This study was supported by National Research Foundation of South Korea: Mid-career scientist grant (2014002855), and Ministry of Industry, Trade, and Natural Resources: World Premier Material Development Program. This work is also supported by in part by Center for Nature-inspired Technology (CNiT) in KAIST Institute for NanoCentury (KINC).

모든 동물 관리 및 실험은 KAIST (한국 과학 기술원)에서 제공하는 윤리적 프로토콜 IRB (임상 시험 심사위원회)에 따라 수행된다. 1. TAPE 형성 <ol><li> 트랜스 용액의 제조 마그네틱 스터에 4 mL의 크기의 유리 병을 놓고, 교반 막대 증류수 1 ㎖를 추가한다. 바이알에 탄닌산 1 g을 넣고, 이상에서 1 시간 동안 200 rpm에서 온화하게 교반하여 물에 녹인다. TA에 완전히 용해 될 때, 혼합물은 갈색 투명해진다. </li><li> 백색 슬러리를 만들기 위해 몇 초 동안 볼 텍싱하여 이들을 혼합하여 증류수 1 ㎖ 다음에 PEG 분말 1g (4 아암 10 kDa의, 리니어, 4.6 kDa의)을 첨가하여 PEG 용액을 제조 하였다. 10 분 동안 60 ° C의 배양기에서 슬러리를 유지한다. 흰색 하나는 완전히 명확해질 때까지. 참고 : 10 kDa의 분자량을 갖는 PEG의 융점이 약 55 인- 60 ℃, 및 4 kDa의 하나는 53 - 58 °의 C. 1g에 물까지의 PEG의 고농도 / ㎖의 투명한 용액으로 얻어 질 수 있도록 용융 된 PEG는 수혼 화성된다. 맑은 PEG 용액을 고온에서 형성되면, 용액을 계속 냉각 한 후, 실온에서 안정하다. </li><li> PEG의 329 μl를 추가 (4 아암 10 kDa의) 용액 4.6 kDa의 갖는 선형 PEG의 경우 1.1 단계에서 얻어진 TA 용액 (1.2 671 μL를 위해 PEG 용액 311 μL를 추가 단계에서 제조 한 마이크로 원심 분리기 튜브에서 TA 용액) 689 μL. 조심스럽게 균일를 혼합 좁은 주걱 두 점성과 꿀 같은 솔루션을 혼합. 주의 : 두 솔루션은 매우 점성, 그래서 과학자가 느리지 만 충분히 끌어와 마이크로 피펫으로 솔루션을 전송해야합니다. </li><li> 고정 된 앵글 로터를 구비 한 원심 분리기에서 3 분 동안 12,300 × g으로 1.3로 공정에서 제조 한 혼합물을 스핀. </li><li> 조심스럽게 렘마이크로 피펫을 사용하여 가능한 한 상등액만큼 비켜하고 침착 한 생성물을 수집 :이 완전히 형성 TAPE이다. . TAPE 형성 한 후, 냉장고에서 보관 - 몇 주까지 NOTE (4 8 °의 C) : 테이프 감마선 또는 외과 적 용도로 사용하기 전에, 전자 빔 처리에 의해 살균 될 수있다. </li></ol> 테이프의 접착 강도의 측정 (2) <ol><li> 피부 조직의 모든 지방을 제거한 후, 생검 펀치로 절단하여 6mm 직경의 돼지의 피부 조직의 두 개를 준비한다. 주 : 돼지 피부 조직이 건강한 돼지 측면 피부에서 얻어진 것으로, 한국 대전에 위치한 지역 고기 시장에서 구입했다. </li><li> 각 조직의 외 측면에 상업 시아 노 아크릴 레이트 접착제를 적용하고, 금속 막대에 조직을 연결합니다. 참고 : 금속로드가 보조 핸들이 그래서를 티슈 사용직접 기계에 의해 사로 잡혔다하지 않았습니다. 따라서, 인장 기의 구성을 다음과 같은 다른 물질로 대체 될 수있다. </li><li> 티슈의 한쪽 - TAPE 방울을 적용 (6 mg의 테이프의 드롭은 약 3 임). 이어서,도 2a에 도시 한 바와 같이 이들이 부착되도록 균일 그 내측에있는 두 개의 다른 조직 사이의 조직을 사용하여 테이프를 확산. </li><li> 그 후, 수동으로 균일하게 혼합하고, 각 조직 테이프 사이의 계면을 극대화 수회 부착 조직의 양쪽 분리. </li><li> UTM을로,로드의주의 그립 각면. 접착 강도는 테이프 부착 개의 조직을 분리하는 데 필요한 힘에 의해 결정된다. 첫째, 1 분 20 N의 힘을 적용합니다. 이어서, 기계로, 1mm / min의 속도로 반대 방향으로 각로드를 당긴다. 때까지 조직을 완전히 분리된다. 주 : 데이터는 힘 - 거리로 주어질 것이다 (FD) 곡선 움직임 검출각로드. </li><li> 즉, 시편 표면적에 의해 단계 250에서 달성 된 FD 곡선에 도시 된 최대 힘 (KN)을 분할하여 테이프의 접착 강도를 계산 3.14 × (0.003 m) 2. </li><li> 물의 존재 하에서 접착 강도를 모니터링하기 위해, 두 조직 사이의 분리 영역 상에 20 μL의 물을 추가하고, 즉시 첨부. 기계로 다시 분리 테스트를 수행합니다. </li></ol> 3. 체외 저하 테스트 <ol><li> 마이크로 원심 분리 튜브의 캡 (d = 8 mm)를 잘라내, 및 W의 C로 정의 캡을 단다. </li><li> TAPE 150 mg을 가진 캡을 입력하고 0 W 총 초기 무게로 설정을 다시 모두 함께 무게. 주의 : 모자 TAPE를 너무 많이 넣지 마십시오. 이 단계 3.4에서 배양 동안 교반 처리에 의해 생성 된 PBS 완충액의 흐름에 대한 물리적 장벽을 될 수있는 테이프의 높이가 상기 캡의 상단보다 낮아야한다. </li><li&gt; 세포 배양 용 플라스크에 테이프를 포함하는 캡을 넣어 (75cm 2) 및 PBS 완충액 50 mL로 (1X, pH 7.4의) 세포 배양 플라스크에 뚜껑 테이프가 완전히 같이, PBS 완충액에 침지되도록 추가 도 3a에 도시 한 (N = 5). </li><li> 부드럽게 교반 (50 RPM)와 생리 학적 조건과 유사한 37 ℃에서 오비탈 진탕 배양기 단계 3.3에서 제조 된 세포 배양 플라스크를 인큐베이션. 주의 : 50 rpm으로 교반 상태를 유지합니다. 높은 RPM은 TAPE의 붕괴가 발생할 수 있습니다. </li><li> 각 시점에서, 세포 배양 플라스크에서 테이프로 뚜껑을 한 후, 질소 가스를 불어 건조한다. 남은 테이프를 포함하는 캡을 단다. W t의 각 시점에서의 무게를 설정한다. 다시 신선한 PBS를 교체하고, 각 시점에서 W (T)를 측정 한 후이를 다시 흔들. </li><li> 상대적인 잔류 중량 (%)은 다음 식을 계산한다. 상대 남은 중량 (%) = (W의 t </suB&gt; - W의 C) / (W 0 - W C) × 100 % </li></ol> TAPE 4. 지혈 능력 주 : 모든 동물 실험은 보건 복지부 한국 정부가 제공하는 가이드 라인과 윤리 프로토콜에 따라 수행되어야한다. <ol><li> REF (24)에 기재된 바와 같이 생체 내 지혈 능력을 평가하기 위해, 마우스를 출혈 간 모델을 검토. </li><li> (그룹 당 n = 5) tiletamine-zolazepam (33.333 ㎎ / ㎏)과 자일 라진 (7.773 ㎎ / ㎏)의 복강 내 주사와 - 십오 마우스 (남성 35g, 정상 ICR 마우스 6 주, 30) 마취. 적절한 마취를 확인하려면, 동물의 발을 부드럽게 꼬집어 어떤 운동이 동물이 수술을하기에 충분히 마취 있음을 나타냅니다 등, 발을 인출하는 등의 움직임을 관찰한다. </li><li> 동물의 눈의 건조를 방지하려면 아래에 충분히 동안 눈에 수의사 연고를 적용하려면esthesia. 중간 선 복부 절개를 통해 간을 노출하고 출혈을 유도하기 위해 18 G 바늘로 간을 찌르. </li><li> 멸균 거즈로 흐르는 혈액을 제거하고 출혈 사이트에 즉시 TAPE 또는 피브린 접착제 (양성 대조군) 100 ㎕를 넣어. 참고 : 더 이상 봉합으로 인해 상처 조직에 대한 고도의 혈액에 강한 접착 특성에 TAPE를 적용한 후 필요하지 않습니다. 음성 대조군의 경우, 치료는 출혈의 사이트에서 발생하지 않는다. </li><li> 각각의 경우, 파손 부위로부터 혈액을 수집 간 아래 공지 질량 여과지 넣어. 새로운 일 4 회 매 30 초 (즉., 2 분)으로 용지를 교체합니다. </li><li> 매 30 초마다 수집 된 여과지에 흡수 된 혈액의 질량을 측정한다. 동물 실험 후, CO 2 질식의 안락사를 통해 쥐를 희생. </li></ol>

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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen-bonded+multilayer+of+pH-responsive+polymeric+micelles+with+tannic+acid+for+surface+drug+delivery.">Hydrogen-bonded multilayer of pH-responsive polymeric micelles with tannic acid for surface drug delivery.</a> Chem Commun. 45 (28), 4194-4196 (2009).</li><li>Murakami, Y., Yokoyama, M., Nishida, H., Tomizawa, Y., Kurosawa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+hemostasis+model+for+the+quantitative+evaluation+of+hydrogel-based+local+hemostatic+biomaterials+on+tissue+surface.">A simple hemostasis model for the quantitative evaluation of hydrogel-based local hemostatic biomaterials on tissue surface.</a> Colloids Surf B Biointerfaces. 65 (2), 186-189 (2008).</li><li>Kim, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TAPE:+A+Medical+Adhesive+Inspired+by+a+Ubiquitous+Compound+in+Plants.">TAPE: A Medical Adhesive Inspired by a Ubiquitous Compound in Plants.</a> Adv Funct Mater. 25 (16), 2402-2410 (2015).</li><li>Suzuki, S., Ikada, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesion+of+cells+and+tissues+to+bioabsorbable+polymeric+materials:+scaffolds,+surgical+tissue+adhesives+and+anti-adhesive+materials.">Adhesion of cells and tissues to bioabsorbable polymeric materials: scaffolds, surgical tissue adhesives and anti-adhesive materials.</a> J Adhes. Sci. Technol. 24 (13), 2059-2077 (2010).</li><li>Lomova, M. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multilayer+Capsules+of+Bovine+Serum+Albumin+and+Tannic+Acid+for+Controlled+Release+by+Enzymatic+Degradation.">Multilayer Capsules of Bovine Serum Albumin and Tannic Acid for Controlled Release by Enzymatic Degradation.</a> ACS Appl Mater Interfaces. 7 (22), 11732-11740 (2015).</li><li>Shin, M., Ryu, J. H., Park, J. P., Kim, K., Yang, J. W., Lee, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA/Tannic+Acid+Hybrid+Gel+Exhibiting+Biodegradability,+Extensibility,+Tissue+Adhesiveness,+and+Hemostatic+Ability.">DNA/Tannic Acid Hybrid Gel Exhibiting Biodegradability, Extensibility, Tissue Adhesiveness, and Hemostatic Ability.</a> Adv Funct Mater. 25 (8), 1270-1278 (2015).</li><li>Kozlovskaya, V., Kharlampieva, E., Drachuk, I., Chenga, D., Tsukruk, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responsive+microcapsule+reactors+based+on+hydrogen-bonded+tannic+acid+layer-by-layer+assemblies.">Responsive microcapsule reactors based on hydrogen-bonded tannic acid layer-by-layer assemblies.</a> Soft Matter. 6 (15), 3596-3608 (2010).</li><li>Oh, D. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid,+efficient,+and+facile+solution+for+dental+hypersensitivity:+The+tannin-iron+complex.">A rapid, efficient, and facile solution for dental hypersensitivity: The tannin-iron complex.</a> Sci Rep. 5, 10884 (2015).</li></ol>

The authors have nothing to disclose.

우리는 식물 유래 폴리 페놀 화합물, TA의 방수 분자 상호 작용에 의해 영감을 지혈 접착제라는 TAPE의 완전히 새로운 클래스를 개발했다. TA 크게 인해 산화 방지제, 항균 항 돌연변이 및 항암 특성으로 주목을 받고 대표 가수 분해성 탄닌이다. 그것은 더 화학 합성 과정없이 다음, 원심 분리하여 두 수용액의 단지 한 단계 혼합 같이 테이프를 제조하는 과정은 매우 간단하고 확...

과거 10 년의 노력으로 인해 수술 적 치료시 사용과 낮은 조직 침입에 자신의 편의에 생분해 성 / 흡수성 접착제와 상처를 닫 현재 수술 봉합 및 스테이플을 교체되었습니다. 시판 조직 접착제는 네 가지 유형으로 분류된다 : (1) 시아 노 아크릴 레이트 유도체 (1) (2) 2,3- 트롬빈에 의해 중합체를 피브린, 피브리노겐으로부터 효소 적 전환에 의해 형성된 피브린 접착제를, (3) 등의 화학적 또는 물리적으로 같은 단백질 계 재료 가교 알부민 및 / 또는 젤라틴 4.5, (4) 합성 폴리머 계 것 6. 그들은 많은 임상 용도에 사용되어 왔지만, 모두 접착제는 자신 고유의 단점들이 널리 사용 장애물이 될 수있는 단점이있다. 시아 노 아크릴 레이트 계 접착제는 조직에 대한 높은 접착 강도를 보여 주지만, 이들 독성 부산물 등의 분해 동안 형성 및 시아 노 포름 종종 부호 원인염증 반응 (7)의 ificant도. 젤라틴 기반의 접착제 8 소, 닭, 돼지, 물고기를 포함하여 피브린 접착제과 동물에 대한 인간의 혈장 : 피브린 접착제 및 알부민 또는 젤라틴 기반 물질은 동물의 소스에서 바이러스와 같은 감염 구성 요소의 전송에 대한 안전 문제가 있습니다. 약간의 합성수지 계 접착제 연방 의약품 안전청 (FDA)에 의해 승인되었지만, 합성 중합체로 만들어진 대부분의 접착제는 제조 공정 단계를 최소화하고 생체 적합성 (9)을 달성하는데 어려움을 계속한다. 가장 중요한 것은, 모든 접착제는 물티슈 10 가난한 기계 및 접착 강도 고통. 최근 해양 홍합 11-13, 도마뱀 붙이 (14), 홍합 15 도마뱀, 그리고 endoparasitic 웜 (16)에 의해 영감을 생체 모방 조직 접착제는 그들의 조정 가능한 기계적으로 현재 의료 접착제에 관한 유망한 대안을 신흥되었습니다생체 적합성과 접착 특성. 그들은 상용 제품 (17)되기 전에 그러나, 오늘날까지 여전히 해결해야 할 문제가 있습니다. 여기서는, 식물 유래 접착 분자, 탄닌산 (TA), 및 생체 불활성 중합체 폴리 (에틸렌 글리콜) (PEG)의 분자간 수소 결합에 의해 제조 TAPE라는 의료용 접착제 완전히 새로운 유형을보고 그 이름에서 알 수 있듯이. TA는 보편적으로 식물의 2 차 대사 중에 발견 대표 가수 분해성 탄닌이다. 또한, 항 돌연변이 및 항암 성질 인하여 항산화에 많은 주목을 받고 있으며, 폴리 (N의 이소 프로필 아크릴 아미드) (PNIPAM) 및 폴리 (N만큼 중합체와 초분자 상호 작용에 참여하는 것으로 나타났다 - 비닐 피 롤리 돈) (PVPON), 계층별로 레이어 (LBL) 영화 18 ~ 20 약물 방출 마이크로 캡슐 21 ~ 23을 형성한다. 본 연구에서는 TA 효율적인 역할을 할 수 있음을 발견방수 접착제 관능기 테이프, 의료 접착제를 형성한다. TA 간단한 혼합하여, 비 오염 중합체 PEG를 갖는 초분자 접착제하게 2.5 배 상업적 피브린 접착제에 비하여 증가 된 접착 강도를,이 밀착성에도 물의 존재 하에서, 착탈의 최대 20주기 동안 유지시켰다 . 그 지혈 능력은 생체 내에서 간 출혈 모델을 테스트하고 몇 초 내에 출혈을 멈추게하는 좋은 지혈 능력을 보여 주었다. TAPE 바이오 영감 접근 방식과 현재 문제의 단점을 해결에 새로운 통찰력을 공개 할 수있는 최초의 식물 유래 접착제로서 관련 분야에서의 중요한 의미를 갖습니다. 우리는 또한 간단한 제조 방법에있어서, 확장 성, 가변 생분해 속도 등의 뮤코 접착제, 약물 방출 패치, 상처 치료 드레싱 등과 같은 의약 다양한 용도 TAPE의 광범위한 사용을 기대할뿐만 아니라 높은 습기에 강한 ADHES이온 특성.

<table>
	<tbody>
		<tr>
			<td>Tannic acid</td>
			<td>Sigma-aldrich</td>
			<td>403040</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly(ethylene oxide), 4-arm, hydroxy terminated</td>
			<td>Aldrich</td>
			<td>565709</td>
			<td>Averge Mn ~10,000</td>
		</tr>
		<tr>
			<td>Poly(ethylene glycol)</td>
			<td>Aldrich</td>
			<td>373001</td>
			<td>Average Mn 4,600</td>
		</tr>
		<tr>
			<td>Biopsy punch</td>
			<td>Miltex</td>
			<td>33-36</td>
			<td>Diameter = 6 mm</td>
		</tr>
		<tr>
			<td>Aron Alpha&#174;</td>
			<td>Toagosei Co., Ltd.</td>
			<td></td>
			<td>Instant glue</td>
		</tr>
		<tr>
			<td>Universal testing machine (UTM)</td>
			<td>Instron</td>
			<td>5583</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes</td>
			<td>SPL life science</td>
			<td>60015</td>
			<td>1.5 mL</td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>SPL life science</td>
			<td>10090</td>
			<td>90 x 15 mm</td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic</td>
			<td>Sigma</td>
			<td>S5011</td>
			<td>1x PBS ingredient</td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic</td>
			<td>Sigma</td>
			<td>S5136</td>
			<td>1x PBS ingredient</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Duchefa biochemie</td>
			<td>S0520.5000</td>
			<td>1x PBS ingredient</td>
		</tr>
		<tr>
			<td>Incubating shaker</td>
			<td>Lab companion</td>
			<td>SIF6000R</td>
			<td></td>
		</tr>
		<tr>
			<td>ICR mice</td>
			<td>Orient bio</td>
			<td>Normal ICR mouse</td>
			<td>6 weeks, 30-35 g, male</td>
		</tr>
		<tr>
			<td>Tiletamine-zolazepam (Zoletil 50)</td>
			<td>Virbac</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zylazine (Rompun)</td>
			<td>Bayer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PrecisionGlideTM needle (18 G)</td>
			<td>BD</td>
			<td>302032</td>
			<td>18 G</td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>Whatman</td>
			<td>1001 125</td>
			<td>Diameter = 125 mm</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Bemis Flexible Pakaging</td>
			<td>PM996</td>
			<td></td>
		</tr>
	</tbody>
</table>

tape: a biodegradable hemostatic glue inspired by a ubiquitous compound in plants for surgical application

이 동영상은 상업 조직 접착제보다 효과적인 지혈 능력과 큰 방수 접착 강도가 생분해 성 수술 용 접착제를 제조하기위한 간단한 프로토콜을 설명합니다. 의료 접착제로 인해 최소한의 침입과 사용에서의 편리 성을 봉합하고 스테이플에 대한 잠재적 인 대안 도구로 큰 관심을 받고있다. 생체 기반 물질의 경우와 같은 피브린 접착제 및 시아 노 아크릴 레이트 계 재료 등의 시판 사람들은 대부분들은 유기 분자 나 복잡한 단백질 정제 방법의 화학 합성의 일련의 요구를 포함한 조직 접착제를 개발하기위한 몇 가지 프로토콜이 있지만 (즉, 피브린 접착제). 또한, 외과 용 접착제의 개발이 생분해 성을 유지하기 때문에 인체의 습윤 환경에서 양호한 성능을 달성하는데 어려움이 여전히 도전하면서 높은 접착 성을 나타내는. 우리는을 준비하는 새로운 방법을 설명식물 유래 습식 강한 접착 분자의 물리적 혼합 한 후, 형성 물 혼화 초분자 집합체의 중량을 기반으로 분리하여, TAPE라고도 의료 접착제, T는 CID (TA), 및 잘 알려진 annic 생체 고분자, 폴리 (에틸렌) 글리콜 (PEG). 우리의 접근 방법으로, TAPE는 물의 존재 상용 피브린 접착제보다 2.5 배 높은 접착 강도를 나타낸다. 또한, TAPE는 생리 학적 조건에서 생분해 성 및 조직의 출혈에 대하여 강력한 지혈 접착제로서 사용될 수있다. 우리는 뮤코 접착 중합체, 약물 저장소 등과 같은 의료 설정 및 약물 전달 다양한 애플리케이션에서 테이프의 광범위한 사용을 예상한다.

1 부피비 (도 1A) TAPE는 TA (1 ㎍ / ml의 증류수)과 2 PEG (1 ㎍ / ml의 증류수)을 포함하는 두 수용액의 혼합물을 원심 분리 한 후 정착 초분자 집합체이다. 혼합 비율이 높은 접착 강도를 달성하는 데 중요한 요소이다 테이프 2로 형성하는 경우 : 1의 혼합비, TA, 25 단위의 수산기 (-OH)의 20 개는 최대 높은 분자간 수소 결합 형성의 결과로, PEG의 각 에테르 기 (-O-)와 상호 ...

Watch this Scientific Journal Video about TAPE: A Biodegradable Hemostatic Glue Inspired by a Ubiquitous Compound in Plants for Surgical Application at JoVE.com

TAPE: A Biodegradable Hemostatic Glue Inspired by a Ubiquitous Compound in Plants for Surgical Application

우리는 효과적인 지혈 기능을 갖는 의료용 생분해 성 접착제를 제조하는 가장 간단한 프로토콜을 설명한다. 테이프 탄닌산 혼합 한 물 - 비혼 화성 초분자 집합체 인 상업용 피브린 접착제에 비해 2.5 배 더 내수성 접착 성을 수득하는 유비쿼터스 화합물은 식물에서 발견 한 폴리 (에틸렌) 글리콜.

TAPE : 외과 응용 프로그램에 대한 식물의 유비쿼터스 화합물에서 영감을 생분해 성 지혈 접착제

This video describes the simplest protocol for preparing biodegradable surgical glue that has an effective hemostatic ability and greater water-resistant ...

tape-biodegradable-hemostatic-glue-inspired-ubiquitous-compound

Research

JoVE Journal

Bioengineering

14.0K 조회수.  Korea Advanced Institute of Science and Technology (KAIST).  우리는 효과적인 지혈 기능을 갖는 의료용 생분해 성 접착제를 제조하는 가장 간단한 프로토콜을 설명한다. 테이프 탄닌산 혼합 한 물 - 비혼 화성 초분자 집합체 인 상업용 피브린 접착제에 비해 2.5 배 더 내수성 접착 성을 수득하는 유비쿼터스 화합물은 식물에서 발견 한 폴리 (에틸렌) 글리콜. 

비디오: TAPE: A Biodegradable Hemostatic Glue Inspired by a Ubiquitous Compound in Plants for Surgical Application

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

단백질과 세포 교도소에 대한 2 차원 문양의 기판 만들기

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

연구

생체공학

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

타겟 안정 동위 원소 희석 액체 크로마 토그래피 - 질량 분광법 분석을위한 세포 지질 추출

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

Constructing a Collagen Hydrogel for the Delivery of Stem Cell-loaded Chitosan Microspheres

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively for tissue regeneration, a number of variables must be taken into account. These variables include: the total volume and surface area of the implantation site, the mechanical properties of the tissue and the tissue microenvironment, which includes the amount of vascularization and the components of the extracellular matrix. Therefore, the materials being used to deliver these cells must be biocompatible with a defined chemical composition while maintaining a mechanical strength that mimics the host tissue. These materials must also be permeable to oxygen and nutrients to provide a favorable microenvironment for cells to attach and proliferate. Chitosan, a cationic polysaccharide with excellent biocompatibility, can be easily chemically modified and has a high affinity to bind with in vivo macromolecules4-5. Chitosan mimics the glycosaminoglycan portion of the extracellular matrix, enabling it to function as a substrate for cell adhesion, migration and proliferation. In this study we utilize chitosan in the form of microspheres to deliver adipose-derived stem cells (ASC) into a collagen based three-dimensional scaffold6. An ideal cell-to-microsphere ratio was determined with respect to incubation time and cell density to achieve maximum number of cells that could be loaded. Once ASC are seeded onto the chitosan microspheres (CSM), they are embedded in a collagen scaffold and can be maintained in culture for extended periods. In summary, this study provides a method to precisely deliver stem cells within a three dimensional biomaterial scaffold.

A major hurdle in current stem cell therapies is determining the most effective method to deliver these cells to host tissues. Here, we describe a chitosan-based delivery method that is efficient and simple in approach, while allowing adipose-derived stem cells to maintain their multipotency.

줄기 세포 - 로드된 키토산 Microspheres의 배달을 위해 콜라겐 히드로겔을 구축

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively ...

Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human gut-on-a-chip microphysiological device. We recapitulate the intestinal lumen-capillary tissue interface in a microfluidic device, where physiological mechanical deformations and fluid shear flow are constantly applied to mimic peristalsis. In the lumen microchannel, human intestinal epithelial Caco-2 cells are cultured to form a &#39;germ-free&#39; villus epithelium and regenerate small intestinal villi. Pre-cultured microbial cells are inoculated into the lumen side to establish a host-microbe ecosystem. After microbial cells adhere to the apical surface of the villi, fluid flow and mechanical deformations are resumed to produce a steady-state microenvironment in which fresh culture medium is constantly supplied and unbound bacteria (as well as bacterial wastes) are continuously removed. After extended co-culture from days to weeks, multiple microcolonies are found to be randomly located between the villi, and both microbial and epithelial cells remain viable and functional for at least one week in culture. Our co-culture protocol can be adapted to provide a versatile platform for other host-microbiome ecosystems that can be found in various human organs, which may facilitate in vitro study of the role of human microbiome in orchestrating health and disease.

We describe an in vitro protocol to co-culture gut microbiome and intestinal villi for an extended period using a human gut-on-a-chip microphysiological system.

에 Microengineered 인간의 장내 융모와 생활 마이크로 바이 옴의 공동 문화 창자 - 온 - 어 - 칩 미세 유체 장치

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human ...

Large-area Scanning Probe Nanolithography Facilitated by Automated Alignment and Its Application to Substrate Fabrication for Cell Culture Studies

Scanning probe microscopy has enabled the creation of a variety of methods for the constructive (&#39;additive&#39;) top-down fabrication of nanometer-scale features. Historically, a major drawback of scanning probe lithography has been the intrinsically low throughput of single probe systems. This has been tackled by the use of arrays of multiple probes to enable increased nanolithography throughput. In order to implement such parallelized nanolithography, the accurate alignment of probe arrays with the substrate surface is vital, so that all probes make contact with the surface simultaneously when lithographic patterning begins. This protocol describes the utilization of polymer pen lithography to produce nanometer-scale features over centimeter-sized areas, facilitated by the use of an algorithm for the rapid, accurate, and automated alignment of probe arrays. Here, nanolithography of thiols on gold substrates demonstrates the generation of features with high uniformity. These patterns are then functionalized with fibronectin for use in the context of surface-directed cell morphology studies.

Here we present a protocol for wide-area scanning probe nanolithography enabled by the iterative alignment of probe arrays, as well as the utilization of lithographic patterns for cell-surface interaction studies.

큰 지역 스캐닝 프로브 나노리 자동된 맞춤 및 셀 문화 연구에 대 한 기판 제작에의 응용에 의해 촉진

Scanning probe microscopy has enabled the creation of a variety of methods for the constructive (&#39;additive&#39;) top-down fabrication of ...

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.

Vivo에서 부분 간 엔지니어링을에서 위한 쥐를 기초로 새로운 수술 기법

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver ...

Scalable Fabrication of Stretchable, Dual Channel, Microfluidic Organ Chips

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human patients. Human Organ-on-a-Chip (Organ Chip) microfluidic cell culture devices, which provide an experimental in vitro platform to assess efficacy, toxicity, and pharmacokinetic (PK) profiles in humans, may be better predictors of therapeutic efficacy and safety in the clinic compared to animal studies. These devices may be used to model the function of virtually any organ type and can be fluidically linked through common endothelium-lined microchannels to perform in vitro studies on human organ-level and whole body-level physiology without having to conduct experiments on people. These Organ Chips consist of two perfused microfluidic channels separated by a permeable elastomeric membrane with organ-specific parenchymal cells on one side and microvascular endothelium on the other, which can be cyclically stretched to provide organ-specific mechanical cues (e.g., breathing motions in lung). This protocol details the fabrication of flexible, dual channel, Organ Chips through casting of parts using 3D printed molds, enabling combination of multiple casting and post-processing steps. Porous poly (dimethyl siloxane) (PDMS) membranes are cast with micrometer sized through-holes using silicon pillar arrays under compression. Fabrication and assembly of Organ Chips involves equipment and steps that can be implemented outside of a traditional cleanroom. This protocol provides researchers with access to Organ Chip technology for in vitro organ- and body-level studies in drug discovery, safety and efficacy testing, as well as mechanistic studies of fundamental biological processes.

Here, we present a protocol that describes the fabrication of stretchable, dual channel, organ chip microfluidic cell culture devices for recapitulating organ-level functionality in vitro.

Stretchable, 듀얼 채널, 미세 기관 칩의 확장 가능한 제작

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human ...

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene expression regulation are commonly disrupted in many diseases, a locus-specific, endogenous, and reversible method to study and control these mechanisms of action has been lacking. To address this issue, we have developed and characterized novel gene-regulating bifunctional molecules. One component of the bifunctional molecule binds to a DNA-protein anchor so that it will be recruited to an allele-specific locus. The other component engages endogenous cellular chromatin-modifying machinery, recruiting these proteins to a gene of interest. These small molecules, called chemical epigenetic modifiers (CEMs), are capable of controlling gene expression and the chromatin environment in a dose-dependent and reversible manner. Here, we detail a CEM approach and its application to decrease gene expression and histone tail acetylation at a Green Fluorescent Protein (GFP) reporter located at the Oct4 locus in mouse embryonic stem cells (mESCs). We characterize the lead CEM (CEM23) using fluorescent microscopy, flow cytometry, and chromatin immunoprecipitation (ChIP), followed by a quantitative polymerase chain reaction (qPCR). While the power of this system is demonstrated at the Oct4 locus, conceptually, the CEM technology is modular and can be applied in other cell types and at other genomic loci.

Regulation of the chromatin environment is an essential process required for proper gene expression. Here, we describe a method for controlling gene expression through the recruitment of chromatin-modifying machinery in a gene-specific and reversible manner.

세포질 기계 화학 Epigenetic 한정자를 리디렉션하여 억압 유전자 전사

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

나노 제형에도 Limulus Amoebocyte Lysate (LAL) 분석 실험을 사용 하 여

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Cell-free Protein Expression Using the Rapidly Growing Bacterium Vibrio natriegens

The marine bacterium Vibrio natriegens has garnered considerable attention as an emerging microbial host for biotechnology due to its fast growth rate. A general protocol is described for the preparation of V. natriegens crude cell extracts using common laboratory equipment. This high yielding protocol has been specifically optimized for user accessibility and reduced cost. Cell-free protein synthesis (CFPS) can be carried out in small scale 10 &#956;L batch reactions in either a 96- or 384-well format and reproducibly yields concentrations of &#62; 260 &#956;g/mL super folder GFP (sfGFP) within 3 h. Overall, crude cell extract preparation and CFPS can be achieved in 1&#8722;2 full days by a single user. This protocol can be easily integrated into existing protein synthesis pipelines to facilitate advances in bio-production and synthetic biology applications.

Cell-free Protein Expression Using the Rapidly Growing Bacterium Vibrio natriegens

Cell-free expression systems are powerful and cost-efficient tools for the high-throughput synthesis and screening of important proteins. Here, we describe the preparation of cell-free protein expression system using Vibrio natriegens for the rapid protein production using plasmid DNA, linear DNA, and mRNA template.

셀 무료 단백질 식을 사용 하 여 빠르게 성장 박테리아 비 브리 오 natriegens

The marine bacterium Vibrio natriegens has garnered considerable attention as an emerging microbial host for biotechnology due to its fast growth rate. A ...