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KR100427557B1 - Bone collagen scaffold - Google Patents

Bone collagen scaffold Download PDF

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KR100427557B1
KR100427557B1 KR10-2001-0015701A KR20010015701A KR100427557B1 KR 100427557 B1 KR100427557 B1 KR 100427557B1 KR 20010015701 A KR20010015701 A KR 20010015701A KR 100427557 B1 KR100427557 B1 KR 100427557B1
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bone collagen
bone
collagen support
fibroblasts
cells
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KR20020075605A (en
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성상철
이명철
김희중
권선방
김혜진
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주식회사 웰스킨
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS 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
    • A61L27/00Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14Macromolecular materials
    • A61L27/22Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/24Collagen
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61FFILTERS 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
    • A61F2/00Filters 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
    • A61F2/02Prostheses implantable into the body
    • A61F2/28Bones
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    • A61L27/00Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36Materials 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
    • A61L27/38Materials 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
    • A61L27/3804Materials 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 characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
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    • A61L27/00Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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    • A61L27/00Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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    • A61L2300/00Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
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    • AHUMAN NECESSITIES
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    • A61L2300/00Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/412Tissue-regenerating or healing or proliferative agents
    • A61L2300/414Growth factors

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Abstract

본 발명은 뼈의 콜라겐 지지체에 관한 것으로, 뼈를 탈석회(decal cification)하여 제조된 5 내지 500 ㎛ 크기의 다공체를 포함하는 뼈 콜라겐 지지체를 제공한다. 본 발명의 뼈 콜라겐 지지체는 성형, 인공피부배양, 또는 진피결손 충진에 있어서, 섬유아세포가 증식할 수 있는 진피대체물로 활용될 수 있을 뿐만 아니라 생리활성인자 전달체로 활용될 수 있다.The present invention relates to a collagen support for bone, and provides a bone collagen support comprising a porous body of 5 to 500 ㎛ size prepared by decal cification of bone. The bone collagen support of the present invention can be used as a bioactive factor transporter as well as a dermal substitute for fibroblasts to proliferate in molding, artificial skin culture, or dermal defect filling.

Description

뼈의 콜라겐 지지체{BONE COLLAGEN SCAFFOLD}Bone COLLAGEN SCAFFOLD}

본 발명은 뼈의 콜라겐 지지체에 관한 것으로서, 더욱 상세하게는 뼈의 콜라겐 지지체를 생체조직공학용 재료 및 인공피부 제조시 진피물질로 사용하는 방법에 관한 것이다.The present invention relates to a collagen support of bone, and more particularly, to a method for using the bone collagen support as a material for biotissue engineering and dermal material in the preparation of artificial skin.

생체조직공학이란 과학의 발달과 함께 등장한 새로운 분야의 하나이며, 생명과학과 공학의 기본 개념과 기술을 통합 응용하여 생체 조직의 구조와 기능 사이의 상관관계를 이해하고 나아가서 생체조직의 대용품을 만들어 이식함으로써 우리 몸의 기능을 유지, 향상 또는 복원하는 것을 목적으로 하는 응용학문이다. 생체조직공학의 이론적 배경은 선택적 세포이식과 인공기질 두 가지로 요약할 수 있다. 선택적 세포이식은 장기 중에서 가장 필요한 조직 또는 조직만을 선택적으로 채취하여 이식하는 것을 의미하며, 인공기질은 세포가 분비하는 세포 외 기질이 조직의 형성에 중요한 역할을 한다는 것에 착안하여 인공으로 합성한 기질을 사용하여 이식하는 것을 의미한다. 이러한 이론적 배경을 바탕으로 여러 종류의 천연 또는 인공 기질에 필요한 세포만을 부착하여 이식함으로써 조직 또는 장기를 형성하려는 실험이 시도되었다.Biotissue engineering is a new field that has emerged with the development of science. By integrating and applying the basic concepts and technologies of bioscience and engineering, it is possible to understand the correlation between the structure and function of biological tissues, and to create and transplant surrogate tissues. This is an applied research aimed at maintaining, improving or restoring the function of our body. The theoretical background of biotissue engineering can be summarized into two categories: selective cell transplantation and artificial substrates. Selective cell transplantation refers to the selective harvesting and transplantation of only the tissues or tissues that are most needed among organs. The artificial substrate is an artificially synthesized substrate based on the fact that the extracellular matrix secreted by cells plays an important role in tissue formation. Means to transplant using. Based on this theoretical background, experiments have been made to form tissues or organs by attaching and transplanting only cells necessary for various kinds of natural or artificial substrates.

생체조직공학 기법에서 가장 중요한 것은 3차원적 세포배양을 위한 지지체 (scaffold)인데, 이는 세포가 3차원적으로 성장 할 수 있는 기반을 제공하는 것이다. 그러나 만일 이러한 3차원적인 지지체가 없이 세포를 배양 할 경우 세포는 오직 배양판의 바닥에 붙어서 자라거나 배양액 내에서 부유 배양하게 된다. 두 가지 모든 경우에 세포는 3차원적인 형상을 잃어버리게 되어 조직으로 발전할 수 없게 된다.The most important in biotissue engineering techniques is the scaffold for three-dimensional cell culture, which provides a foundation for cells to grow three-dimensionally. However, if cells are cultured without these three-dimensional supports, the cells will only grow on the bottom of the plate and grow or float in culture. In both cases, the cell loses its three-dimensional shape and cannot develop into tissue.

하지만 재료과학의 발달로 생분해성 폴리머를 생체조직공학에 접목시키게 되었고 생분해성 폴리머는 인체내에서 일정시간이 흐르면 스스로 분해되어 없어지는 특성을 가지고 있어 이식된 세포가 조직을 형성한 후에 분해되어 없어지므로 생체조직공학의 목적에 적합한 생체재료로 현재까지 많이 사용되고 있다.However, with the development of materials science, biodegradable polymers have been incorporated into biotissue engineering, and biodegradable polymers have the property of self-decomposing and disappearing after a certain time in the human body. It is widely used as a biomaterial suitable for the purpose of biotissue engineering.

초창기의 생분해성 폴리머를 이용한 세포배양은 폴리머판에 세포를 심어 배양하는 방법으로 시도되었으나 만족할 만한 결과를 얻지 못하였다. 그 뒤 폴리머를 가는 실과 같은 형태로 제작한 뒤 그것을 서로 엮어 망사와 같은 모양으로 만든 후 그 위에 세포를 심어 배양하였다. 상기한 방법은 세포사이에 충분한 공간이 확보되어 체액의 확산에 의해 산소나 영양분의 공급도 쉬워지고 신생혈관의 형성도 원활히 이루어져서 성공적으로 세포가 성장, 분화하고 조직을 형성하는 결과를 얻게 되었다. 또한 사용되는 폴리머는 인공적으로 합성하는 것이기 때문에 만들기에 따라 구성성분과 형태 등을 마음대로 조절할 수 있다. 재료의 장력이나 분해속도, 세포부착 능력 등을 미리 계산하여 제조할 수도 있고, 폴리머에 세포의 성장에 관여하는 인자를 함유하여 폴리머가 분해되면서 그런 인자들이 서서히 흘러나오게도 할 수 있는 등의 기법도 가능하게 되었다.Initially, cell cultures using biodegradable polymers were attempted by culturing cells on polymer plates, but they did not yield satisfactory results. After that, the polymer was made into a thin thread-like form and then woven together to form a mesh-like shape, and the cells were planted and cultured thereon. In the above method, sufficient space is secured between cells to facilitate the supply of oxygen and nutrients due to the diffusion of body fluids, and the formation of neovascularization is facilitated, resulting in successful cell growth, differentiation, and tissue formation. In addition, since the polymer used is artificially synthesized, the composition and shape of the polymer can be adjusted at will according to the preparation. It can be prepared by calculating the tension, decomposition rate, and cell adhesion ability of the material in advance. Also, a technique that contains the factors involved in cell growth in the polymer can cause such factors to flow out gradually as the polymer is degraded. It became possible.

기본적인 생체조직의 기법을 요약해 보면 먼저 환자의 몸에서 필요한 조직을 조금 채취하고 그 조직 편으로부터 세포를 분리한 다음 분리된 세포를 배양을 통하여 필요한 양만큼 증식시키고 생분해성 폴리머에 심어 일정기간 체외 배양한 뒤 이 세포-폴리머 구조물을 다시 인체 내로 이식한다. 이식후의 세포는 인체 내에서 혈관이 자라 들어와 혈관신생과 함께 혈액의 공급이 이루어지며 세포들은 증식 분화하여 새로운 조직 및 장기를 형성하고 폴리머는 분해되어 없어지게 되는 것이다.To summarize the basic biotissue technique, we first collect a small amount of tissue from the patient's body, separate the cells from the tissues, and then proliferate the separated cells in the required amount through the culture, and incubate them in a biodegradable polymer for a period of time. This cell-polymer construct is then implanted back into the body. After transplantation, blood vessels grow in the human body and blood is supplied with angiogenesis. Cells proliferate and differentiate to form new tissues and organs, and polymers are broken down.

한편 콜라겐은 세포 적합성이 우수하여 생분해 폴리머와 인공피부재생에 보조제로 많이 사용되고 있다. 콜라겐은 동물의 몸속에 가장 많이 들어있는 섬유상의 단백질로, 피부의 진피층과 결합조직의 주성분이며, 또한 뼈 중량의 20 %가 콜라겐이다.On the other hand, collagen has been widely used as an adjuvant for biodegradable polymer and artificial skin regeneration due to its excellent cell compatibility. Collagen is the most fibrous protein contained in the animal's body. It is the main component of the dermis and connective tissue of the skin, and 20% of the bone weight is collagen.

일반적으로 콜라겐은 의료용이나 화장품용으로 사용되고 있으며, 콜라겐은 생체 내에서 세포 접착기능, 몸과 장기의 구조유지, 세포기능의 활성화, 세포 증식작용, 지혈작용, 면역강화 등에 작용하는 것으로 알려져 있다.In general, collagen is used for medical or cosmetics, collagen is known to act on the cell adhesion function, maintaining the structure of the body and organs, activation of cell function, cell proliferation, hemostatic action, immune strengthening in vivo.

또한 대한민국 특허공고 제 90-14547호의 콜라겐을 함유하는 생조직 등가물, 대한민국 특허공고 제 96-6788호의 인공피부용 키틴계 상호침투구조 필름의 제조방법, 대한민국 특허공개 제 91-8999호에서 생체 적합성 천공막 및 이의 제조방법, 대한민국 특허공고 제 97-60646호에서 삼차원적으로 재생된 인공 피부 조직 및 그 제조방법 등의 인공피부 제조방법에 대하여 콜라겐을 이용하거나 생분해성 폴리머를 이용하여 제시하고 있으나 단지 인공피부의 3차원적인 배양에만 국한된 채 인체피부의 성상을 재현 할 수 있는 진피 대체물질을 제공하지 못하고 있다.Also, a biotissue equivalent containing collagen of Korean Patent Publication No. 90-14547, a method for producing a chitin-based interpenetrating structure film for artificial skin of Korean Patent Publication No. 96-6788, and a biocompatible perforated membrane in Korean Patent Publication No. 91-8999 And a method of manufacturing the same, artificial skin tissue such as three-dimensional regenerated artificial skin tissue and a method of manufacturing the same in Korean Patent Publication No. 97-60646, but using collagen or a biodegradable polymer, but only artificial skin It has not been able to provide dermal substitutes that can reproduce the appearance of human skin, confined to the three-dimensional culture.

따라서, 체외에서 배양한 인공피부가 인체 피부의 성상을 유지하여 학문적인 연구나, 의학적인 치료용으로 적합하기 위한 진피 대체물을 개발하는 것이 요구되어진다.Therefore, it is required to develop dermal substitutes that are suitable for academic research or medical treatment by maintaining artificial skin cultured in vitro.

따라서, 본 발명은 진피 충전물질로 사용할 수 있는 뼈 콜라겐 지지체를 제공하는 것을 목적으로 한다.Accordingly, an object of the present invention is to provide a bone collagen support that can be used as a dermal filler material.

도 1은 뼈 콜라겐에 섬유아세포를 뿌린 후 3일 후에 270배에서 주사전자현미경으로 관찰한 사진이고,1 is a photograph taken by scanning electron microscopy at 270-fold 3 days after sprinkling fibroblasts to bone collagen,

도 2는 뼈 콜라겐에 섬유아세포를 뿌린 후 10일 후에 250배에서 주사전자현미경으로 관찰한 사진이고,Figure 2 is a photograph observed with a scanning electron microscope at 250 times 10 days after sprinkling fibroblasts to bone collagen,

도 3은 뼈 콜라겐에 섬유아세포를 뿌린 후 3일 후에 1000배의 배율에서 주사전자현미경으로 관찰한 사진이고,3 is a photograph observed with a scanning electron microscope at a magnification of 1000 times 3 days after sprinkling fibroblasts to bone collagen,

도 4는 뼈 콜라겐에 섬유아세포를 뿌린 후 10일 후에 700배의 배율에서 주사전자현미경으로 관찰한 사진이고,4 is a photograph taken with a scanning electron microscope at a magnification of 700 times after 10 days after sprinkling fibroblasts to bone collagen,

도 5는 항생제를 포함하는 뼈 콜라겐 지지체가 항생제를 용출하는 양을 시간에 따라 측정한 것이다.FIG. 5 shows the amount of bone collagen scaffold comprising antibiotics eluting antibiotics over time.

상기의 목적을 달성하기 위하여 본 발명은 진피 충전물질용 뼈 콜라겐 지지체를 제공한다.In order to achieve the above object, the present invention provides a bone collagen support for dermal filler material.

또한 본 발명은 뼈를 콜라겐 지지체를 포함하는 생리활성인자 전달체로서 제공한다.In another aspect, the present invention provides a bone as a physiologically active factor carrier comprising a collagen support.

이하 본 발명을 상세하게 설명한다.Hereinafter, the present invention will be described in detail.

본 발명의 뼈 콜라겐 지지체는 동물의 뼈를 탈석회화하여(decalcification) 수득되는 것을 특징으로 한다. 상기 탈석회화 방법은 뼈를 HCl내에서 실시하는 것이 바람직하고, 더욱 바람직하게는 0.5 N HCl내에 뼈를 침지한 후 12시간 내지 24시간 반응시키고, 상기 반응물을 PBS(Phosphate buffered saline) 완충액으로 세척하여 pH 7.4로 적정하는 것이다.The bone collagen support of the present invention is characterized in that it is obtained by decalcification of bone of an animal. The decalcification method is preferably carried out in HCl bone, more preferably 12 hours to 24 hours after immersing the bone in 0.5 N HCl, the reaction is washed with PBS (Phosphate buffered saline) buffer Titration to pH 7.4.

본 발명의 뼈 콜라겐 지지체는 다공체를 포함하는 것을 특징으로 한다. 지지체 내에 형성된 다공체는 세포사이에 충분한 공간을 제공하여 체액의 확산에 의해 산소나 영양분의 공급을 원활히 유지시키고 신생혈관의 형성을 도와, 성공적으로 세포가 성장하여 분화하고 조직을 형성하는 결과를 얻게 한다. 따라서 다공도 역시 지지체의 생체적합성 만큼 중요하다. 본 발명의 뼈 콜라겐 스폰지의 다공체는 5 내지 500 ㎛가 바람직하고, 더욱 바람직하게는 10 내지 100 ㎛ 이다. 다공체가 5 ㎛미만인 경우는 세포사이의 충분한 공간 확보가 이루어지지 않아 세포 성장을 억제시킬 수 있으며, 500 ㎛초과인 경우 세포사이의 간격이 너무 넓어 정상 피부조직으로의 발전이 어렵고 진피를 구성하는 지지체로도 인장강도 등에 문제점이 있다. 본 발명의 지지체내의 다공체는 바람직하게 10 내지 100 ㎛로서 생체적합성이 매우 뛰어난 지지체이다.The bone collagen support of the present invention is characterized by including a porous body. The porous body formed in the support provides sufficient space between the cells to facilitate the supply of oxygen and nutrients by the diffusion of body fluids and help to form new blood vessels, resulting in the successful growth and differentiation of cells and the formation of tissues. . Therefore porosity is also as important as the biocompatibility of the support. As for the porous body of the bone collagen sponge of this invention, 5-500 micrometers is preferable, More preferably, it is 10-100 micrometers. When the porous body is less than 5 ㎛, sufficient space between cells is not secured, and cell growth can be suppressed.In the case of more than 500 ㎛, the spacing between cells is too wide, which makes it difficult to develop normal skin tissue and supports the dermis. There is also a problem with tensile strength. The porous body in the support of the present invention is preferably a support having an excellent biocompatibility as 10 to 100 m.

본 발명의 뼈 콜라겐 지지체는 세포가 증식하기에 적합하고, 장기간 보관이 용이하여 상품화 할 수 있다. 또한 뼈 콜라겐 지지체는 중심까지 잘 발달된 다양한 형태와 크기를 가진 유공형의 스펀지 형태로서 지지체에 세포를 접종시 내부까지 세포가 잘 유입 될 수 있는 형태를 지녔으며, 또한 생물체에서 직접 얻을 물질이기 때문에 생체적합성도 매우 뛰어나다.The bone collagen support of the present invention is suitable for proliferation of cells, can be stored for a long time, and can be commercialized. In addition, the bone collagen scaffold is a pore-type sponge having various shapes and sizes well developed to the center, and has a form in which cells can be well introduced into the cell when inoculating the scaffold, and it is a substance directly obtained from an organism. The biocompatibility is also very good.

또한 본 발명은 뼈 콜라겐 지지체를 진피 충전물질로 제공한다. 상기 진피 충전물질은 피부반흔의 치료 또는 진피의 결손이 있는 부위에 적용하여 피부를 재생시킬 수 있으며, 음경 확대술과 같은 신체의 기관을 성형할 경우 사용될 수 있다.The present invention also provides a bone collagen support as a dermal filler. The dermal filler may be applied to the treatment of skin scars or to areas where there is a defect in the dermis to regenerate the skin, and may be used when shaping organs of the body such as penis enlargement.

또한 본 발명의 뼈 콜라겐 지지체는 상기 뼈 콜라겐 지지체에 섬유아세포를 배양하여 제조된 인공피부를 제공할 수 있다.In addition, the bone collagen support of the present invention may provide an artificial skin prepared by culturing fibroblasts on the bone collagen support.

상기 뼈 콜라겐 지지체에 섬유아세포를 배양할 경우, 섬유아세포를 100 내지 1000 개/mg으로 접종하여 5일 내지 7일 배양하는 것이 바람직하며, 배양후 10일 정도 되면 섬유아세포가 층을 이루며 증식된다.When culturing fibroblasts on the bone collagen support, fibroblasts are inoculated at 100 to 1000 / mg and preferably cultured for 5 days to 7 days, and after about 10 days, the fibroblasts form a layer and proliferate.

또한 본 발명은 뼈 콜라겐 지지체를 생리활성인자 전달체로 활용할 수 있다. 더욱 바람직하게는 본 발명의 뼈 콜라겐 지지체는 생체 또는 시험관내에서 성장인자 봉입, 성장인자 방출용, 혈관성장인자 봉입용, 혈관성장인자 방출용, 항생제 봉입용, 항생제 방출용, 약물전달체 등으로 활용될 수 있다. 즉 뼈 콜라겐 지지체는 상기 인자(성장인자, 약물 등)들을 함유하여 봉입방법에 따라 일정기간에 걸쳐 약물을 방출하는 약물전달체로서의 역할을 수행할 수 있다.In addition, the present invention can utilize the bone collagen support as a physiologically active factor carrier. More preferably, the bone collagen support of the present invention may be used as a growth factor encapsulation, growth factor release, vascular growth factor encapsulation, vascular growth factor release, antibiotic encapsulation, antibiotic release, drug delivery agent, etc. in vivo or in vitro. Can be. In other words, the bone collagen support may contain the above factors (growth factor, drug, etc.) to serve as a drug carrier to release the drug over a period of time according to the encapsulation method.

이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐 본 발명이 하기의 실시예에 한정되는 것은 아니다.Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are provided only to more easily understand the present invention, and the present invention is not limited to the following examples.

[실시예 1]Example 1

섬유아세포의 배양Fibroblast Culture

포피에서 수득한 피부의 피하지방층을 완전히 제거시키고, 콜나게나제를 처리하여 진피를 분리한 다음 트립신(trypsin)-EDTA를 처리하여 진피로부터 섬유아세포를 분리시켰다. 분리된 섬유아세포는 10 % FBS(fetal bovine serum)와 항생제를 넣은 DMEM(Dulbeccos modified eagles medium) 배지를 사용하여 5 % CO2, 37 ℃ 배양기에서 배양하였다.The subcutaneous fat layer of the skin obtained from the foreskin was completely removed, the dermis was separated by treatment with colnase, and then fibroblasts were separated from the dermis by treatment with trypsin-EDTA. The isolated fibroblasts were cultured in 5% CO 2 , 37 ℃ incubator using Dulbeccos modified eagles medium (DMEM) medium containing 10% FBS (fetal bovine serum) and antibiotics.

뼈 콜라겐의 제조Preparation of Bone Collagen

동물에서 수득한 뼈 100 g을 0.5 N HCl에 넣고 탈석회(decalcification)하였다. 탈석회 후 PBS(phosphate buffered saline)로 세척하여 pH 7.4까지 적정하였다. 탈석회된 콜라겐 스펀지는 일반적인 알코올 농도 구배에 의해 탈수 한 후 공기 중에서 건조하여 밀폐용기에 넣어 보관하며 필요시 꺼내어 사용하였다. 건조된 콜라겐 지지체는 중심부까지 잘 발달된 다양한 형태와 크기를 가진 유공형의 스펀지 형태로서 지지체에 세포를 접종시 내부까지 세포가 잘 유입 될 수 있는 형태를 지녔으며, 또한 생물체에서 직접 얻을 물질이기 때문에 생체적합성도 매우 뛰어났다.100 g of bone obtained from the animal was placed in 0.5 N HCl and decalcified. After deliming it was washed with PBS (phosphate buffered saline) and titrated to pH 7.4. The demineralized collagen sponge was dehydrated by a general alcohol concentration gradient, dried in air, stored in an airtight container, and taken out if necessary. The dried collagen scaffold is a porous sponge with various shapes and sizes that are well developed to the center, and when the cells are inoculated into the scaffold, the cells can be well introduced into the scaffold. The biocompatibility was also very good.

[실시예 2]Example 2

상기 실시예 1에서 제조된 뼈 콜라겐 지지체를 저온 에틸렌 옥사이드 가스로 소독한 다음 PBS로 수회 세척하고 배양용기에 넣어 같은 수의 섬유아세포(100~1000개/mg)를 뿌려주었다. 10 % FBS와 항생제를 포함한 DMEM 배지에서 3일 또는 10일 동안 배양하였다.The bone collagen support prepared in Example 1 was sterilized with low temperature ethylene oxide gas, washed several times with PBS, and sprinkled with the same number of fibroblasts (100-1000 / mg). Incubated for 3 or 10 days in DMEM medium containing 10% FBS and antibiotics.

[실험예 1]Experimental Example 1

상기 실시예 2에서 배양한 섬유아세포는 주사전자현미경(Scanning Electron Microscope)으로 관찰하였다.The fibroblasts cultured in Example 2 were observed with a scanning electron microscope.

먼저, 섬유아세포를 뿌리고 3일 또는 10일 동안 배양한 뼈 콜라겐을 4 ℃의 파라-글루타알데하이드(para-glutaraldehyde) 용액에 2시간 동안 고정한 다음 증류수로 여러 번 세척하였다. 시료내의 수분을 제거하기 위해 농도별 아세톤으로 탈수시킨 후 양면 테이프가 붙은 시료대위에 올려 건조시켰다. 건조된 시료는 이온 코팅기(EiKO, 1B-3)로 금 코팅하여 주사전자현미경(AKAShi, ISI-SS40)으로 관찰하였다. First, fibroblasts were seeded and cultured for 3 or 10 days, bone collagen was fixed in para-glutaraldehyde solution at 4 ° C. for 2 hours, and then washed several times with distilled water. In order to remove moisture in the sample, dehydrated with acetone for each concentration, and dried on a sample bed with double-sided tape. The dried samples were gold-coated with an ion coater (EiKO, 1B-3) and observed with a scanning electron microscope (AKAShi, ISI-SS40).

도 1은 뼈 콜라겐에 섬유아세포를 뿌린 후 3일 후에 270배에서 주사전자현미경으로 관찰한 사진이고, 도 2는 10일 후에 250배에서 관찰한 그림이다. 또한 도 3은 뼈 콜라겐에 섬유아세포를 뿌린 후 3일 후에 1000배의 배율에서 주사전자현미경으로 관찰한 사진이고, 도 4는 10일 후에 700배의 배율로 관찰한 사진이다. 도 1 및 도 3에 나타난 바와 같이, 배양 3일째에는 섬유아세포가 뼈 콜라겐에 착상하여 콜라겐 스펀지에 형성된 구멍(pore)을 막아가고 있는 양상을 나타내었고, 도 2 및 도 4에 나타난 바와 같이, 배양 10일째에는 섬유아세포가 더욱 증식하여 고배율에서 관찰한 결과 세포가 층을 이루고 있음을 확인할 수 있었다.1 is a photograph observed by scanning electron microscopy at 270 times 3 days after sprinkling fibroblasts to bone collagen, Figure 2 is a picture observed at 250 times after 10 days. In addition, Figure 3 is a photograph observed with a scanning electron microscope at a magnification of 1000 times 3 days after sprinkling fibroblasts to bone collagen, Figure 4 is a photograph observed at a magnification of 700 times 10 days. As shown in Figures 1 and 3, on the third day of culture, fibroblasts were implanted in bone collagen and blocked pores formed in the collagen sponge, and as shown in FIGS. 2 and 4, the culture On day 10, fibroblasts were further proliferated and observed at high magnification, indicating that the cells were layered.

[실시예 3]Example 3

키토산 스폰지와 상기 실시예 1의 뼈 콜라겐 지지체를 이용하여 항생제의 방출실험을 실시하였다. 항생제의 용출은 항생제를 포함하는 스폰지 또는 뼈콜라겐 지지체를 수용액에 담근 후 일정시간대별로 시료를 채취하여 용액내의 농도를 결정한 후 전체 용액의 양으로 환산 결정하였다. 그 결과는 하기 표 1 및 도 5에 나타내었고, 뼈 콜라겐 지지체는 키토산 스폰지에 의한 항생제 용출 속도에 비해 낮은 용출속도를 나타내어, 장기간 항생제에 의한 치료효과를 지속시킬 수 있을 것으로 여겨졌다.The release experiment of antibiotics was performed using the chitosan sponge and the bone collagen support of Example 1. The elution of the antibiotic was determined by dipping the sponge or bone collagen scaffold containing the antibiotic in an aqueous solution and taking a sample at a predetermined time to determine the concentration in the solution, and then converted to the total amount of the solution. The results are shown in Table 1 and FIG. 5, and the bone collagen support showed a lower dissolution rate compared to the antibiotic dissolution rate by the chitosan sponge, and it was thought that the therapeutic effect by the antibiotic for a long time could be maintained.

방출된 항생제의 양(%)% Of antibiotic released 0분0 min 1분1 minute 5분5 minutes 10분10 minutes 30분30 minutes 키토산스폰지Chitosan Sponge 10.8 %10.8% 13.1 %13.1% 35.3 %35.3% 55.2 %55.2% 102.1 %102.1% 실시예 1Example 1 4.8 %4.8% 7.5 %7.5% 28.4 %28.4% 44.8 %44.8% 70.9 %70.9%

상기에 언급한 바와 같이, 본 발명의 뼈 콜라겐 지지체는 쉽게 제조할 수 있을 뿐만 아니라 다공성을 포함하여 세포 적합성이 우수하여 세포배양을 쉽고 간편하게 할 수 있다. 또한 피부결손, 성형, 생리활성인자 전달체로 활용될 수 있다.As mentioned above, the bone collagen support of the present invention can be easily prepared, as well as excellent cell suitability, including porosity, to facilitate cell culture. It can also be used as a skin defect, cosmetic, bioactive factor transporter.

Claims (6)

5 내지 500 ㎛ 크기의 다공체가 형성되도록 뼈를 탈석회하여 제조되는 진피 충전물질용 뼈 콜라겐 지지체.Bone collagen support for dermal filler material prepared by deliming bone to form a porous body of 5 to 500 ㎛ size. 삭제delete 삭제delete 제 1항에 있어서, 상기 뼈 콜라겐 지지체에 섬유아세포를 증식하여 성형, 인공피부배양, 또는 진피결손 충전시 진피대체물로 사용하는 것을 특징으로 하는 뼈 콜라겐 지지체.The bone collagen support according to claim 1, wherein the bone collagen support is used as a dermal substitute during proliferation, molding, artificial skin culture, or filling of dermal defects by proliferating fibroblasts. 제 1항에 있어서, 상기 뼈 콜라겐 지지체는 생리활성인자를 더욱 담지하여 생리활성인자 전달체로 사용하는 것을 특징으로 하는 뼈 콜라겐 지지체.The bone collagen support according to claim 1, wherein the bone collagen support further carries a bioactive factor and is used as a bioactive factor carrier. 제 5항에 있어서, 상기 생리활성인자는 성장인자, 항생제 또는 약제인 것을 특징으로 하는 뼈 콜라겐 지지체.6. The bone collagen support according to claim 5, wherein the bioactive factor is a growth factor, an antibiotic or a medicament.
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