KR102180865B1 - Photocurable bioink with electroconductivity and a preparation thereof - Google Patents
Photocurable bioink with electroconductivity and a preparation thereof Download PDFInfo
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- KR102180865B1 KR102180865B1 KR1020190120465A KR20190120465A KR102180865B1 KR 102180865 B1 KR102180865 B1 KR 102180865B1 KR 1020190120465 A KR1020190120465 A KR 1020190120465A KR 20190120465 A KR20190120465 A KR 20190120465A KR 102180865 B1 KR102180865 B1 KR 102180865B1
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- bio
- polymer
- ink composition
- silk fibroin
- graphene oxide
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- C09D11/00—Inks
- C09D11/52—Electrically conductive inks
Abstract
Description
본 발명은 전기전도성을 갖는 바이오 잉크 조성물 및 이의 제조방법, 그리고 이를 사용하여 구조체를 제조하는 방법에 관한 것이다. The present invention relates to a bio-ink composition having electrical conductivity, a method for manufacturing the same, and a method for manufacturing a structure using the same.
그래핀은 2004년 Dr. Geim과 Dr. Novoselov가 스카치 테이프를 이용하여 흑연으로부터 얻어내어 우수한 전기적 성질이 최초로 보고된 이후 트랜지스터, 고분자 복합체 등 여러 분야에서 그래핀 기반 소재 응용 연구가 진행되고 있다. Graphene was introduced by Dr. Geim and Dr. Since Novoselov's excellent electrical properties obtained from graphite using scotch tape were first reported, research on application of graphene-based materials in various fields such as transistors and polymer composites has been conducted.
그래핀을 대량 생산할 수 있는 방법으로 흑연으로부터 화학적 산화를 통한 그래핀(Graphene Oxide: GO) 제조 방법을 들 수 있는데, 공정비용이 낮고 다양한 환원 방법으로 표면 성질을 제어할 수 있는 장점이 있다. As a method for mass-producing graphene, there is a method of manufacturing graphene (Graphene Oxide: GO) through chemical oxidation from graphite, which has the advantage of low process cost and control of surface properties through various reduction methods.
이때 얻어진 그래핀 표면에 층간 삽입되었던 산화물의 영향으로 표면에 수산기 (-OH)와 에폭시기 (>O)가 결합되어 가장자리에 카르복실기 (-COOH)가 결합된 형태로 존재하게 되며, 이로 인해 친수성이 높아지지만 그래핀의 높은 전기전도 특성을 상실하게 된다. 하지만. 높은 열이나 환원제 처리를 통해 GO가 환원(reduction Graphene Oxide: rGO)될 경우에는, oxygen-containing groups들이 제거가 되어 그래핀 고유의 뛰어난 물리적 특성을 복원시킬 수 있다. At this time, the hydroxyl group (-OH) and the epoxy group (>O) are bonded to the surface due to the influence of the oxide intercalated on the obtained graphene surface, and the carboxyl group (-COOH) is bonded to the edge, thereby increasing hydrophilicity. However, graphene loses its high electrical conductivity. But. When GO is reduced (reduction graphene oxide: rGO) through high heat or a reducing agent treatment, oxygen-containing groups are removed and the excellent physical properties inherent in graphene can be restored.
이러한 rGO는 중간엽줄기세포의 분화에 적절한 것으로 보고되었다. 그러나 생명공학의 응용에 있어서 물에 대한 용해도는 매우 중요한데, rGO는 특성상 소수성을 띄므로 생체재료로의 제작 및 적용에 있어서 문제가 될 수 있으며, 분화 이외에 다양한 화합물, 재료, 단백질, 성장인자등을 결합하기 위한 사이트가 부족한 단점도 있다.These rGOs have been reported to be suitable for differentiation of mesenchymal stem cells. However, in the application of biotechnology, solubility in water is very important.Since rGO is hydrophobic in nature, it can be a problem in the production and application of biomaterials.In addition to differentiation, various compounds, materials, proteins, growth factors, etc. There is also the disadvantage of lack of a site to combine.
3차원 프린팅 (3D printing) 기술은, 컴퓨터 이용 설계 (CAD)로 제작된 도면을 바탕으로 3차원 형상을 구현하는 기술로서 프린팅에 사용되는 재료 및 프린터 구동원리에 따라 광중합 방식, 재료 압출 방식, 접착제 분사방식, 재료분사방식, 고에너지 직접조사방식, 분말 적층 용융방식, 시트 라미네이션 방식 등으로 분류될 수 있다(ASTM, F2792-12a). 3D printing technology is a technology that realizes a three-dimensional shape based on a drawing produced by computer-aided design (CAD), depending on the material used for printing and the printer driving principle, light polymerization method, material extrusion method, adhesive. It can be classified into a spraying method, a material spraying method, a high-energy direct irradiation method, a powder lamination method, and a sheet lamination method (ASTM, F2792-12a).
3차원 프린팅 기술이 점차 발전함에 따라 좀 더 정밀하고 세밀한 3차원 형상을 제조할 수 있게 되면서, 이를 의료·바이오 분야에 접목시켜 의료기기 부품이나 실제 인간의 조직을 그대로 모방하여 신체 장기 재생을 위해 활용되고 있다. 즉, 3차원 프린팅 기술은 조직공학용 지지체는 물론 수술 시뮬레이션 및 수술 임플란트 제작, 개인별 맞춤형 보형물 제작, 인공 혈관, 인공 장기 등으로 활용될 수 있도록 연구개발이 되고 있다. As 3D printing technology gradually develops, it is possible to manufacture more precise and detailed 3D shapes.By incorporating this into the medical and bio fields, it is used for regeneration of body organs by imitating medical device parts or actual human tissues. Has become. In other words, 3D printing technology is being researched and developed so that it can be used as a tissue engineering scaffold, as well as surgical simulation and surgical implant production, personalized implant production, artificial blood vessels, and artificial organs.
이 중에서도 세포를 포함하여 함께 프린팅 할 수 있는 기법은 ‘바이오프린팅’이라 불리우며, 잉크젯 프린팅, 압출방식, 레이저 방식 혹은 광중합 방식으로 출력 가능하다. 광중합 방식은 다시 SLA(stereolithography apparatus)와 DLP(digital light processing) 방식으로 구분되는데, SLA는 광경화성 수지가 들어있는 수조에 저전력, 고밀도의 UV 레이저를 주사하여 수지를 경화시켜 모델을 조형하는 방식이고, DLP는 DMD(Digital micromirror device) 칩을 이용해 이미지의 고정밀 표시를 구현하는 기술로 광원으로 레이저 대신 digital light projector를 이용한다. 따라서 DLP의 경우에는 한층을 한꺼번에 경화할 수 있어 출력시간을 단축시킬 수 있다. Among them, the technique that can print together, including cells, is called “bioprinting,” and it can be printed by inkjet printing, extrusion, laser or light polymerization. The photopolymerization method is further divided into SLA (stereolithography apparatus) and DLP (digital light processing) methods.SLA is a method of modeling a model by curing the resin by scanning a low-power, high-density UV laser into a tank containing a photocurable resin. , DLP is a technology that implements high-precision display of images using a digital micromirror device (DMD) chip, and uses a digital light projector instead of a laser as a light source. Therefore, in the case of DLP, the printing time can be shortened because one layer can be cured at once.
통상적으로 세포를 포함하여 3D 프린팅을 가능하게 하는 재료를 바이오잉크라고 칭하며, 바이오잉크로는 하이드로젤, 마이크로 캐리어, 세포가 제거된 세포외 기질, 또는 세포집합체 등을 재료로 사용할 수 있다. 이중 하이드로젤은 생체적합성이 우수하고 인체의 조직과 유사한 구조를 가지며, 세포 및 생리활성물질의 캡슐화가 용이하다. In general, a material that enables 3D printing including cells is referred to as bioink, and as the bioink, a hydrogel, a microcarrier, an extracellular matrix from which cells have been removed, or a cell aggregate may be used as a material. Among them, the hydrogel is excellent in biocompatibility, has a structure similar to that of the human body, and is easy to encapsulate cells and bioactive substances.
세포를 포함하는 3차원 인쇄된 구조체가 구조적 측면과 생물학적 측면에서 기능을 발휘하기 위하여 바이오잉크는, 인쇄적성, 세포적합성, 생분해성, 젤화 특성/기계적 물성, 그리고 세포의 성장과 분화를 조절할 수 있는 특성을 가져야 한다. 즉, 출력된 구조물은 재생기간 동안 원하는 모양을 유지해야 하고, 생체 내에서 적절한 속도로 분해되어야 한다. In order for the three-dimensional printed structure including cells to function in the structural and biological aspects, bioink is capable of controlling printability, cell compatibility, biodegradability, gelation/mechanical properties, and cell growth and differentiation. Must have characteristics. In other words, the printed structure must maintain the desired shape during the regeneration period and must be decomposed at an appropriate speed in the living body.
DLP 프린팅에 적용하기 위한 바이오잉크로 천연 유래(gelatin methacryloyl;GelMA, collagen methacryloyl;collagen MA) 또는 합성 하이드로젤(poly(ethylene glycol) diacrylate;PEGDA, polyvinyl alchohol methacrylate;PVAMA 등)의 하이드로젤이 개발되어 사용되고 있으나, 생체적합성, 프린팅 적합성, 기하학적 정밀성 등 물리적 및 생물학적 측면에서 한계가 있다. Hydrogels of natural origin (gelatin methacryloyl; GelMA, collagen methacryloyl; collagen MA) or synthetic hydrogels (poly(ethylene glycol) diacrylate; PEGDA, polyvinyl alchohol methacrylate; PVAMA, etc.) have been developed as bio-inks for DLP printing. Although used, there are limitations in physical and biological aspects such as biocompatibility, printing compatibility, and geometric precision.
본 발명의 발명자들은 DLP 바이오 프린터에 적용 가능한 실크피브로인(silk fibroin)의 바이오잉크를 개발하여, 이의 광조형성, 프린팅성, 세포적합성 등을 확인하였다(등록특허 제10-1983741호). The inventors of the present invention developed a bio-ink of silk fibroin applicable to a DLP bio-printer, and confirmed its photosynthesis, printability, and cellular compatibility (Registration Patent No. 10-1983741).
최근 전기전도성 물질을 이용하여 생체재료를 개발하는 연구가 활발히 진행되고 있다. 신경세포, 심근세포, 근육세포, 줄기세포 등은 전기장에 직접적인 반응을 하고, 혈관형성, 세포분열, 신경세포신호, 신경발아, 상처 치료 등 영향을 미친다. 따라서 전기전도성 스캐폴드는 내인성 및 외인성 전계를 전기 감응 세포내로 유도함으로써 세포 분화를 촉진시킬 수 있는 기능이 있어야 한다. Recently, researches on developing biomaterials using electrically conductive materials have been actively conducted. Neurons, cardiomyocytes, muscle cells, stem cells, etc. react directly to the electric field and have an effect on blood vessel formation, cell division, nerve cell signals, nerve germination, and wound healing. Therefore, the electrically conductive scaffold must have a function capable of promoting cell differentiation by inducing endogenous and exogenous electric fields into the electric-sensitive cells.
이러한 종래의 기술을 토대로 본 발명자는 실크피브로인을 이용하여 GO를 rGO로 제조하였으며, 광가교가 가능한 하이드로젤 형태의 바이오잉크를 제조하여 본 발명은 완성하였다.Based on this conventional technology, the present inventors prepared GO as rGO using silk fibroin, and produced a bio-ink in the form of a hydrogel capable of photocrosslinking, thereby completing the present invention.
본 발명에 따른 바이오잉크는 실크피브로인으로 인하여 생체적합성이 향상되고, 친수성이 증진되며, 이에 따른 용해도가 향상되어, 반응중 도입되는 광가교 그룹으로 인하여 광조형 프린팅이 가능하다. 또한, 완성된 rGO는 전기전도성을 갖기 때문에 전기 감응세포의 성장을 촉진할 수 있다. 본 명세서에서는 실크피브로인으로 제작된 rGO의 물리적 특성, 전기적 특성을 확인하였으며, 전기 감응 세포를 포함하여 3D 프린팅을 진행함으로써, 세포적합성과 분화정도를 확인하였다.The bio-ink according to the present invention improves biocompatibility due to silk fibroin, improves hydrophilicity, and thus improves solubility, and enables photolithography printing due to the photocrosslinking group introduced during the reaction. In addition, since the finished rGO has electrical conductivity, it can promote the growth of electrical sensitive cells. In the present specification, physical and electrical properties of rGO made of silk fibroin were confirmed, and cell suitability and degree of differentiation were confirmed by performing 3D printing including electrosensitive cells.
본 발명에서는, 그래핀 산화물(Graphene Oxide: GO)을 유독한 화학적 처리 또는 고온 어닐링을 하지 않고 단지 천연 단백질 고분자인 실크 피브로인(silk fibroin: SF)으로 화학적 개질하여 환원된 그래핀 산화물(reduction Graphene Oxide: rGO)을 제조하고, 이를 포함하는 바이오 잉크를 제공하고자 한다.In the present invention, graphene oxide (GO) is chemically modified with silk fibroin (SF), a natural protein polymer without toxic chemical treatment or high-temperature annealing, and reduced graphene oxide (reduction graphene oxide). : rGO), and to provide bio-inks containing it.
또한, 본 발명은 SF의 생체적합성과 GO의 전기전도성을 동시에 갖는 바이오잉크의 제조방법을 제공하고자 한다.In addition, the present invention is to provide a method for producing a bio-ink having both the biocompatibility of SF and the electrical conductivity of GO.
본 발명에 따른 바이오 잉크는 SF과 GO의 복합체를 포함함으로써, 신경세포의 성장을 촉진시킬 수 있다.The bio-ink according to the present invention may promote the growth of nerve cells by including a complex of SF and GO.
분 발명의 일 실시 형태에 따른 바이오 잉크 조성물은, 실크피브로인(Silk fibroin, SF), 메타크릴레이트(methacrylate) 화합물 및 그래핀 산화물(Graphene Oxide, GO)이 중합된 고분자 중합체;와 광개시제(photo initiator);를 포함한다.The bio-ink composition according to an embodiment of the present invention includes a polymer polymer in which a silk fibroin (SF), a methacrylate compound, and a graphene oxide (GO) are polymerized; and a photo initiator ); includes.
이때 사용되는 고분자 중합체는, SF의 아미노산 잔기에 적어도 하나 이상의 메타크릴레이트 화합물과 적어도 하나 이상의 GO가 공중합된 것이 바람직하다.The polymer polymer used at this time is preferably copolymerized with at least one methacrylate compound and at least one GO on an amino acid residue of SF.
본 발명의 다른 실시 형태로, 이러한 바이오 잉크 조성물의 제조 방법을 들 수 있는데, 용매에 실크피브로인(SF)을 용해시키는 제1단계; 실크피브로인(SF) 용액에 메타크릴레이트 화합물을 투입한 후, 교반하여 개질된 실크피브로인(SB)를 제조하는 제2단계; 상기 제2단계를 통해 얻어진 개질된 실크피브로인(SB)에 그래핀 산화물(GO)을 투입한 후, 교반하여 고분자 중합체(SGOB1)를 제조하는 제3단계; 상기 제3단계를 통해 얻어진 고분자 중합체(SGOB1)가 포함된 용액을 건조하여 분말화시키는 제4단계; 및 물에 상기 고분자 중합체(SGOB1) 분말과 광개시제를 혼합하는 제5단계;를 포함한다.In another embodiment of the present invention, there may be mentioned a method of preparing such a bio-ink composition, comprising: a first step of dissolving silk fibroin (SF) in a solvent; A second step of preparing a modified silk fibroin (SB) by adding a methacrylate compound to the silk fibroin (SF) solution and stirring; A third step of preparing a polymer polymer (SGOB1) by adding graphene oxide (GO) to the modified silk fibroin (SB) obtained through the second step and stirring; A fourth step of drying and powdering the solution containing the polymer polymer (SGOB1) obtained through the third step; And a fifth step of mixing the polymer polymer (SGOB1) powder and a photoinitiator in water.
상기 제1단계는, 분쇄된 누에고치를 0.05M NaCO3 수용액에 넣고 끓인 후 세척을 통해 세리신을 제거함으로써 실크피브로인만을 얻는 정련단계;를 포함한다.The first step includes a refining step of obtaining only silk fibroin by removing sericin through washing after boiling the crushed cocoon in a 0.05M NaCO 3 aqueous solution and washing.
또한, 상기 제1단계는 정련단계를 거친 실크피브로인을, 9.3M 브롬화리튬(LiBr)용액에 0.05 ~ 0.35g/ml 농도로 용해시킨 후, 60℃의 온도로 가열하는 과정을 더 포함할 수 있다.In addition, the first step may further include dissolving the silk fibroin that has undergone the refining step at a concentration of 0.05 to 0.35 g/ml in a 9.3 M lithium bromide (LiBr) solution, and heating it to a temperature of 60°C. .
상기 제2단계의 메타크릴레이트 화합물은, 글리시딜메타크릴레이트(glycidyl methacrylate: GMA) 또는 메타크릴 무수물(methacrylic anhydride; MA)이 사용될 수 있으며, 좀 더 구체적으로는 상기 제1단계에서 얻어진 실크피브로인이 용해된 용액에 글리시딜메타크릴레이트(glycidyl methacrylate: GMA) 4 ~ 6mL를 혼합한 후, 50 ~ 70℃의 온도에서 200 ~ 400rpm의 속도로 교반하되, 상기 GMA의 농도가 352mM 이하인 것이 바람직하다.As the methacrylate compound of the second step, glycidyl methacrylate (GMA) or methacrylic anhydride (MA) may be used, and more specifically, the silk obtained in the first step After mixing 4 to 6 mL of glycidyl methacrylate (GMA) in a solution in which fibroin is dissolved, stirring at a speed of 200 to 400 rpm at a temperature of 50 to 70 °C, but the concentration of the GMA is 352 mM or less. desirable.
상기 제3단계의 그래핀 산화물(GO)은, 0.25 ~ 3.5wt%의 농도로 물에 분산된 후, 초음파 처리되는 것이 바람직하다. 또한, 이러한 그래핀 산화물(GO)에 1-ethyl-3-(3-dimethylamino propyl) carbodiimide hydrochloric acid(EDC)를 0.15 ~ 1.96g을 반응시킨 후, 상온에서 0.21 ~ 2.8g의 N-hydroxysuccinimide(NHS)을 혼합하여 반응시키고, mercaptoethanol 용액을 추가로 더 혼합하는 과정을 통해 상기 제3단계가 수행될 수 있다.The graphene oxide (GO) of the third step is preferably dispersed in water at a concentration of 0.25 to 3.5 wt% and then subjected to ultrasonic treatment. In addition, 0.15 to 1.96 g of 1-ethyl-3-(3-dimethylamino propyl) carbodiimide hydrochloric acid (EDC) was reacted with the graphene oxide (GO), and then 0.21 to 2.8 g of N-hydroxysuccinimide (NHS ), the third step may be performed by mixing and reacting, and further mixing a mercaptoethanol solution.
특히 상기 제3단계에서는, 개질된 실크피브로인(SB)과 그래핀 산화물(GO)을 1:1의 부피비로 혼합하여 carbodiimidation 반응이 수행되는 것이 바람직하며, 상기 제3단계와 제4단계 사이에, 고분자 중합체가 포함된 용액을 12 ~ 14kDa cutoff 투석튜브에 넣은 후, 불순물을 제거하는 과정을 추가로 더 수행하는 것이 더욱 바람직하다.In particular, in the third step, it is preferable to perform a carbodiimidation reaction by mixing the modified silk fibroin (SB) and graphene oxide (GO) in a volume ratio of 1:1, and between the third step and the fourth step, It is more preferable to further perform a process of removing impurities after putting the solution containing the polymer polymer into a 12 ~ 14kDa cutoff dialysis tube.
상기 제4단계의 분말화 과정은, 상기 고분자 중합체가 포함된 용액을 -90 ~ -70℃ 온도로 동결하는 단계; 및 -90 ~ -70℃ 온도에서 약 40 ~ 60시간 동안 동결건조하는 것이 바람직하다.The powdering process of the fourth step may include freezing the solution containing the polymer polymer at a temperature of -90 to -70°C; And freeze-drying at a temperature of -90 to -70°C for about 40 to 60 hours.
상기 제5단계에서 혼합되는 광개시제는, 벤질디메틸케탈(benzyl dimethyl ketal), 아세토페논(acetophenone), 벤조인메틸에테르(benzoin methyl ether), 디에톡시아세토페논(diethoxyacetophenone), 벤조일 포스핀 옥사이드(benzoyl phosphine oxide) 및 1-하이드록시사이클로헥실 페닐 케톤(1-hydroxycyclohexyl phenyl ketone)으로 이루어진 군 중에서 선택되는 적어도 하나 이상을 포함할 수 있다.The photoinitiators mixed in the fifth step, benzyl dimethyl ketal, acetophenone, benzoin methyl ether, diethoxyacetophenone, benzoyl phosphine oxide (benzoyl phosphine). oxide) and 1-hydroxycyclohexyl phenyl ketone, and at least one selected from the group consisting of.
이렇게 제1단계 내지 제5단계의 과정을 거쳐 얻어지는 바이오 잉크 조성물에는, 고분자 중합체 분말 15 ~ 25wt% 및 광개시제 0.2 ~ 0.4wt%가 포함될 수 있다.In the bio-ink composition obtained through the process of the first to fifth steps, 15 to 25 wt% of a polymer powder and 0.2 to 0.4 wt% of a photoinitiator may be included.
또한, 이러한 바이오 잉크 조성물의 제조 과정 중에서, 상기 실크피브로인(SF)를 대신하여 히알루론산, 콜라겐, 알지네이트, 녹말, 키토산, 젤라틴, 덱스트린, 셀룰로오스, 알긴산, 콘드로이틴 설페이트, 헤파린 및 헤파란으로 이루어진 군에서 선택된 어느 하나 이상을 사용하는 것도 가능하다.In addition, in the manufacturing process of such a bio-ink composition, in the group consisting of hyaluronic acid, collagen, alginate, starch, chitosan, gelatin, dextrin, cellulose, alginic acid, chondroitin sulfate, heparin and heparan in place of the silk fibroin (SF) It is also possible to use any one or more selected.
본 발명의 또 다른 실시 형태로, 앞서 설명한 바이오 잉크 조성물을 사용하여 fused deposition modeling(FDM) 방식 또는 Digital light processing(DLP) 방식의 3D 바이오 프린터를 통해 구조체를 제조하는 방법을 들 수 있으며, 이러한 바이오 잉크 조성물의 제조 방법으로 제조된 바이오 잉크 조성물을 사용하여 fused deposition modeling(FDM) 방식 또는 Digital light processing(DLP) 방식의 3D 바이오 프린터를 통해 구조체를 제조하는 방법을 더 포함한다.In another embodiment of the present invention, there may be mentioned a method of manufacturing a structure through a fused deposition modeling (FDM) method or a digital light processing (DLP) type 3D bio printer using the bio ink composition described above. It further includes a method of manufacturing a structure through a 3D bioprinter of a fused deposition modeling (FDM) method or a digital light processing (DLP) method using the bio ink composition prepared by the method of manufacturing the ink composition.
본 발명의 생체적합성과 전기전도성을 갖는 광가교 바이오잉크는 누에고치유래 실크단백질과 그래핀 산화물(Graphene Oxide: GO)을 화학적으로 결합시킨 환원된 그래핀 산화물(reduction Graphene Oxide: rGO)로 메타크릴레이트계 화합물의 도입과 프린팅시 혼합되는 광개시제를 통하여 광가교가 가능하며, GO를 함유시킬 실크피브로인(silk fibroin: SF)는 FDA승인을 받은 재료로 세포적합성이 높아 의료용 재료로 활발히 활용되어오고 있으며, 우수한 강도를 나타낸다. 따라서 신경세포 또는 근육세포등 전기전도성을 요구하는 세포를 포함하여 다양한 조직공학용 지지체 재룟로 활용이 가능하다. The photo-crosslinked bioink having biocompatibility and electrical conductivity of the present invention is a reduced graphene oxide (reduction graphene oxide: rGO) that chemically combines silk protein derived from cocoon and graphene oxide (GO). Photocrosslinking is possible through the introduction of a rate-based compound and a photoinitiator that is mixed during printing.Silk fibroin (SF) to contain GO is a material approved by the FDA and has been actively used as a medical material due to its high cellular compatibility. , Shows excellent strength. Therefore, it can be used as a support material for various tissue engineering including cells that require electrical conductivity such as nerve cells or muscle cells.
본 발명에서 제시하는 방법으로 3D 프린팅시에 구조체가 적절하게 제조되므로 스캐폴드 제작에 유용하다. GO를 단순히 물리적으로 혼합하거나 각 주요 성분을 반응시키는 순서에 따라 그래핀 산화물의 분산도 및 용해도가 떨어질 수 있어 결과적으로 3차원 프린터를 통한 구조체 제작 결과에 영향을 준다.Since the structure is properly manufactured during 3D printing by the method presented in the present invention, it is useful for scaffold manufacturing. Depending on the order of simply physically mixing GO or reacting each major component, the degree of dispersion and solubility of graphene oxide may decrease, and as a result, it affects the result of fabrication of the structure through a 3D printer.
다만, 본 발명의 효과들은 이상에서 언급한 효과로 제한되지 않으며, 언급되지 않은 또 다른 효과들은 아래의 기재로부터 통상의 기술자에게 명확하게 이해될 수 있을 것이다.However, the effects of the present invention are not limited to the above-mentioned effects, and other effects not mentioned will be clearly understood by those skilled in the art from the following description.
도 1(a)는 본 발명에 따른 바이오 잉크 조성물의 제조 과정을 도식적으로 나타낸 그림이고, 도 1(b)는 본 발명의 원료물질로부터 고분자 중합체가 형성되는 과정을 도식적으로 나타낸 그림이다.
도 2는 본 발명으로 만들어진 바이오잉크의 FT-IR을 통한 관능기 분석 결과이다.
도 3은 바이오잉크에서 SF와 GO의 결합정도를 TNBS 어세이를 통해 분석한 결과이다.
도 4는 바이오잉크에서 실제 결합된 그래핀 산화물의 양을 확인하기 위한 TGA 분석 결과이다.
도 5는 바이오잉크의 물에 대한 용해도 및 분산도를 측정한 결과이다.
도 6은 바이오잉크의 프린팅 가능성을 보여주는 결과이다.
도 7은 바이오잉크로 제작된 하이드로젤의 향상된 강도를 나타내는 결과이다.
도 8은 바이오잉크로 제작된 하이드로젤의 전기전도도을 측정한 결과로, 8(a)는 하이드로젤의 전기전도성 측정을 위한 셀을 레진으로 3D 프린팅하여 제작하여 하이드로젤을 장착한 모습이고, 8(b)는 이러한 하이드로젤의 전기전도도 측정결과이다.
도 9(a)와 9(b)는 각각 바이오잉크로 제작된 하이드로젤이 전도체로서의 기능성을 3.5V의 전압에서 LED 회로에서 확인한 결과로, 고분자 중합체의 농도가 각각 0.25wt%, 2.5wt%이다.
도 10은 바이오잉크 내에 전기 감응 세포(Neuro2a)를 포함하여 하이드로젤 프린팅 후 세포 증식도를 확인한 결과이다.
도 11은 바이오잉크내에 전기 감응 세포(Neuro2a)가 포함된 바이오 잉크 조성물에 포함된 하이드로젤 프린팅 후 세포의 생존도를 날짜별(Day 0 내지 7: 도 11)로 확인한 결과이다.
도 12는 하이드로젤 내에 파종된 Neuro2a 세포에서 alpha-tubulin의 발현 여부를 분화인자가 들어가지 않은 성장배양액내에서 확인한 결과이다.
도 13은 하이드로젤 내에 파종된 Neuro2a 세포에서 alpha-tubulin의 발현 여부를 신경세포분화인자가 들어간 분화배양액내에서 확인한 결과이다. 1(a) is a diagram schematically showing a manufacturing process of a bio-ink composition according to the present invention, and FIG. 1(b) is a diagram schematically showing a process of forming a polymer polymer from the raw material of the present invention.
2 is a result of functional group analysis through FT-IR of the bio-ink made by the present invention.
3 is a result of analyzing the binding degree of SF and GO in the bio-ink through TNBS assay.
4 is a TGA analysis result for confirming the amount of actually bound graphene oxide in the bio-ink.
5 is a result of measuring the solubility and dispersibility of bio-ink in water.
6 is a result showing the possibility of printing bio-ink.
7 is a result showing the improved strength of the hydrogel produced with bio-ink.
Figure 8 is a result of measuring the electrical conductivity of the hydrogel made of bio-ink, 8 (a) is a state in which the hydrogel is mounted by 3D printing a cell for measuring the electrical conductivity of the hydrogel with resin. b) is the result of measuring the electrical conductivity of this hydrogel.
9(a) and 9(b) are results of confirming the functionality of the hydrogel made of bio-ink as a conductor in the LED circuit at a voltage of 3.5V, respectively, and the concentrations of the polymer polymer are 0.25wt% and 2.5wt%, respectively .
10 is a result of confirming the degree of cell proliferation after hydrogel printing including electro-sensitized cells (Neuro2a) in the bio-ink.
FIG. 11 is a result of confirming the viability of cells by day (
12 is a result of confirming the expression of alpha-tubulin in Neuro2a cells seeded in the hydrogel in the growth culture solution to which no differentiation factor was added.
13 is a result of confirming the expression of alpha-tubulin in Neuro2a cells seeded in the hydrogel in the differentiation culture medium containing the neuronal differentiation factor.
이하에서는 본 발명의 바람직한 실시예를 통해 상세히 설명하기에 앞서, 본 명세서 및 청구범위에 사용된 용어나 단어는 통상적이거나 사전적인 의미로 한정하여 해석되어서는 아니 되며, 본 발명의 기술적 사상에 부합하는 의미와 개념으로 해석되어야 함을 밝혀둔다.Hereinafter, before describing in detail through a preferred embodiment of the present invention, terms or words used in the present specification and claims should not be construed as being limited to a conventional or dictionary meaning, and conforming to the technical idea of the present invention. It must be interpreted as meaning and concept.
본 명세서 전체에서, 어떤 부분이 어떤 구성요소를 "포함" 한다고 할 때, 이는 특별히 반대되는 기재가 없는 한 다른 구성요소를 제외하는 것이 아니라 다른 구성 요소를 더 포함할 수 있는 것을 의미한다.Throughout this specification, when a certain part "includes" a certain component, it means that other components may be further included rather than excluding other components unless otherwise stated.
한편, 본 발명에서 개시되는 각각의 설명 및 실시형태는 각각의 다른 설명 및 실시 형태에도 적용될 수 있다. 즉, 본 발명에서 개시된 다양한 요소들의 모든 조합이 본 발명의 범주에 속한다. 또한, 하기 기술되는 구체적인 서술에 의하여 본 발명의 범주가 제한된다고 할 수 없다.Meanwhile, each description and embodiment disclosed in the present invention can be applied to each other description and embodiment. That is, all combinations of various elements disclosed in the present invention belong to the scope of the present invention. In addition, it cannot be said that the scope of the present invention is limited by the specific description described below.
또한, 당해 기술분야의 통상의 지식을 가진 자는 통상의 실험만을 사용하여 본 출원에 기재된 본 발명의 특정 양태에 대한 다수의 등가물을 인지하거나 확인할 수 있다. 또한, 이러한 등가물은 본 발명에 포함되는 것으로 해석되어야 한다.In addition, those of ordinary skill in the art can recognize or ascertain using only routine experimentation a number of equivalents to the specific aspects of the invention described in this application. In addition, these equivalents should be construed as being included in the present invention.
상술한 바와 같은 목적을 달성하기 위한 본 발명의 일 실시 형태로는 바이오 3D 프린팅에 사용될 수 있는 전기전도성을 갖는 바이오잉크를 들 수 있다.As an embodiment of the present invention for achieving the above-described object, a bio-ink having electrical conductivity that can be used for bio 3D printing may be mentioned.
이러한 바이오잉크는, 실크 피브로인(silk fibroin: SF), 그래핀 산화물 (Graphene Oxide: GO)과 메타크릴레이트(methacrylate) 화합물이 중합된 고분자 중합체; 및 광개시제;를 포함하며, 상기 고분자 중합체는 SF의 아미노산 잔기에 하나 이상의 methacrylate계 화합물과 하나 이상의 GO가 공중합 되어 형성된 것이 바람직하다.Such bio-inks include a high molecular weight polymer in which silk fibroin (SF), graphene oxide (GO) and methacrylate compounds are polymerized; And a photoinitiator, wherein the polymer is preferably formed by copolymerizing at least one methacrylate-based compound and at least one GO on an amino acid residue of SF.
도 1(b)에 제시된 것처럼, SF의 아민 그룹과 GMA의 에폭시그룹간의 친핵체첨가 반응으로 개질된 실크피브로인인 SB를 제조한다. 한편, EDC로 GO의 카르복실그룹을 활성화시킨 후, NHS하에서 상기 개질된 실크피브로인인 SB와 반응시켜 고분자 중합체인 SGOB1를 제조하며, 이러한 고분자 중합체와 광 개시제를 포함하는 전기전도성을 갖는 광가교 바이오 잉크 조성물을 제조한다.As shown in Fig. 1(b), a modified silk fibroin SB was prepared by a nucleophile addition reaction between the amine group of SF and the epoxy group of GMA. On the other hand, after activating the carboxyl group of GO with EDC, it reacts with the modified silk fibroin SB under NHS to produce SGOB1, a polymer polymer, and photo-crosslinking biotechnology having electrical conductivity including such a polymer polymer and a photoinitiator. Prepare an ink composition.
본 발명의 또 다른 실시 형태로 이러한 전기전도성을 갖는 바이오 잉크의 제조 방법을 들 수 있는데, 도 1(a)에 제시된 것처럼 용매에 실크피브로인(SF)을 용해시키는 제1단계; 실크피브로인(SF) 용액에 메타크릴레이트 화합물을 투입한 후, 교반하여 개질된 실크피브로인(SB)를 제조하는 제2단계; 상기 제2단계를 통해 얻어진 개질된 실크피브로인(SB)에 그래핀 산화물(GO)을 투입한 후, 교반하여 고분자 중합체(SGOB1)를 제조하는 제3단계; 상기 제3단계를 통해 얻어진 고분자 중합체(SGOB1)가 포함된 용액을 건조하여 분말화시키는 제4단계; 및 물에 상기 고분자 중합체(SGOB1) 분말과 광개시제를 혼합하는 제5단계;를 포함한다.Another embodiment of the present invention is a method of manufacturing a bio-ink having such electrical conductivity, including a first step of dissolving silk fibroin (SF) in a solvent as shown in FIG. 1(a); A second step of preparing a modified silk fibroin (SB) by adding a methacrylate compound to the silk fibroin (SF) solution and stirring; A third step of preparing a polymer polymer (SGOB1) by adding graphene oxide (GO) to the modified silk fibroin (SB) obtained through the second step and stirring; A fourth step of drying and powdering the solution containing the polymer polymer (SGOB1) obtained through the third step; And a fifth step of mixing the polymer polymer (SGOB1) powder and a photoinitiator in water.
상기 제1단계는, 실크피브로인(SF) 용액을 제조하는 단계로, 약 40g의 잘게 잘려진 누에고치를 1L의 0.05M NaCO3 solution에 넣고 약 30분간 끓인 후 세척을 통해 세리신을 제거함으로써 SF만을 얻는 정련단계를 거친다. 이후, 9.3M 브롬화리튬(LiBr)용액에 SF를 20g 용해시킨 후, 40 ~ 80분 동안 50 ~ 70℃ 온도로 가열할 수 있다. 이러한 SF의 용액화 과정은 반드시 LiBr을 사용해야만 하는 것은 아니며, 염화칼슘(CaCl2) 역시 사용 가능하다.The first step is a step of preparing a silk fibroin (SF) solution, in which about 40 g of chopped cocoon is put in 1 L of 0.05M NaCO 3 solution, boiled for about 30 minutes, and then sericin is removed through washing to obtain only SF. Go through the refining stage. Thereafter, 20 g of SF is dissolved in a 9.3 M lithium bromide (LiBr) solution, and then heated to a temperature of 50 to 70° C. for 40 to 80 minutes. In this process of solutionization of SF, it is not necessary to use LiBr, and calcium chloride (CaCl 2 ) can also be used.
상기 제2단계는, SF가 용해된 용액에 4~6mL(352 mM)의 글리시딜메타크릴레이트(glycidyl methacrylate: GMA)를 투입한 후, 50 ~ 70 ℃ 온도에서 약 2 ~ 4시간 동안 약 200 ~ 400rpm의 회전속도로 교반하는 것이 바람직하다.In the second step, 4 to 6 mL (352 mM) of glycidyl methacrylate (GMA) is added to the solution in which SF is dissolved, and then at a temperature of 50 to 70° C. for about 2 to 4 hours. It is preferable to stir at a rotation speed of 200 to 400 rpm.
상기 제2단계에서 GMA가 6mL를 초과하여 사용될 경우에는 그래핀 산화물의 결합력이 저하될 수 있고, 4mL 미만으로 사용될 경우에는 GMA처리시 광가교능이 저하될 수 있으므로, 바람직하지 않다. 또한, 이때 사용되는 GMA의 농도가 352mM 이하인 것이 더욱 바람직하며, SF에 광가교 그룹을 도입하기 위해 사용되는 methacrylate 화합물은 GMA 외에도 메타크릴 무수물(methacrylic anhydride: MA) 역시 사용 가능하다.In the second step, when GMA is used in excess of 6 mL, the bonding strength of graphene oxide may be reduced, and when it is used in an amount of less than 4 mL, the photo-crosslinking ability may decrease during GMA treatment, which is not preferable. In addition, it is more preferable that the concentration of GMA used at this time is 352 mM or less, and the methacrylate compound used to introduce a photo-crosslinking group into SF may be used in addition to GMA, but also methacrylic anhydride (MA).
상기 제3단계는, 광경화 그룹이 도입된 SF에 GO를 도입하는 단계로, 0.25 ~ 3.5wt%의 농도로 GO를 물에 분산시킨 후, 약 1시간 동안 초음파 세척한다. 여기에 1-ethyl-3-(3-dimethylamino propyl) carbodiimide hydrochloric acid(EDC)를 0.15 ~ 1.96g(좀 더 바람직하게는, 0.15g, 1.4g 혹은 1.96g)을 넣어 반응시켜 GO의 카르복실기가 활성화되도록 한다. 이후 상온에서 0.21 ~ 2.8g의 N-hydroxysuccinimide(NHS)을 첨가하여 약 30분간 저어주면서 반응 중간물의 안정성을 높여주는 것이 바람직하며, 반응을 종료시키기 위하여 2mL의 mercaptoethanol solution을 넣어준다. The third step is a step of introducing GO to SF to which a photocuring group has been introduced, and after dispersing GO in water at a concentration of 0.25 to 3.5 wt%, ultrasonic cleaning is performed for about 1 hour. 0.15 ~ 1.96g (more preferably, 0.15g, 1.4g or 1.96g) of 1-ethyl-3-(3-dimethylamino propyl) carbodiimide hydrochloric acid (EDC) is added and reacted to activate the carboxyl group of GO. Make it possible. After that, it is desirable to increase the stability of the reaction intermediate by adding 0.21 to 2.8 g of N-hydroxysuccinimide (NHS) at room temperature and stirring for about 30 minutes. To terminate the reaction, 2 mL of mercaptoethanol solution is added.
이렇게 카르복실기가 활성화된 GO와 앞선 제2단계에서 준비된 개질된 실크피브로인(SB)을 1:1의 부피비로 혼합하여 carbodiimidation reaction을 통한 아마이드 결합의 개시를 유도한다. 이 반응은 상온에서 24시간 유지시키며, 상기 제3단계 후에 제조된 고분자 중합체가 포함된 용액을 투석 튜브에 넣고, 물에 침지시켜 불순물을 제거하는 투석단계를 더 포함할 수 있다.In this way, the carboxyl group activated GO and the modified silk fibroin (SB) prepared in the second step are mixed in a volume ratio of 1:1 to induce the initiation of amide binding through a carbodiimidation reaction. This reaction is maintained at room temperature for 24 hours, and a dialysis step of removing impurities by placing the solution containing the polymer polymer prepared after the third step into a dialysis tube and immersing it in water may be further included.
상기 투석단계는, 제조된 고분자 중합체가 포함된 용액을 12 ~ 14 kDa cutoff 투석튜브에 넣은 후, 물에 3 ~ 7일간 침지시켜 불순물을 제거하는 것이 바람직하다. In the dialysis step, it is preferable to remove impurities by putting the prepared solution containing the polymer polymer into a 12 to 14 kDa cutoff dialysis tube and immersing it in water for 3 to 7 days.
상기 제4단계는, 상기 고분자 중합체가 포함된 용액을 -90 ~ -70℃ 온도로 10 ~ 14시간 동안 동결한 다음, 상기 동결 온도와 동일한 온도에서 약 40 ~ 60시간 동안 동결건조 하는 것이 바람직하다. 투석된 용액은 miracloth에 통과시켜 걸러주고 5일간 동결 건조 시킨다. In the fourth step, it is preferable to freeze the solution containing the polymer polymer at a temperature of -90 to -70°C for 10 to 14 hours, and then freeze-dry for about 40 to 60 hours at the same temperature as the freezing temperature. . The dialyzed solution is filtered through miracloth and freeze-dried for 5 days.
이러한 투석시간이 6일 미만일 경우 제조된 중합체 내 포함된 이온 성분의 제거가 충분하지 못하여 생체 적합성이 저하되거나 프린팅으로 제조된 구조체의 기계적 강도가 저하되며, 투석시간이 8일을 초과하게 되는 경우 시간 초과에 따른 이익이 없어 경제성이 저하된다.If the dialysis time is less than 6 days, the removal of the ionic component contained in the prepared polymer is insufficient, resulting in reduced biocompatibility or the mechanical strength of the printed structure, and if the dialysis time exceeds 8 days, the time There is no profit from excess, which reduces economic feasibility.
사용되는 GO의 농도는 다양하게 변화시킬 수 있으나 젤레이션의 효율성에 따라 약 3.5wt% 이내로 제한하는 것이 바람직하다. The concentration of GO used can be varied in various ways, but it is desirable to limit it to within about 3.5wt% depending on the efficiency of gelation.
상기 제5단계는, 바이오잉크 제조 과정의 마무리 단계로, 광개시제를 혼합하는 단계이다. 광개시제는 빛 혹은 광에 노출될 경우, 바이오잉크 내에서 라디칼 반응을 일으켜 광가교 그룹간의 공유결합을 형성시킴으로써 하이드로젤의 광가교를 유도한다. The fifth step is a step of mixing a photoinitiator as a finishing step of the bioink manufacturing process. When the photoinitiator is exposed to light or light, it induces photocrosslinking of the hydrogel by causing a radical reaction in the bioink to form covalent bonds between the photocrosslinking groups.
화학적으로 안정하고 인체에 독성이 없는 화합물이면 특별히 한정되지 않고 사용 가능하고, 바람직하게는, 벤질디메틸케탈(benzyl dimethyl ketal), 아세토페논(acetophenone), 벤조인메틸에테르(benzoin methyl ether), 디에톡시아세토페논(diethoxyacetophenone), 벤조일 포스핀 옥사이드(benzoyl phosphine oxide) 및 1-하이드록시사이클로헥실 페닐 케톤(1-hydroxycyclohexyl phenyl ketone)으로 이루어진 군 중에서 선택된 적어도 하나 이상이 사용될 수 있다.If it is a chemically stable and non-toxic compound, it is not particularly limited and can be used, and preferably, benzyl dimethyl ketal, acetophenone, benzoin methyl ether, diethoxy At least one selected from the group consisting of acetophenone (diethoxyacetophenone), benzoyl phosphine oxide (benzoyl phosphine oxide), and 1-hydroxycyclohexyl phenyl ketone (1-hydroxycyclohexyl phenyl ketone) may be used.
특히 본 발명에 따른 바이오잉크에 포함될 수 있는 광개시제로는, 자유 라디컬계이며 생체적합한 광개시제가 바람직한데, 4-(2-히드록시에톡시)페닐-(2-히드록시-2-프로필)케톤(Irgacure 2959)을 포함한 알파 히드록시알킬페논류(irgacure 184, Irgacure 651 포함), 리튬 페닐-2,4,6-트리메틸벤조일포스피네이트(lithium phenyl-(2,4,6-trimethyl benzoyl) phosphinate, LAP)와 같은 제1형 광개시제를 300~400nm 광원에서 사용될 수 있고, 가시광선 영역(400~700nm)의 광원을 사용할 경우에는 에오신 와이(Eoxin-Y), 로즈벵갈(rose bengal), 리보플라빈(riboflavin vitamin B2)과 같은 제2형 광개시제가 사용될 수 있다. 그러나 3D 프린팅의 빠른 출력에 적용하기 위하여 300-400nm 파장의 광원과 함께 상기 제1형 광개시제를 사용하는 것이 바람직하다. In particular, as a photoinitiator that can be included in the bioink according to the present invention, a free radical-based and biocompatible photoinitiator is preferable, and 4-(2-hydroxyethoxy)phenyl-(2-hydroxy-2-propyl)ketone ( Alpha hydroxyalkylphenones (including irgacure 184, Irgacure 651) including Irgacure 2959), lithium phenyl-2,4,6-trimethylbenzoyl phosphinate (lithium phenyl-(2,4,6-trimethyl benzoyl) phosphinate,
이상에서는 본 발명에서 바이오잉크의 기반을 이루는 재료로 실크피브로인을 중심으로 설명하였으나, 반드시 이에 제한되는 것은 아니며, 생체적합성과 친수성을 지닌 히알루론산, 콜라겐, 알지네이트, 녹말, 키토산, 젤라틴, 덱스트린, 셀룰로오스, 알긴산, 콘드로이틴 설페이트, 헤파린 및 헤파란으로 이루어진 군에서 선택된 어느 하나 이상이 상기 실크피브로인을 대체하여 대신 사용되는 것도 가능하다.In the above, silk fibroin has been described as a material that forms the basis of the bioink in the present invention, but is not necessarily limited thereto, and hyaluronic acid, collagen, alginate, starch, chitosan, gelatin, dextrin, and cellulose having biocompatibility and hydrophilicity , Alginic acid, chondroitin sulfate, heparin, and any one or more selected from the group consisting of heparan may be used instead of the silk fibroin.
본 발명에 따른 전기전도성을 갖는 바이오잉크는 광원을 처리할 수 있는 모든 3D 바이오 프린터에 사용될 수 있으며, 바람직하게는 DLP(Digital light processing) 방식의 3D 바이오 프린터에 사용될 수 있다.The bio-ink having electrical conductivity according to the present invention may be used in any 3D bio printer capable of processing a light source, and preferably may be used in a 3D bio printer of a digital light processing (DLP) method.
이하에서는 이상의 내용을 하기 실시예/비교예/실험예를 통해 보다 상세하게 설명하고자 한다. 다만, 이러한 설명은 본 발명을 좀 더 쉽게 이해할 수 있도록 예시하기 위한 것일 뿐 본 발명의 범위가 이들로 한정되는 것은 아님을 밝혀둔다. Hereinafter, the above will be described in more detail through the following Examples/Comparative Examples/Experimental Examples. However, it should be noted that this description is for illustrative purposes only so that the present invention can be more easily understood, and the scope of the present invention is not limited thereto.
[[ 제조예Manufacturing example ] ] 실크에서From silk 실크피브로인Silk fibroin (( SFSF ) 용액을 제조 A) prepare a solution
40g의 잘게 잘려진 누에고치를 1L의 0.05M NaCO3 solution에 넣고 30분간 끓인 후 세척을 통해 세리신을 제거함으로써 실크피브로인만을 얻는 정련단계를 거친다. 9.3M 브롬화리튬(LiBr)용액에 실크 피브로인 20g을 용해시킨 후, 60분간 60℃온도로 가열한다. 40g of finely chopped cocoon is added to 1L of 0.05M NaCO 3 solution, boiled for 30 minutes, and then washed to remove sericin to obtain only silk fibroin. After dissolving 20 g of silk fibroin in a 9.3 M lithium bromide (LiBr) solution, it is heated at 60°C for 60 minutes.
[[ 실시예Example 1] One] 실크피브로인Silk fibroin (( SFSF ), ), 메타크릴레이트Methacrylate 화합물( compound( GMAGMA ), ), 그래핀Graphene 산화물 (GO)의 순서로 중합된 고분자 중합체 제조( Preparation of polymer polymer polymerized in the order of oxide (GO) ( SGOB1SGOB1 ))
먼저, SF에 광경화 그룹을 도입하기 위해, 실험예 1에서 얻어진 SF에 5mL (352 mM)의 glycidyl methacrylates(GMA)를 투입한 후, 60℃ 온도에서 2 ~ 4시간 동안 200 ~ 400rpm의 회전속도로 교반하며 충분히 반응을 시킨다(개질된 실크피브로인 SB 제조).First, in order to introduce the photocuring group into SF, 5 mL (352 mM) of glycidyl methacrylates (GMA) was added to the SF obtained in Experimental Example 1, and then the rotation speed of 200 to 400 rpm for 2 to 4 hours at 60°C. Stir with and sufficiently react (manufactured by modified silk fibroin SB).
GO를 도입하는 단계로, 각각 0.25wt%(0.25%SGOB1), 2.5wt%(2.5%SGOB1), 3.5wt%(3.5%SGOB1)의 농도로 그래핀 산화물을 물에 분산시킨 후, 1시간 동안 초음파 세척 과정을 수행한다. 여기에 1-ethyl-3-(3-dimethylamino propyl) carbodiimide hydrochloric acid(EDC)를 각각 0.15g(0.25%SGOB1), 1.4g(2.5%SGOB1), 1.96g(3.5%SGOB1)씩 넣어 반응시켜 GO의 카르복실기가 활성화되도록 하였다.In the step of introducing GO, graphene oxide was dispersed in water at a concentration of 0.25wt% (0.25%SGOB1), 2.5wt% (2.5%SGOB1), and 3.5wt% (3.5%SGOB1), respectively, for 1 hour. Perform an ultrasonic cleaning process. Here, 1-ethyl-3-(3-dimethylamino propyl) carbodiimide hydrochloric acid (EDC) was added 0.15g (0.25%SGOB1), 1.4g (2.5%SGOB1), and 1.96g (3.5%SGOB1) each to react. The carboxyl group of was made to be activated.
반응 직후 0.21g(0.25%SGOB1), 2g(2.5%SGOB1), 2.8g(3.5%SGOB1)의 N-hydroxysuccinimide(NHS)을 첨가하여 30분간 저어주면서 반응 중간물의 안정성을 높여준다. 반응을 정지시키기 위하여 2mL의 mercaptoethanol solution을 넣어준다.Immediately after the reaction, 0.21g (0.25%SGOB1), 2g (2.5%SGOB1), 2.8g (3.5%SGOB1) of N-hydroxysuccinimide (NHS) was added and stirred for 30 minutes to increase the stability of the reaction intermediate. Add 2mL of mercaptoethanol solution to stop the reaction.
이렇게 카르볼실기가 활성화된 GO 각각에 광경화 그룹이 도입되어 개질된 실크피브로인(SB)를 1:1의 부피비로 넣어주어 carbodiimidation reaction을 통해 아마이드 결합의 개시를 유도한다. 이 반응은 상온에서 24시간 유지시킨다. In this way, a photocuring group is introduced into each of the activated GOs in which the carbolyl group is introduced to induce the initiation of amide bonds through a carbodiimidation reaction by putting the modified silk fibroin (SB) in a volume ratio of 1:1. This reaction is maintained at room temperature for 24 hours.
[[ 비교예Comparative example 1] One] 실크피브로인(SF)에Silk fibroin (SF) 그래핀Graphene 산화물(GO)을 결합시킨 중합체 제조 (SGO) Preparation of polymers incorporating oxides (GO) (SGO)
광경화 그룹이 도입되지 않은 SF에 GO를 직접 도입하여 중합체를 제조하였으며, 앞서 실험예 2의 2.5%SGOB1의 제조 과정과 동일하되, SF에 glycidyl methacrylates(GMA)를 투입하는 과정 없이 직접 SF와 카르복실기가 활성화된 GO를 1:1의 부피비율로 혼합하여 carbodiimidation reaction을 통한 아마이드 결합의 개시를 유도한다. 이 반응은 상온에서 24시간 유지시킨다.A polymer was prepared by directly introducing GO into SF where the photocuring group was not introduced, and the same as the manufacturing process of 2.5% SGOB1 in Experimental Example 2 above, but without adding glycidyl methacrylates (GMA) to SF, directly SF and carboxyl groups The activated GO is mixed in a volume ratio of 1:1 to induce the initiation of amide binding through carbodiimidation reaction. This reaction is maintained at room temperature for 24 hours.
[[ 비교예Comparative example 2] 2] 실크피브로인Silk fibroin (( SFSF ), ), 그래핀Graphene 산화물(GO), Oxide (GO), 메타크릴레이트Methacrylate 화합물(GMA)의 순서로 중합된 고분자 중합체 제조( Preparation of polymer polymer polymerized in order of compound (GMA) ( SGOB2SGOB2 ))
앞서 비교예 1에서 제조된 SGO에 메타크릴레이트 화합물을 사용하여 광가교그룹을 도입하였으며, 비교예 1의 SGO 용액에 6mL(424 mM)의 GMA를 투입한 후, 60℃ 온도에서 2 ~ 4시간 동안 200 ~ 400rpm의 회전속도로 교반하여 충분히 반응을 시킨다. A photocrosslinking group was introduced into the SGO prepared in Comparative Example 1 using a methacrylate compound, and 6 mL (424 mM) of GMA was added to the SGO solution of Comparative Example 1, and then 2 to 4 hours at a temperature of 60°C. While stirring at a rotation speed of 200 ~ 400rpm to sufficiently react.
[[ 비교예Comparative example 3] 3] 실크피브로인(SF)에Silk fibroin (SF) 메타크릴레이트계Methacrylate 화합물( compound( GMAGMA )을 중합시킨 고분자 중합체 제조() Polymerized polymer polymer production ( SBSB ))
앞선 제조예에서 제조된 SF에 실시예 1과 동일한 방식으로 광가교 그룹을 도입하는 하여 시료(SB)를 제조하였으며, SF 용액에 6mL(424 mM)의 GMA를 투입한 후, 60℃ 온도에서 2 ~ 4시간 동안 200 ~ 400rpm 회전속도로 교반하며 충분히 반응을 시킨다. A sample (SB) was prepared by introducing a photocrosslinking group to the SF prepared in the previous preparation example in the same manner as in Example 1, and 6 mL (424 mM) of GMA was added to the SF solution, and then 2 at 60°C. Stir at a rotation speed of 200 ~ 400rpm for ~ 4 hours to sufficiently react.
[[ 제조예Manufacturing example 2] 바이오잉크 제조 및 3D 프린팅 단계 2] Bio-ink manufacturing and 3D printing step
실시예 1 및 비교예 1 내지 3에서 제조된 시료 각각에 대해서 동결 건조 단계를 동일하게 수행하였다. 상기 실시예 1 및 비교예 1 내지 3에서 제조된 모든 고분자 중합체들(SGOB1, SGO, SGOB2, SB)은 반응 후 12 ~ 14kDa cutoff 투석튜브에 넣은 후, 물에 6일간 침지시켜 불순물을 제거하였다. The freeze-drying step was performed in the same manner for each of the samples prepared in Example 1 and Comparative Examples 1 to 3. All polymer polymers (SGOB1, SGO, SGOB2, SB) prepared in Example 1 and Comparative Examples 1 to 3 were placed in a 12 to 14 kDa cutoff dialysis tube after reaction, and then immersed in water for 6 days to remove impurities.
투석된 용액은 miracloth에 통과시켜 걸러주고 걸러준 용액은 -80℃ 온도로 10 ~ 14시간 동안 동결한 다음, 동결온도와 동일 온도하에서 40 ~ 60시간 동안 동결건조 한다. 실시예 1에서 제조된 SGOB1 시료의 경우, 사용된 GO의 양에 따라 0.25%SGOB1, 2.5%SGOB1, 3.5%SGOB1로 각각 구분하였다. The dialyzed solution is filtered through miracloth, and the filtered solution is frozen at -80°C for 10 to 14 hours, and then freeze-dried at the same temperature as the freezing temperature for 40 to 60 hours. In the case of the SGOB1 sample prepared in Example 1, it was classified into 0.25%SGOB1, 2.5%SGOB1, and 3.5%SGOB1 according to the amount of GO used.
바이오잉크로 프린팅 하기 직전 단계로 SGOB1, 또는 SGOB2 용액을 20%의 농도로 제조하고 여기에 광개시제 LAP가 0.3%(w/v) 포함되도록 섞어준다. As a step just before printing with bio-ink, a SGOB1 or SGOB2 solution was prepared at a concentration of 20%, and the photoinitiator LAP was mixed to contain 0.3% (w/v).
바이오잉크로 DLP (digital light processing)을 시행하는 단계로, 상기의 용액을 DLP 프린터 bath에 넣고 출력을 수행하였다. In a step of performing digital light processing (DLP) with bio-ink, the above solution was put into a DLP printer bath and printing was performed.
[[ 실험예Experimental example 1] 바이오잉크 내 1] In bio ink 그래핀Graphene 산화물과 Oxide and 실크피브로인Silk fibroin 용액의 화학적 결합 여부 분석 Analysis of whether the solution is chemically bound
바이오 잉크 조성물 내에 포함된 물질의 메타크릴레이션 반응이 성공적으로 수행되었는지 여부를 확인하기 위하여 동결건조된 스폰지 형태의 시료에 대해서 FT-IR을 측정하였고 그 결과를 도 2에 나타내었다. In order to confirm whether the methacrylate reaction of the material contained in the bio-ink composition was successfully performed, FT-IR was measured for a lyophilized sponge-shaped sample, and the results are shown in FIG. 2.
실크피브로인의 결합은 실크피브로인의 amide I (1645 cm-1), II(1513cm-1), III(1229cm-1)로 확인된다. 실크피브로인에 그래핀 산화물의 도입은 amide III 밴드 및 OH밴드 크기의 감소로 이어진다. SB, SGOB1 및 SGOB2에서 에폭시 그룹의 감소를 통해 메타크릴레이션 결과를 확인할 수 있었다. The binding of silk fibroin is identified as silk fibroin amide I (1645 cm -1 ), II (1513cm -1 ), III (1229cm -1 ). The introduction of graphene oxide into silk fibroin leads to a decrease in the size of the amide III band and the OH band. In SB, SGOB1 and SGOB2, the methacrylate results were confirmed through the reduction of the epoxy group.
NMR 분석으로 메타크릴레이트 비닐그룹을 나타내는 피크(6.5 ~ 7ppm)와, 메틸그룹을 나타내는 피크(2.69 ~ 2.91ppm)를 확인하였고 하기의 표 1에 정리하였다.By NMR analysis, a peak representing a vinyl methacrylate group (6.5 ~ 7ppm) and a peak representing a methyl group (2.69 ~ 2.91ppm) were identified, and are summarized in Table 1 below.
각 시료는 1mL D2O에 5mg의 건조된 재료를 용해시켜 0.45μm 필터에 걸러 700μL NMR 튜브에 넣어 준비하였고 600 MHz H-NMR로 분석하였다. GMA와 같은 메타크릴레이트 화합물이 처리되지 않은 그룹인 SF, SGO에는 해당 피크가 나타나지 않았으며, SB, SGOB1, SGOB2의 경우에만 피크가 확인되었다. 특히 SGOB1의 경우에는 상기 표 1에서 확인되듯이, GMA의 결합정도가 상대적으로 높음을 알 수 있다. Each sample was prepared by dissolving 5 mg of dried material in 1 mL D 2 O, filtered through a 0.45 μm filter, and put in a 700 μL NMR tube, and analyzed by 600 MHz H-NMR. Corresponding peaks did not appear in SF and SGO, which are groups that were not treated with a methacrylate compound such as GMA, and peaks were observed only in SB, SGOB1 and SGOB2. In particular, in the case of SGOB1, it can be seen that the degree of binding of GMA is relatively high, as confirmed in Table 1 above.
바이오 잉크 조성물 내에서 실크피브로인과 그래핀 산화물의 결합 정도를 TNBS 어세이를 통해 분석하였으며, 그 결과를 도 3에 나타내었다. The degree of binding between silk fibroin and graphene oxide in the bio-ink composition was analyzed through TNBS assay, and the results are shown in FIG. 3.
TNBS 어세이는 샘플내의 1차 아민기를 정량하는데 사용되며, 각 시료는 10mg/ml의 저장용액에서 100μL을 취하여 0.1 M sodium bicarbonate(pH=8.5) 1mL(반응버퍼)에 희석시켰다. 반응버퍼 내에 TNBS를 0.01%(w/v)로 준비한다. 여기서 500μL을 취하여 워킹용액 250μL과 혼합하고 37℃에서 2시간 배양한다. 250μL의 10wt% SDS와 125μL의 1N HCl로 혼합물을 섞어 반응을 정지시킨 후, 355nm의 파장에서 흡광도를 측정하였으며, 이를 1차 아민기로 해석하였다. 도 3의 결과에서 확인되듯이, 실크피브로인(SF)과 비교하여 SGO, SGOB2, SGOB1, SB 순으로 흡광도가 낮아졌으며, SGOB1그룹에서 상당량의 SF-GMA 결합이 존재함을 알 수 있었다. The TNBS assay is used to quantify the primary amine group in the sample, and each sample was diluted in 1 mL (reaction buffer) of 0.1 M sodium bicarbonate (pH = 8.5) by taking 100 μL from a 10 mg/ml stock solution. TNBS is prepared in 0.01% (w/v) in the reaction buffer. Here, 500 μL is taken, mixed with 250 μL of working solution, and incubated at 37°C for 2 hours. After stopping the reaction by mixing the mixture with 250 μL of 10wt% SDS and 125 μL of 1N HCl, the absorbance was measured at a wavelength of 355 nm, which was interpreted as a primary amine group. As can be seen from the results of Figure 3, compared to silk fibroin (SF), the absorbance was lowered in the order of SGO, SGOB2, SGOB1, and SB, and it was found that there is a significant amount of SF-GMA binding in the SGOB1 group.
도 4는 바이오 잉크 조성물 내에서 실제 결합된 그래핀 산화물의 양을 확인하기 위한 TGA 분석 결과이다. 질소 분위기(100mL/min) 및 10℃/min의 승온 속도 하에서 측정하였다. 그래핀 산화물(GO), SB, SGOB1 각각에서 60.21wt%, 66.31wt%, 61.45wt%의 감소가 확인되었으며, 이를 통하여 SGOB1 내에 약 4.8wt%의 그래핀 산화물이 함유되어있음이 확인할 수 있었다.4 is a TGA analysis result for confirming the amount of actually bound graphene oxide in the bio-ink composition. It was measured under a nitrogen atmosphere (100 mL/min) and a temperature increase rate of 10° C./min. In each of graphene oxide (GO), SB, and SGOB1, reductions of 60.21wt%, 66.31wt%, and 61.45wt% were confirmed, and it was confirmed that about 4.8wt% of graphene oxide was contained in SGOB1.
[[ 실험예Experimental example 2] 바이오잉크 내에서 2] In bioink 그래핀Graphene 산화물의 용해도 및 분산도 측정 Measurement of oxide solubility and dispersion
도 5는 바이오 잉크 조성물의 물에 대한 용해도 및 분산도를 관찰한 결과이다. 물에 순수 GO(I) 혹은 rGO(II)를 분산시킨 경우, SB내에 GO를 단순 분산시킨 경우(III), 0.25% SGOB1(IV), 2.5% SGOB1(V), 3.5% SGOB1(VI)을 0.05%로 용해시킨 후 60분 동안의 변화를 관찰한 결과이다(단, (III)의 경우에는 0.001% GO와 0.049% SB를 혼합하였음). 도 5a는 혼합 즉시(t = 0min), 도 5b 내지 도 5d는 각각 혼합 후 20분(t = 20min), 40분(t = 40min) 및 60분(t = 60min) 경과 후 관찰한 결과이다. 시료 (I) 내지 (III)은 바닥에 침전되었으나, 시료 (IV) 내지 (VI)는 농도와 관계없이 용매상에 고르게 분산된 것을 확인할 수 있다. 5 is a result of observing the solubility and dispersibility of a bio ink composition in water. When pure GO(I) or rGO(II) is dispersed in water, GO is simply dispersed in SB (III), 0.25% SGOB1(IV), 2.5% SGOB1(V), 3.5% SGOB1(VI) This is the result of observing the change for 60 minutes after dissolving at 0.05% (however, in the case of (III), 0.001% GO and 0.049% SB were mixed). 5A shows the results observed immediately after mixing (t = 0min), and FIGS. 5B to 5D are observed after 20 minutes (t = 20min), 40 minutes (t = 40min) and 60 minutes (t = 60min) after mixing, respectively. Samples (I) to (III) precipitated on the bottom, but it can be seen that the samples (IV) to (VI) were evenly dispersed in the solvent regardless of the concentration.
[[ 실험예Experimental example 3] 바이오잉크의 프린팅 가능성 측정 3] Measurement of bio-ink printability
도 6은 각 시료인 바이오 잉크 조성물의 프린팅 가능성을 보여주는 결과이다. SGOB1의 경우에는 농도가 증가할수록 고에너지량의 UV를 처리하여 프린팅을 완성할 수 있었다. 이는 바이오잉크의 농도가 증가할수록 광가교사이트가 증가하였기 때문인 것으로 해석된다. 반면 SGOB2 바이오잉크의 경우는 UV 조사량의 증가와 무관하게 프린팅이 되지 않았으며, 이는 SGOB2에 GMA의 결합이 잘 이루어지지 않았기 때문인 것으로 해석된다.6 is a result showing the possibility of printing the bio-ink composition as each sample. In the case of SGOB1, as the concentration increased, the printing could be completed by processing high energy amount of UV. This is interpreted to be due to the increase in photo-crosslinking sites as the concentration of the bio-ink increases. On the other hand, in the case of SGOB2 bioink, printing was not performed regardless of the increase in UV irradiation, which is interpreted to be due to poor binding of GMA to SGOB2.
[[ 실험예Experimental example 4] 바이오잉크로 제작된 4] Made with bio-ink 하이드로젤의Hydrogel 강도 측정 Strength measurement
제조예에서 제조된 바이오 잉크 조성물 시료를 사용하여 DLP 프린팅으로 출력된 하이드로젤의 샘플(직경 10mm, 높이 10mm) 각각에 대해서 10kg 로드셀을 이용하여 압축강도를 측정하였고(압축속도 = 5mm/min), 인장강도는 덤벨 모양의 시편(길이 7mm, 두께 1.5mm)을 출력하여 측정하였다(인장속도 = 5mm/min).Compressive strength was measured using a 10 kg load cell for each of the hydrogel samples (
이렇게 측정된 인강강도와 압축강도 결과는 각각 도 7(a)와 7(b)에 제시하였다. SGOB1의 경우에는 농도가 증가함에 따라 인장강도(a)와 압축강도(b) 모두 공통적으로 증가하였으며, SB 바이오 잉크 조성물 대비 인장강도는 약 2.6배, 압축강도는 약 1.3배 증가하였다.The measured tensile strength and compressive strength results are presented in Figs. 7(a) and 7(b), respectively. In the case of SGOB1, as the concentration increased, both tensile strength (a) and compressive strength (b) increased in common, and compared to SB bioink composition, tensile strength increased by about 2.6 times and compressive strength by about 1.3 times.
[[ 실험예Experimental example 5] 바이오잉크로 제작된 5] Made with bio-ink 하이드로젤의Hydrogel 전기전도성 측정 Electrical conductivity measurement
SGOB의 전기전도성을 측정하기 위하여 증류수에 바이오잉크를 준비하고 DLP 프린팅하여 하이드로젤을 제작하였다(직경 5mm, 두께 5mm). 프린팅 직후의 하이드로젤을, 4개의 프로브가 장착된 셀 내에 장착한 후, 즉시 하이드로젤의 전기 전도성을 측정하였으며, 측정 툴을 도 8(a)에 나타내었다. In order to measure the electrical conductivity of SGOB, bio-ink was prepared in distilled water and DLP-printed to produce a hydrogel (
전기전도성은 20Hz ~ 1kHz의 범위에서 frequency sweep하면서 측정하였고, 그 결과를 도 8(b)에 제시하였다. SGOB 시료의 경우에는 frequency를 높일수록 전기전도성이 증가하였고, SGOB1 내의 GO농도가 높아질수록 전기전도성이 증가하였다.Electrical conductivity was measured while frequency sweeping in the range of 20Hz ~ 1kHz, and the results are presented in FIG. 8(b). In the case of the SGOB sample, the electrical conductivity increased as the frequency increased, and the electrical conductivity increased as the GO concentration in SGOB1 increased.
도 9는 바이오잉크로 제작된 하이드로젤이 전도체로서의 기능성을 3.5V의 전압에서 LED 회로에서 확인한 결과이다. 하이드로젤은 가로, 세로, 높이 10mm x 5mm x 3mm의 크기로 프린팅 되었다. SGOB1 하이드로젤을 통하여 전기가 잘 흐르는 것을 LED 램프가 켜짐을 통해 알 수 있다. 9 is a result of confirming the functionality of a hydrogel made of bio-ink as a conductor in an LED circuit at a voltage of 3.5V. The hydrogel was printed in dimensions of 10mm x 5mm x 3mm width, length, and height. It can be seen by turning on the LED lamp that electricity flows well through the SGOB1 hydrogel.
[[ 실험예Experimental example 6] 바이오잉크로 제작된 6] Made with bio-ink 하이드로젤의Hydrogel 세포적합성 측정 Cell suitability measurement
바이오 잉크 조성물 내에 세포(Neuro2a)를 포함시킨 후, 하이드로젤 프린팅 하여 세포 증식도를 측정하기 위해 CCK-8 assay를 시행하였고, 그 결과를 도 10에 나타내었다. 세포(1x10^7 cells/mL)를 혼합하기 전 바이오잉크는 60℃에서 30분 동안 저온멸균하였다. After including cells (Neuro2a) in the bio-ink composition, the CCK-8 assay was performed to measure the degree of cell proliferation by hydrogel printing, and the results are shown in FIG. 10. Before mixing the cells (1x10^7 cells/mL), the bioink was pasteurized at 60°C for 30 minutes.
세포 프린팅 후 성장배양액(10% FBS, 1% Sodium Pyruvate, 1% Penicillin/Streptomycin, 1% non-essential amino acid가 함유된 Welgene’s High Glucose Dulbecco’s Modified Eagle’s Medium(DMEM))에서 30℃, 5% CO2에서 배양하였으며, CCK-8 용액을 4시간 처리후에 증식률을 7일간 측정하였다. After cell printing, in growth culture solution (10% FBS, 1% Sodium Pyruvate, 1% Penicillin/Streptomycin, Welgene's High Glucose Dulbecco's Modified Eagle's Medium (DMEM) containing 1% non-essential amino acid) at 30℃, 5% CO2 The culture was carried out, and the proliferation rate was measured for 7 days after treatment with the CCK-8 solution for 4 hours.
7일간 세포증식률은 그래핀 산화물이 포함되거나 포함되지 않은 그룹 모두에서 비슷한 수준으로 증가하는 것을 볼 수 있었으며, 이러한 시료 범위 내로 포함된 그래핀 산화물의 농도 범위에서는 세포 독성에 영향을 주지 않았음을 확인할 수 있었다. It was found that the cell proliferation rate for 7 days increased to a similar level in both groups with or without graphene oxide, and it was confirmed that the concentration range of graphene oxide contained within the sample range did not affect cytotoxicity. Could
도 11은 바이오 잉크 조성물 내에 세포(Neuro2a)를 포함하여 하이드로젤 프린팅 후 세포의 생존도를 Live/Dead assay로 확인한 결과를 도시한 것이다. 앞서 설명한 배양 조건과 동일하며, 7일간 하이드로젤 내에서 죽은 세포(빨간색)는 거의 발견되지 않았으며 살아있는 세포(녹색)가 점차 증가하는 것을 확인할 수 있다(D0: 배양 시작시, D1, D3, D5 및 D7은 각각 배양 기간(D1은 배양 1일 경과, D7은 배양 7일 경과를 의미함. 이하에서도 동일함)을 의미한다.11 shows the results of confirming the viability of cells after hydrogel printing including cells (Neuro2a) in a bio-ink composition by Live/Dead assay. It is the same as the culture conditions described above, and it can be seen that almost no dead cells (red) were found in the hydrogel for 7 days, and the live cells (green) gradually increased (D0: at the beginning of culture, D1, D3, D5). And D7 each mean a culture period (D1 means 1 day of culture and D7 means 7 days of culture. The same applies hereinafter).
[[ 실험예Experimental example 7] 바이오잉크로 제작된 7] Made with bio-ink 하이드로젤의Hydrogel 신경세포 Nerve cell 분화능Differentiation 확인 Confirm
하이드로젤 내에 파종된 Neuro2a 세포에서 alpha-tubulin의 발현 여부를 분화인자가 들어가지 않은 성장배양액내에서 확인하였다. 프린팅, 세포혼합, 배양조건은 앞선 실험예 6과 동일하다. The expression of alpha-tubulin in Neuro2a cells seeded in the hydrogel was confirmed in the growth culture medium without differentiation factors. Printing, cell mixing, and culture conditions were the same as in Experimental Example 6.
하이드로젤은 10㎛로 동결박절하였으며 통상적인 면역형광염색법을 시행하였다. mouse monoclonal alpha-tubulin antibody를 1차 항체 (1:150 diluted)로, goat anti-mouse FITC conjugated 2차 항체 (1:200 diluted)를 사용하였고, 결과를 도 12에 나타내었다.The hydrogel was frozen and sliced to 10 μm and subjected to conventional immunofluorescence staining. Mouse monoclonal alpha-tubulin antibody was used as the primary antibody (1:150 diluted), goat anti-mouse FITC conjugated secondary antibody (1:200 diluted) was used, and the results are shown in FIG. 12.
성장배양액 내에 그래핀 산화물이 포함된 경우가, 그렇지 않은 경우에 비해 alpha-tubulin 발현정도가 높음을 알 수 있다.It can be seen that the expression level of alpha-tubulin is higher in the case where graphene oxide is contained in the growth culture medium than in the case where it is not.
도 13에는 하이드로젤 내에 파종된 Neuro2a 세포에서 alpha-tubulin의 발현 여부를 신경세포분화인자가 들어간 분화배양액 내에서 확인한 결과를 정리하였다.FIG. 13 summarizes the results of confirming the expression of alpha-tubulin in Neuro2a cells seeded in the hydrogel in the differentiation culture solution containing the neuronal differentiation factor.
분화배양액의 조성은 1% FBS, 1% Sodium Pyruvate, 1% Non-essential amino acid, 1% Penicillin-Streptomycin, and 10 μRetinoic acid이 함유된 DMEM 이고, 상기와 같은 조건에서 배양 및 분석하였다. The composition of the differentiation culture solution was DMEM containing 1% FBS, 1% Sodium Pyruvate, 1% Non-essential amino acid, 1% Penicillin-Streptomycin, and 10 μRetinoic acid, and cultured and analyzed under the same conditions as described above.
그래핀 산화물이 포함된 경우가 그렇지 않은 경우에 비해 alpha-tubulin 발현정도가 높았으며 그 정도는 성장배양액으로 배양했을 때보다 더 향상되었다.In the case where graphene oxide was included, the level of alpha-tubulin expression was higher than in the case where it was not, and the level was improved more than that in the growth culture medium.
Claims (17)
광개시제(photo initiator);를 포함하는, 바이오 잉크 조성물.A polymer polymer in which silk fibroin (SF), a methacrylate compound, and graphene oxide (GO) are polymerized; and
Photo initiator (photo initiator); containing, bio ink composition.
SF의 아미노산 잔기에 적어도 하나 이상의 메타크릴레이트 화합물과 적어도 하나 이상의 GO가 공중합된 것을 특징으로 하는, 바이오 잉크 조성물.The method of claim 1, wherein the polymer polymer,
A bio ink composition, characterized in that at least one methacrylate compound and at least one GO are copolymerized with an amino acid residue of SF.
실크피브로인(SF) 용액에 메타크릴레이트 화합물을 투입한 후, 교반하여 개질된 실크피브로인(SB)을 제조하는 제2단계;
상기 제2단계를 통해 얻어진 개질된 실크피브로인(SB)과 그래핀 산화물(GO)을 혼합한 후, 교반하여 고분자 중합체(SGOB1)를 제조하는 제3단계;
상기 제3단계를 통해 얻어진 고분자 중합체(SGOB1)가 포함된 용액을 건조한 후, 분말화시키는 제4단계; 및
물에 상기 고분자 중합체(SGOB1) 분말과 광개시제를 혼합하는 제5단계;를 포함하는 바이오 잉크 조성물의 제조방법The first step of dissolving silk fibroin (SF) in a solvent;
A second step of preparing a modified silk fibroin (SB) by adding a methacrylate compound to the silk fibroin (SF) solution and stirring;
A third step of mixing the modified silk fibroin (SB) and graphene oxide (GO) obtained through the second step, and then stirring to prepare a polymer polymer (SGOB1);
A fourth step of drying and powdering the solution containing the polymer polymer (SGOB1) obtained through the third step; And
A method for producing a bio-ink composition comprising a fifth step of mixing the polymer polymer (SGOB1) powder and a photoinitiator in water
분쇄된 누에고치를 0.05M NaCO3 수용액에 넣고 끓인 후 세척을 통해 세리신을 제거함으로써 실크피브로인을 얻는 정련단계;를 포함하는 것을 특징으로 하는, 바이오 잉크 조성물의 제조방법. The method of claim 3, wherein the first step,
Refining step of obtaining silk fibroin by removing sericin through washing after boiling the pulverized cocoon in 0.05M NaCO 3 aqueous solution and washing. The method for producing a bio-ink composition comprising:
상기 정련단계를 거친 실크피브로인을, 9.3M 브롬화리튬(LiBr)용액에 0.05 ~ 0.35 g/ml 농도로 용해시킨 후, 60 ℃온도로 가열하는 과정을 더 포함하는 것을 특징으로 하는, 바이오 잉크 조성물의 제조방법.The method of claim 4, wherein the first step,
After dissolving the silk fibroin that has undergone the refining step at a concentration of 0.05 to 0.35 g/ml in a 9.3 M lithium bromide (LiBr) solution, the bio-ink composition further comprises a step of heating to a temperature of 60°C. Manufacturing method.
상기 제2단계의 메타크릴레이트 화합물은, 글리시딜메타크릴레이트(glycidyl methacrylate: GMA) 또는 메타크릴 무수물(methacrylic anhydride; MA)인 것을 특징으로 하는, 바이오 잉크 조성물의 제조방법. The method of claim 3,
The methacrylate compound of the second step is glycidyl methacrylate (GMA) or methacrylic anhydride (MA).
실크피브로인이 용해된 용액에 글리시딜메타크릴레이트(glycidyl methacrylate: GMA) 4~6 mL를 혼합한 후, 50 ~ 70 ℃의 온도에서 200~400rpm의 속도로 교반하되는 것을 특징으로 하는, 바이오 잉크 조성물의 제조방법.The method of claim 6, wherein the second step,
After mixing 4-6 mL of glycidyl methacrylate (GMA) in a solution in which silk fibroin is dissolved, the biotechnology is stirred at a speed of 200 to 400 rpm at a temperature of 50 to 70 °C. Method for producing an ink composition.
0.25 ~ 3.5%의 농도로 물에 분산된 후, 초음파 처리된 것을 특징으로 하는, 바이오 잉크 조성물의 제조방법.The method of claim 3, wherein the graphene oxide (GO) in the third step,
After being dispersed in water at a concentration of 0.25 to 3.5%, it characterized in that the ultrasonic treatment, the method for producing a bio ink composition.
1-ethyl-3-(3-dimethylamino propyl) carbodiimide hydrochloric acid(EDC)를 0.15~1.96g를 반응시킨 후, 상온에서 0.21~2.8g의 N-hydroxysuccinimide(NHS)을 혼합하여 반응시키고, mercaptoethanol 용액을 추가로 더 혼합하여 GO의 카르복실기가 활성화되도록 하는 것을 특징으로 하는, 바이오 잉크 조성물의 제조 방법.The method of claim 3, wherein the graphene oxide (GO) in the third step,
1-ethyl-3-(3-dimethylamino propyl) carbodiimide hydrochloric acid (EDC) was reacted with 0.15 to 1.96 g, followed by reaction with 0.21 to 2.8 g of N-hydroxysuccinimide (NHS) at room temperature, and a mercaptoethanol solution A method for producing a bio-ink composition, characterized in that the carboxyl group of GO is activated by further mixing.
상기 개질된 실크피브로인(SB)과 그래핀 산화물(GO)를 1:1의 부피비로 혼합하여 carbodiimidation 반응을 수행하는 것을 특징으로 하는, 바이오 잉크 조성물의 제조방법.The method of claim 3, wherein the third step,
A method for producing a bio-ink composition, characterized in that a carbodiimidation reaction is performed by mixing the modified silk fibroin (SB) and graphene oxide (GO) in a volume ratio of 1:1.
상기 제3단계와 제4단계 사이에, 고분자 중합체가 포함된 용액을 12 ~ 14 kDa cutoff 투석튜브에 넣은 후, 불순물을 제거하는 것을 특징으로 하는, 바이오 잉크 조성물의 제조방법.The method of claim 3,
Between the third and fourth steps, a solution containing a polymer polymer is added to a 12 to 14 kDa cutoff dialysis tube, and then impurities are removed.
상기 고분자 중합체가 포함된 용액을 -90 ~ -70℃ 온도로 동결하는 단계; 및
-90 ~ -70℃ 온도에서 40 ~ 60 시간 동안 동결건조하는 것을 특징으로 하는, 바이오 잉크 조성물의 제조방법.The method of claim 3, wherein the fourth step,
Freezing the solution containing the polymer polymer at a temperature of -90 to -70°C; And
A method for producing a bio-ink composition, characterized in that lyophilized at a temperature of -90 to -70°C for 40 to 60 hours.
벤질디메틸케탈(benzyl dimethyl ketal), 아세토페논(acetophenone), 벤조인메틸에테르(benzoin methyl ether), 디에톡시아세토페논(diethoxyacetophenone), 벤조일 포스핀 옥사이드(benzoyl phosphine oxide) 및 1-하이드록시사이클로헥실 페닐 케톤(1-hydroxycyclohexyl phenyl ketone)으로 이루어진 군 중에서 선택되는 적어도 하나 이상을 포함하는, 바이오 잉크 조성물의 제조방법.The method of claim 3, wherein the photoinitiator,
Benzyl dimethyl ketal, acetophenone, benzoin methyl ether, diethoxyacetophenone, benzoyl phosphine oxide and 1-hydroxycyclohexyl phenyl Ketone (1-hydroxycyclohexyl phenyl ketone) comprising at least one selected from the group consisting of, a method for producing a bio ink composition.
상기 실크피브로인(SF)를 대신하여 히알루론산, 콜라겐, 알지네이트, 녹말, 키토산, 젤라틴, 덱스트린, 셀룰로오스, 알긴산, 콘드로이틴 설페이트, 헤파린 및 헤파란으로 이루어진 군에서 선택된 어느 하나 이상을 사용하는 것을 특징으로 하는, 바이오 잉크의 제조방법The method according to any one of claims 3 to 14,
In place of the silk fibroin (SF), hyaluronic acid, collagen, alginate, starch, chitosan, gelatin, dextrin, cellulose, alginic acid, chondroitin sulfate, heparin, and at least one selected from the group consisting of heparan is used. , Manufacturing method of bio ink
A method of manufacturing a structure through a fused deposition modeling (FDM) method or a digital light processing (DLP) type 3D bioprinter using the bio-ink composition prepared by the method according to any one of claims 3 to 14.
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| WO2022034462A1 (en) * | 2020-08-13 | 2022-02-17 | Universidad De Los Andes | Extrudable photocrosslinkable hydrogel and method for its preparation |
| CN113527709A (en) * | 2021-08-18 | 2021-10-22 | 四川大学华西医院 | Modified tussah silk protein and 3D printing ink based on modified tussah silk protein |
| WO2023113354A1 (en) * | 2021-12-13 | 2023-06-22 | 전남대학교산학협력단 | Bioink composition kit for tubular biotissue, fabrication method therefor, method for constructing tubular biotissue, using same, and tubular biotissue constructed by same method |
| KR20240013507A (en) | 2022-07-22 | 2024-01-30 | 한림대학교 산학협력단 | A photo-curable bioink to fabricate ultra-strong, electroconductive, and biocompatible hydrogel for regenerative medicine and preparation method thereof |
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