a tissue cleaner containing a surfactant to remove impurities such as soft tissue, fat, bone marrow, Cut in half.
  • the cut bones were partially demineralized in 20 ml of 0.6N HCl aqueous solution per gram of bone weight for 3 hours.
  • the partially demineralized bone was separated and soaked in 20 ml of distilled water per gram of bone to remove the demineralized solution in the bone.
  • the resulting bone was sliced in the form of a sheet about 0.5 mm thick using a bone slicer (YOU IL MC / CO. KR).
  • the obtained bone in the form of a sheet is the same as in FIG.
  • the resulting sheet-shaped bone was completely demineralized by soaking in 30 ml of an aqueous 0.6N HCl solution per gram of bone for 3 hours. Precipitate and separate the demineralized bone substrate, put in a grinder (IKA, M20 Univeral mill, GR) for about 10 minutes, neutralized with phosphate buffer (PBS), washed with distilled water, and then lyophilized 31 g of demineralized bone matrix was prepared.
  • the photograph of observing the obtained demineralized bone matrix with an optical microscope is as shown in FIG. From the results of A of FIG. 2, it can be seen that the obtained demineralized bone matrix is a fibrous demineralized bone matrix.
  • the bone (weight: about 165 g) donated from the human body is removed using surgical instruments to remove the soft tissues attached to the bones, and the tissue cleaner containing the surfactant is used to remove impurities such as soft tissues, fat, and bone marrow. Cut in half.
  • the cut bones were partially demineralized in 20 ml of 0.6N HCl aqueous solution per gram of bone weight for 3 hours.
  • the partially demineralized bone was recovered and soaked in 20 ml of distilled water per gram of bone to remove the demineralized solution in the bone.
  • the obtained bone was soaked in 30 ml of 0.6N HCl aqueous solution per g of bone weight (g) for 3 hours, and then demineralized.
  • demineralization did not proceed so that demineralized bone matrix could not be obtained.
  • the bone (weight: about 192 g) donated from the human body is removed using surgical instruments to remove the soft tissues attached to the bones, and the tissue cleaner containing the surfactant is used to remove impurities such as soft tissues, fat, and bone marrow. Cut in half.
  • the cut bones were partially demineralized in 20 ml of 0.6N HCl aqueous solution per gram of bone weight for 3 hours.
  • the partially demineralized bone was separated and soaked in 20 ml of distilled water per gram of bone to remove the demineralized solution in the bone.
  • the obtained bone is put into a grinder (IKA, M20 Universal Mill, GR) for about 60 minutes to obtain a ground bone, the shape is the same as B of FIG.
  • the ground bone was completely demineralized by soaking in 30 ml of an aqueous 0.6N HCl solution per gram of bone weight for 3 hours.
  • the demineralized bone substrate was precipitated and separated, neutralized with phosphate buffered solution (PBS), washed with distilled water, and lyophilized to prepare about 36.4 g of demineralized bone substrate.
  • PBS phosphate buffered solution
  • the photograph of observing the obtained demineralized bone matrix with an optical microscope is shown in FIG. From the result of B of FIG. 2, it can be seen that the obtained demineralized bone matrix is a particulate demineralized bone matrix.

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Abstract

본 발명은 (a) 체외로 분리된 뼈를 산성 용액 중에서 1 ∼ 5 시간 동안 1차 탈회시키는 단계; (b) 단계(a)로부터 얻어진 뼈를 0.1 ∼ 3 mm의 두께로 슬라이싱(slicing)하여 시트 형태의 뼈를 형성시키는 단계; (c) 단계(b)에서 얻어진 시트 형태의 뼈를 산성 용액 중에서 2 ∼ 6 시간 동안 2차 탈회하는 단계; 및 (d) 단계(c)에서 얻어진 탈회된 뼈를 분쇄하는 단계를 포함하는, 섬유상 탈회골 기질의 제조방법에 관한 것이다.

Description

섬유상 탈회골 기질의 제조방법
본 발명은 섬유상 탈회골 기질의 개선된 제조방법에 관한 것으로, 더욱 상세하게는 부분 탈회, 슬라이싱, 완전 탈회, 및 분쇄의 공정을 포함하는 섬유상 탈회골 기질의 개선된 제조방법에 관한 것이다.
골을 산에 담가 골 중의 무기질 성분을 제거한 것을 탈회골 기질(demineralized bone matrix, DBM)이라 한다. 탈회골 기질은 대부분 고도로 가교된 콜라겐으로 구성되며, 잔존하는 비 콜라겐성 단백질로는 TGF-β, PDGF, 오스테오폰틴, 오스테오넥틴, 골 형태형성 단백질(bone morphogenetic protein, BMP) 등이 포함된다. 탈회골 기질은 골 결손의 수선 등을 목적으로 한 골 이식용 조성물의 형태로 사용된다.
탈회골 기질은 체외로 분리된 뼈에 대하여 탈회 공정을 수행한 후, 이를 적절한 크기로 분쇄함으로써, 입자 형태로 제조된다. 본 발명자들은 0.05 내지 250 ㎛의 입자크기를 갖는 탈회골 기질; 250 내지 2000㎛의 입자크기를 갖는 탈회골 기질; 및 수화물질을 특정 비율로 혼합한 골 재생용 조성물을 개시한 바 있으며, 상기 조성물은 우수한 주입성 및 형태유지성(취급성)을 갖는다(대한민국 특허등록 제10-1041784호).
한편, 피질성 골(cortical bone)은 골의 장축에 평행하게 콜라겐 섬유 다발(fiber bundles)로 구성되어 있으며, 이로부터 얻어진 섬유상의 탈회골 기질은 골 수선 및 다른 정형외과적 적용을 위한 임플란트에 있어서 유용한 성질을 나타내는 것으로 알려져 있다. 섬유상 탈회골 기질은 체외로 분리된 피질성 뼈를 특수 분쇄기(예를 들어, 미국 특허 제5,607,269호)를 사용하여 섬유 형태의 입자가 얻어질 때까지 분쇄한 후, 탈회시켜 제조된다. 그러나, 이러한 제조방법은 분쇄 기기의 기계적 한계로 인하여, 골 원료로서 천연의 피질성 샤프트(intact cortical shafts)만을 사용하여야 하며, 또한 얻어지는 섬유상 탈회골 수율이 낮은 문제점이 있다. 이러한 문제점을 개선하기 위하여 미국특허 제7,323,193호 및 제7,939,108호는 체외로 분리된 뼈를 적당한 크기로 잘라 산성 용액 중에서 6시간 동안 탈회시킨 후, 다시 2일 동안 산성 용액에서 탈회를 수행한 후, 이를 분쇄하여 섬유상 탈회골 기질을 제조하는 방법을 개시한 바 있다. 그러나, 상기 개선된 미국특허 제7,323,193호 및 제7,939,108호에 따른 제조방법은 2일 즉, 48시간의 장기간 동안 탈회 공정을 수행하여야 하는 문제가 있다.
본 발명자들은 유용한 성질을 갖는 섬유상 탈회골 기질의 제조방법, 특히 장시간의 탈회 공정을 개선할 수 있는 제조방법을 개발하기 위하여 다양한 연구를 수행하였다. 그 결과, 부분 탈회시켜 얻어진 골을 슬라이싱(slicing)을 통하여 얇은 시트 형태로 제조한 후, 이를 탈회시켰을 때, 놀랍게도 탈회 공정을 약 6시간 정도로 현저하게 단축시킬 수 있을 뿐만 아니라, 탈회골 기질이 섬유 형태를 가지면서 높은 수율로 얻어진다는 것을 발견하였다.
따라서, 본 발명은 부분 탈회, 슬라이싱, 완전 탈회, 및 분쇄의 공정을 순차적으로 수행을 통한 섬유상 탈회골 기질의 제조방법을 제공하는 것을 목적으로 한다.
본 발명의 일 태양에 따라, (a) 체외로 분리된 뼈를 산성 용액 중에서 1 ∼ 5 시간 동안 1차 탈회시키는 단계; (b) 단계(a)로부터 얻어진 뼈를 0.1 ∼ 3 mm의 두께로 슬라이싱(slicing)하여 시트 형태의 뼈를 형성시키는 단계; (c) 단계(b)에서 얻어진 시트 형태의 뼈를 산성 용액 중에서 2 ∼ 6 시간 동안 2차 탈회하는 단계; 및 (d) 단계(c)에서 얻어진 탈회된 뼈를 분쇄하는 단계를 포함하는, 섬유상 탈회골 기질의 제조방법이 제공된다.
본 발명에 따른 섬유상 탈회골 기질의 제조방법에 있어서, 단계(a) 및 단계(c)의 상기 산성 용액은 서로 독립적으로 0.1 ∼ 3 N HCl 수용액일 수 있으며, 바람직하게는 단계(a) 및 단계(c)의 상기 산성 용액은 약 0.6 N HCl 수용액일 수 있다. 또한, 상기 분쇄는 1000 내지 5000 ㎛의 범위의 길이를 갖는 섬유상 탈회골 기질이 얻어지도록 수행될 수 있다.
본 발명에 의해, 부분 탈회, 슬라이싱, 완전 탈회, 및 분쇄의 공정을 순차적으로 수행할 경우, 탈회 공정을 약 6 시간 정도로 현저하게 단축시킬 수 있다는 것이 발견되었다. 특히 슬라이싱 공정을 도입함으로써, 탈회 공정의 단축뿐만 아니라, 최종적으로 얻어지는 탈회골 기질이 섬유 형태를 갖는 탈회골 기질로 높은 수율로얻어진다는 것이 밝혀졌다. 따라서, 본 발명에 따른 제조방법은 산업적 규모의 대량생산에 적합하게 적용될 수 있다.
도 1은 부분 탈회된 뼈를 슬라이싱한 후에 얻어진 뼈(A) 및 탈회된 뼈를 분쇄한 후 얻어진 뼈(B)를 나타낸다.
도 2는 섬유상 탈회골 기질(A) 및 입자상 탈회골 기질(B)의 형태를 광학현미경으로 측정한 사진이다.
본 발명은 (a) 체외로 분리된 뼈를 산성 용액 중에서 1 ∼ 5 시간 동안 1차 탈회시키는 단계; (b) 단계(a)로부터 얻어진 뼈를 0.1 ∼ 3 mm의 두께로 슬라이싱(slicing)하여 시트 형태의 뼈를 형성시키는 단계; (c) 단계(b)에서 얻어진 시트 형태의 뼈를 산성 용액 중에서 2 ∼ 6 시간 동안 2차 탈회하는 단계; 및 (d) 단계(c)에서 얻어진 탈회된 뼈를 분쇄하는 단계를 포함하는, 섬유상 탈회골 기질의 제조방법을 제공한다.
본 발명의 제조방법은 부분 탈회, 슬라이싱, 완전 탈회, 및 분쇄의 공정을 순차적으로 수행하며, 특히 부분 탈회 및 슬라이싱 공정을 통하여, 이어지는 탈회 공정을 약 6 시간 정도로 현저하게 단축시킬 수 있을 뿐만 아니라, 섬유 형태를 갖는 탈회골 기질로 높은 수율로 제조할 수 있다.
본 발명의 제조방법은 부분 탈회 공정, 즉 체외로 분리된 뼈를 산성 용액 중에서 1 ∼ 5 시간 동안 1차 탈회시키는 단계[즉, 단계(a)]를 포함한다. 상기 체외로 분리된 뼈는 인간을 포함한 포유동물의 뼈일 수 있다. 상기 체외로 분리된 뼈는 통상의 방법에 따라 뼈에 붙어있는 연조직, 지방, 골수 등을 제거한 후 사용하는 것이 바람직하다. 상기 제거는 통상 60 내지 90 중량%의 에탄올 수용액을 사용하여 수행될 수 있으며, 필요에 따라 계면활성제를 추가로 사용할 수도 있다. 상기 산성 용액으로는 전형적으로 HCl 수용액을 사용할 수 있으며, 0.1 ∼ 3 N HCl 수용액, 바람직하게는 약 0.6 N HCl 수용액이 사용될 수 있다. 상기 1차 탈회 시간은 1 ∼ 5 시간, 바람직하게는 약 3 시간일 수 있다.
본 발명의 제조방법은 부분 탈회된 뼈를 시트 형태로 슬라이싱하는 공정 즉, 부분 탈회 공정으로부터 얻어진 뼈를 0.1 ∼ 3 mm_의 두께로 슬라이싱(slicing)하여 시트 형태의 뼈를 형성시키는 단계[즉, 단계(b)]를 포함한다. 상기 슬라이싱은 뼈를 얇게 잘라낼 수 있는 기기라면 제한 없이 사용될 수 있으며, 예를 들어 Bone Slicer(YOU IL MC/CO. KR) 등의 본 슬라이서(bone slicer)가 사용될 수 있다. 상기 슬라이싱을 통하여 얻어지는 시트 형태의 뼈의 두께는 0.1 ∼ 3 mm, 바람직하게는 0.2 ∼ 1.0 mm, 더욱 바람직하게는 0.3 ∼ 0.6 mm, 가장 바람직하게는 약 0.5 mm일 수 있다. 상기 두께가 3.0 mm를 초과할 경우에는 시트형태가 아닌 부셔진 입자형태가 얻어지고, 0.1 mm 미만의 경우 시트 형태로 얻어지나 탈회해도 섬유상이 아닌 입자상이 얻어진다.
본 발명의 제조방법은 부분 탈회된 시트 형태의 뼈를 완전 탈회시키는 공정, 즉 상기에서 얻어진 시트 형태의 뼈를 산성 용액 중에서 2 ∼ 6 시간 동안 2차 탈회하는 단계[즉, 단계(c)]를 포함한다. 상기 산성 용액으로는 전형적으로 HCl 수용액을 사용할 수 있으며, 0.1 ∼ 3 N HCl 수용액, 바람직하게는 약 0.6 N HCl 수용액이 사용될 수 있다. 상기 2차 탈회 즉, 완전 탈회는 종래의 탈회 시간에 비하여 현저하게 단축된 시간, 즉 2 ∼ 6 시간, 바람직하게는 약 3 시간 이내에 수행될 수 있다는 것이 본 발명에 의해 밝혀졌다.
본 발명은 완전 탈회된 뼈를 분쇄하는 단계[즉, 단계(d)]를 포함한다. 상기 분쇄 공정은 통상의 분쇄기를 사용하여 수행될 수 있다. 바람직하게는, 상기 분쇄는 1000 내지 5000 ㎛의 범위, 더욱 바람직하게는 2,000 내지 4,000 ㎛의 범위의 길이를 갖는 섬유상 탈회골 기질이 얻어지도록 수행될 수 있으며, 이는 분쇄기의 종류에 따라 적절한 분쇄 조건을 설정함으로써 얻어질 수 있다.
이하, 본 발명을 실시예를 통하여 더욱 상세히 설명한다. 그러나, 하기 실시예는 본 발명을 예시하기 위한 것으로 본 발명이 이에 제한되는 것은 아니다.
실시예 1.
인체로부터 공여된 뼈(중량: 약 172 g)를 수술기구를 사용하여 뼈에 붙어있는 연조직을 제거하고, 계면활성제가 포함된 조직세정제를 사용하여 연조직, 지방, 및 골수 등의 불순물을 제거한 다음, 절반으로 절단하였다. 절단된 뼈들을 뼈 무게(g)당 0.6N HCl 수용액 20 ml에 3 시간 동안 담가 부분 탈회시켰다. 부분 탈회된 뼈를 분리하여, 뼈 무게(g)당 증류수 20 ml에 담가 뼈 중의 탈회 용액을 제거하였다. 얻어진 뼈를 본 슬라이서(bone slicer)(YOU IL MC/CO. KR)를 사용하여 약 0.5 mm 두께의 시트(sheet) 형태로 슬라이싱하였다. 얻어진 시트 형태의 뼈는 도 1의 A와 같다. 얻어진 시트 형태의 뼈를 뼈 무게(g)당 0.6N HCl 수용액 30 ml에 3시간 동안 담가 완전 탈회시켰다. 탈회골 기질을 침전시켜 분리한 후, 분쇄기(IKA, M20 Univeral mill, GR)에 넣어 약 10 분 동안 분쇄한 후, 인산완충용액(PBS)으로 중화시키고, 증류수로 세척한 후, 동결건조하여 약 31 g의 탈회골 기질을 제조하였다. 얻어진 탈회골 기질을 광학현미경으로 관찰한 사진은 도 2의 A와 같다. 도 2의 A의 결과로부터, 얻어진 탈회골 기질은 섬유상 탈회골 기질임을 알 수 있다.
비교예 1.
인체로부터 공여된 뼈(중량: 약 165 g)를 수술기구를 사용하여 뼈에 붙어있는 연조직을 제거하고, 계면활성제가 포함된 조직세정제를 사용하여 연조직, 지방, 및 골수 등의 불순물을 제거한 다음, 절반으로 절단하였다. 절단된 뼈들을 뼈 무게(g)당 0.6N HCl 수용액 20 ml에 3 시간 동안 담가 부분 탈회시켰다. 부분 탈회된 뼈를 회수하여, 뼈 무게(g)당 증류수 20 ml에 담가 뼈 중의 탈회 용액을 제거하였다. 얻어진 뼈를 뼈 무게(g)당 0.6N HCl 수용액 30 ml에 3시간 동안 담가 다시 탈회 공정을 수행하였으나, 탈회가 거의 진행되지 않아 탈회골 기질을 얻을 수 없었다.
비교예 2.
인체로부터 공여된 뼈(중량: 약 192 g)를 수술기구를 사용하여 뼈에 붙어있는 연조직을 제거하고, 계면활성제가 포함된 조직세정제를 사용하여 연조직, 지방, 및 골수 등의 불순물을 제거한 다음, 절반으로 절단하였다. 절단된 뼈들을 뼈 무게(g)당 0.6N HCl 수용액 20 ml에 3시간 동안 담가 부분 탈회시켰다. 부분 탈회된 뼈를 분리하여, 뼈 무게(g)당 증류수 20 ml에 담가 뼈 중의 탈회 용액을 제거하였다. 얻어진 뼈를 분쇄기(IKA, M20 Universal Mill, GR)에 넣어 약 60분 동안 분쇄하여 분쇄된 뼈를 얻으며, 그 형태는 도 1의 B와 같다. 상기 분쇄된 뼈를 뼈 무게(g)당 0.6N HCl 수용액 30 ml에 3시간 동안 담가 완전 탈회시켰다. 탈회골 기질을 침전시켜 분리한 후, 인산완충용액(PBS)으로 중화시키고, 증류수로 세척한 후, 동결건조하여 약 36.4g의 탈회골 기질을 제조하였다. 얻어진 탈회골 기질을 광학현미경으로 관찰한 사진은 도 2의 B와 같다. 도 2의 B의 결과로부터, 얻어진 탈회골 기질은 입자상 탈회골 기질임을 알 수 있다.

Claims (4)

  1. (a) 체외로 분리된 뼈를 산성 용액 중에서 1 ∼ 5 시간 동안 1차 탈회시키는 단계; (b) 단계(a)로부터 얻어진 뼈를 0.1 ∼ 3 mm의 두께로 슬라이싱(slicing)하여 시트 형태의 뼈를 형성시키는 단계; (c) 단계(b)에서 얻어진 시트 형태의 뼈를 산성 용액 중에서 2 ∼ 6 시간 동안 2차 탈회하는 단계; 및 (d) 단계(c)에서 얻어진 탈회된 뼈를 분쇄하는 단계를 포함하는, 섬유상 탈회골 기질의 제조방법.
  2. 제1항에 있어서, 단계(a) 및 단계(c)의 상기 산성 용액이 서로 독립적으로 0.1 ∼ 3 N HCl 수용액인 것을 특징으로 하는 섬유상 탈회골 기질의 제조방법.
  3. 제2항에 있어서, 단계(a) 및 단계(c)의 상기 산성 용액이 0.6 N HCl 수용액인 것을 특징으로 하는 섬유상 탈회골 기질의 제조방법.
  4. 제1항 내지 제3항 중 어느 한 항에 있어서, 상기 분쇄가 1000 내지 5000 ㎛의 범위의 길이를 갖는 섬유상 탈회골 기질이 얻어지도록 수행되는 것을 특징으로 하는 섬유상 탈회골 기질의 제조방법.
PCT/KR2011/007517 2011-09-29 2011-10-11 섬유상 탈회골 기질의 제조방법 WO2013047936A1 (ko)

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CN201180073750.6A CN103998070B (zh) 2011-09-29 2011-10-11 纤维状脱钙骨基质的制备方法
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US20140208980A1 (en) 2014-07-31
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