저자는 생체 모방 공학 및 Bioinspired 공학에 대한 GW 센터 (코 브레)에서 NSF 부여 CBET-0909678 및 자금 지원을 인정합니다. 우리의 지원을위한 실험실 씨 마티유 Barraja에 도움 씨 크리스토퍼 Popma 씨 리앤 Penna 씨 섀넌 칼라 씨 Shadman 후세인 씨 모하메드 R. Najjari 및 양 제시카 Hinke을 학생들에게 감사 CAD 도면.

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관심 없음 충돌 선언하지 않습니다.

이 논문에 제시된 프로토콜은 입자 영상 속도계 기술 (PIV)과 일관된 구조 검출 방법, 즉., 연속 웨이블릿 변환을 사용하여 고 충실도 실험 데이터의 취득을 설명합니다 <img alt="식 (1)" src="https://neuron2.jove.com/files/ftp_upload/51288/51288eq1.jpg" /> , 소용돌이 및 전단 지배 흐름의 식별에 적합합니다. 이상화 &quot;유형 IV&quot;골절의 존재 생리 유입에서 실험 데이터의 분석 밝혀 그 단순한 유체 다이내믹 이론에...

보조 유동 구조는 만곡 된 파이프 및 채널과 같은 곡률 내부 유동 형상에서 발생하는 선회 흐름 패턴이다. 이러한 선회 구조 인한 원심력, 역 압력 구배 유입 특성의 결합 작용으로 발생한다. . 3 차 유동 모폴로지 크게 박동에 의해 영향 - 일반적으로, 보조 유동 구조 및 진동형 유입 조건에서 대칭 Dean- 및 라인 씨 형 와류가 1 정상 유입 하에서 대칭 딘 형 와류 같은 만곡 관의 평면 단면에 나타나는 타악기의 여러 고조파, 생리 유입 조건. 이러한 구조는 1 유동 비 생리적 (정상 및 진동)에 비해 현저하게 다른 크기 강도 형상 특성을 확보 -. 6 동맥 죽상 경화 병변의 발달 영역에 고주파 전단 진동의 존재에 의해 영향을 낮은 평균 전단 (27) (28)를 경험 </suP&gt;. 보조 유동 구조는 질병 예컨대 아테롬성 동맥 경화증 및 가능의 진행에 영향 인해 벽 전단 응력과 혈액 유래 입자의 노출 시간을 변경함으로써 박동성 혈류 내피 반응을 매개 할 수있다. 죽상 경화증에 대한 일반적인 치료는 폐색 병변 동맥 협착에 기인하는 합병증은 스텐트 이식이다. 스텐트 골절은 또한 의료 등의 스텐트 내 재 협착과 같은 합병증 (ISR), 스텐트 혈전증과 동맥류 형성 9로 이어질 이식 된 스텐트의 구조적인 실패입니다 -. 13 스텐트 골절 다양한 실패 &quot;유형 I-에-IV&quot;로 분류되어, 특징 &quot;유형 IV&quot;가 가장 높은 임상 심각도를 특징 및 스텐트 조각 (14)의 선형 ​​변위와 함께 스텐트 스트럿의 전체 횡 골절로 정의된다. 본 연구에서 제시된 프로토콜은 experimenta 설명곡선 동맥 모델의 이상화 된 &quot;타입 IV&quot;스텐트 골절 하류 보조 유동 구조의 시각화 리터 방법. 제안 된 프로토콜은 다음과 같은 네 가지 필수 기능을 가지고 있습니다 : 설계 및 실험실 규모 스텐트 모델 제작 : 스텐트의 기하학적 설명 니티놀 와이어 (29) (니켈 및 티타늄의 합금)을 사용하여 자체 팽창 나선 (스프링 또는 나선) 세트 얽혀 연관 될 수있다. 스텐트와 지주 지름의 길이는 임상 주입 5 중에 발생한 동맥 병변의 길이 규모에 좌우된다. 파라 메트릭 지주 직경의 변화와 권선 (또는 피치)의 상승은 다양한 기하학적 구성의 스텐트에 연결됩니다. 3D 인쇄를 위해 선택된 스텐트 설계 파라미터의 요약은 표 1에 제시되어있다. 혈액 아날로그 작동 유체의 제조는 유사한만곡 동맥 검사부 광학 액세스 속도 비 침습성 측정을 위해 요구된다 : 혈액의 동점도 시험 부의 굴절률. 따라서, 인간의 혈액을 일치하는 이상적인 굴절률 혈관 모델 인덱스 동적 점도와 함께 작동 유체 뉴턴 혈액 모방 16 정확한 혈류 측정을 얻기 위해 사용된다 -. (18) (30)를 본 연구에 사용 된 작동 유체는보고 독일어 등으로. (2006), (부피 기준) 79 % 포화 수성 요오드화 나트륨 (NaI를), 20 % 순수 글리세롤, 1 %의 물 (16)로 구성된 것이다. 두 개의 구성 요소의 2 차원 입자 화상 속도계 (2C-2D PIV)를 사용하여 코 히어 런트 보조 유동 구조의 검출을위한 실험 구성은 : 실험 하류 다양한 평면의 단면 위치에서의 위상 평균 이차 유동 속도 데이터를 수집하도록 설계되었다 STR의 조합aight 및 구현 만곡 스텐트 부분 이상화 된 &quot;타입 IV&quot;스텐트 골절 5, 6, 9, 14. 입자상 속도계 (PIV) 기술을 이용하여 이차 유속 필드의 취득에 관한 프로토콜 단계로 이루어지는 PIV 시스템을 포함 레이저 (광 시트) 소스 초점 흐름 특별한 상호 상관 전하 결합 소자 (CCD 센서 또는 카메라)과 트레이서 입자의 영역을 조명하기위한 광학 장치의 Δt (짧은 시간 간격 내에서 광 시트에 의해 조명 될 32, 31) 표 4를 참조하십시오. 프로토콜의 단계에서는 가정 다음 우선, 2 화면 단일 노출 기록하여 화상을 평가하는 이성, 이차원 (2C-2D) PIV 시스템의 교정, 실험 장치. 둘째, 2C-2D PIV 시스템은 각각의 기록 동안에 획득 개의 영상 프레임 사이의 상호 상관을 수행하여 추적 된 입자의 평균 변위를 계산한다. BRPIV 사양 및 이미지 수집 소프트웨어의 IEF 요약 재료 및 장비 표에 표시됩니다. 셋째, 레이저를 작동하는 데 필요한 모든 안전 예방 조치는 호스트 기관에 의해 제공되는 가이드 라인에 따라 훈련 기관 담당자가 따른다. 저자는 참고 문헌을 제시한다. , 에어로 하이드로 미세 유체 역학 상관 피크 검출 변위 추정 물질 추적 입자의 밀도, 측정 잡음 및 정확도의 구현 기능 및 PIV 기술의 응용에 대한 전체적인 이해를 위해 31, 32. 또한, 레이저 및 카메라가 PIV 데이터 취득 시스템 (도 3a) 및 데이터 처리 소프트웨어에 의해 제어 될 수 있다는 점에 유의하라. 데이터 수집 및 후 처리 히어 런트 검출 구조의 경우 : 2C-2D PIV를 사용하여 위상 차의 평균 유속의 측정은 이하의 프로토콜 정보를 이용하여 생성 하였다. 후 처리<html> 연속 웨이블렛 변환을 상기 데이터의 ING는 다음과 같은 세 가지 방법을 이용하여 간섭 보조 유동 구조 검출을 포함 <img alt="식 (1)" src="/files/ftp_upload/51288/51288eq1.jpg" /> 5, 6, 19-24, 26. 저자는 속도 구배 텐서 본질적으로하는 3 × 3 행렬을 참고 <img alt="식 (2)" src="/files/ftp_upload/51288/51288eq2.jpg" /> . 이 프로토콜 (2C-2D PIV 기술)에서 이차원 실험 측정치를 획득하는 방법을 제시한다. 따라서, 속도 구배 텐서에 대한 모든 실험적인 접근이 방법을 사용하여 달성 할 수 없습니다. 각 화소의 속도 구배 텐서 <img alt="식 (3)" src="/files/ftp_upload/51288/51288eq3.jpg" /> PIV 이미지 <img alt="식 (4)" src="/files/ftp_upload/51288/51288eq4.jpg" /> 2 × 2 행렬이어야한다, <img alt="식 (5)" src="/files/ftp_upload/51288/51288eq5.jpg" /> . 는 z 성분 와도quation 6 &quot;SRC =&quot;/ 파일 / ftp_upload / 51288 / 51288eq6.jpg &quot;&gt; 각 픽셀에 대한 / <img alt="식 (7)" src="/files/ftp_upload/51288/51288eq7.jpg" /> 속도 구배 텐서의 반대 칭 부분을 사용하여 계산된다 <img alt="식 (8)" src="/files/ftp_upload/51288/51288eq8.jpg" /> . 그 결과 와도의 2 차원 배열이됩니다 <img alt="식 (9)" src="/files/ftp_upload/51288/51288eq9.jpg" /> 그는 등고선 플롯으로 시각화 할 수 있습니다. 저자는 강력하게 심판을 제안한다. 소용돌이 발산, 변형율 및 코히 런트 검출 구조의 기술 향상을 향한 속도 구배 텐서에 웅변 토의 실험 액세스 25. 또한 저자들은 참조를 상기 간섭 구조 검출 방법 간의 상호 관계를 탐구하고 제안하지 않는다. 그 주제에 대한 종합 토론 23, 24. 프로토콜의 단계의 초점은 보조 흐름 (와류)의 양적 식별이다 일(또한 일관된 구조라고도 함) ructures. 코 히어 런트 검출 구조 즉 세 가지 방법., <img alt="식 (10)" src="/files/ftp_upload/51288/51288eq10.jpg" /> 그리고 잔물결이 와도을 변환 <img alt="식 (11)" src="/files/ftp_upload/51288/51288eq11.jpg" /> 이상화 된 &quot;타입 IV&quot;스텐트 골절 하류 보조 유동 구조의 멀티 - 스케일 멀티 강도 발생의 검출을 향해 속도 필드 데이터에 적용된다. 그만큼 <img alt="식 (12)" src="/files/ftp_upload/51288/51288eq12.jpg" /> 와도 텐서의 유클리드 놈이 균주 19, 23, 속도 24 국지적 속도 구배 행렬은 대칭 적 (스트레인 비율)과 항 - 대칭 (회전) 부분으로 분해되는 것을 지배하는 경우, 공간 영역으로서 와류를 정의한다. 변형율 행렬의 고유 값 계산된다; <img alt="식 (13)" src="/files/ftp_upload/51288/51288eq13.jpg" /> . 변형율의 규범이어서 계산된다; <img의 고도 = &quot;식 (14)&quot;SRC = &quot;/ 파일 / ftp_upload / 51288 / 51288eq14.jpg&quot;/&gt;. 소용돌이는 반 대칭 부분에서 계산된다. Enstrophy 또는 z 성분의 소용돌이의 광장, <img alt="식 (15)" src="/files/ftp_upload/51288/51288eq15.jpg" /> )를 계산한다. 그만큼 <img alt="식 (16)" src="/files/ftp_upload/51288/51288eq16.jpg" /> 마지막으로 계산이고; <img alt="식 (17)" src="/files/ftp_upload/51288/51288eq17.jpg" /> . 전체 집합의 윤곽 플롯 <img alt="식 (18)" src="/files/ftp_upload/51288/51288eq18.jpg" /> 의 이소 - 지역으로 <img alt="식 (19)" src="/files/ftp_upload/51288/51288eq19.jpg" /> 이차 흐름 구조 (19)가 표시됩니다. 그만큼 <img alt="식 (20)" src="/files/ftp_upload/51288/51288eq20.jpg" /> 또한 &#39;소용돌이 힘 &quot;으로 알려진 로컬 속도 구배 텐서의 중요한 점 분석에 의해 수행되는 와류 식별 방법 및 해당 고유 20-24 <strong&gt;. 각 화소의 속도 구배 텐서의 고유치 <img alt="식 (21)" src="/files/ftp_upload/51288/51288eq21.jpg" /> 계산된다. 고유 값은 형식이어야합니다 <img alt="식 (22)" src="/files/ftp_upload/51288/51288eq22.jpg" /> . 의 윤곽 플롯 <img alt="식 (23)" src="/files/ftp_upload/51288/51288eq23.jpg" /> 의 이소 - 지역으로 <img alt="식 (24)" src="/files/ftp_upload/51288/51288eq24.jpg" /> (22) - 이차 흐름 구조 (20)가 표시됩니다. 방법을 웨이블릿 변환하는 것은 물리적 및 스펙트럼 공간에서 부드러움이있는 분석 기능 (또는 웨이브)를 사용, 허용 (또는 제로 평균을 가지고)와 유한이 <img alt="식 (25)" src="/files/ftp_upload/51288/51288eq25.jpg" /> 5, 6, 26. A는 팽창 된 또는 컨벌루션 저자는 차원 와도 필드 웨이블릿 수축 웨이블릿 변환 와도 <img alt="식 (26)" src="/files/ftp_upload/51288/51288eq26.jpg" /> 필드 c를 생성한다저울과 장점 (5, 6)의 넓은 범위, 2 차원 웨이블릿 변환 와도 필드 (26). 섀넌 엔트로피와 일관된 구조 omprising 모든 간섭 구조가 적절하게 해결하는 최적의 웨이블렛 스케일을 추정하기 위해 계산된다. 이 엔트로피 추정 확률의 세트를 포함 <img alt="식 (27)" src="/files/ftp_upload/51288/51288eq27.jpg" /> 각 픽셀에 대한 <img alt="식 (21)" src="/files/ftp_upload/51288/51288eq21.jpg" /> 되도록 <img alt="식 (28)" src="/files/ftp_upload/51288/51288eq28.jpg" /> , 정규화 된 사각형 위치 m의 픽셀과 관련된 와도 계수, n은 5, 6. 절차상의 단계는 그림 6에 그래픽으로 표시됩니다. 웨이블릿의 선택에 배치 제한은 참고 문헌에 자세히 표시됩니다. 26이 프로토콜 단계는 2D Ricker 웨이블릿을 이용하여 코 히어 런트 검출 구조를위한 절차를 설명한다. 이 사용 w에 대한 정당성선회 패턴 매칭을위한 avelet은 참고 문헌에 제시되어있다. 도 5,도 6과 관련 참조 내에 들었다.

<table border="0" cellpadding="0" cellspacing="0" width="698">
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Acrylic tubes and sheet</td>
			<td style="width:155px;">McMaster-Carr Supply Company</td>
			<td style="width:119px;"></td>
			<td style="width:208px;">Inlet and outlet pipes and, material of the curved artery test section</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Object24 Desktop 3D printer</td>
			<td style="width:155px;">Stratasys</td>
			<td style="width:119px;"></td>
			<td style="width:208px;">Desktop rapid prototyping machine. http://www.stratasys.com/.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">VeroWhitePlus Opaque material</td>
			<td style="width:155px;">Stratasys</td>
			<td style="width:119px;"></td>
			<td style="width:208px;">Building material for Object24 Desktop 3D printer</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Fullcure 705</td>
			<td style="width:155px;">Stratasys</td>
			<td style="width:119px;"></td>
			<td style="width:208px;">&#160;Non-toxic gel-like photopolymer Support material for Object24 Desktop 3D printer</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Ubbelhode viscometer</td>
			<td style="width:155px;">Cole Parmer</td>
			<td>YO-98934-12</td>
			<td style="width:208px;">Toward measurement of kinematic viscosity of the blood-analog fluid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">VELP scientifica - ESP stirrer&#160;</td>
			<td style="width:155px;">VELP Scientifica</td>
			<td>F206A0179</td>
			<td style="width:208px;">Magnetic stirrer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ohaus Scout Pro SP 601&#160;</td>
			<td style="width:155px;">The Lab Depot</td>
			<td>SP4001</td>
			<td style="width:208px;">Weigh scale</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Refractometer</td>
			<td>Atago</td>
			<td style="width:119px;">PAL-RI</td>
			<td style="width:208px;">Toward measurement of refractive index of blood-analog fluid</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Beakers, pipettes, syringes and spatula</td>
			<td style="width:155px;">Sigma-Aldrich&#160;</td>
			<td style="width:119px;">CLS710110,&#160; CLS10031L, CLS71015, CLS71011 Z193216</td>
			<td style="width:208px;">Toward handling materials required for blood-analog solution preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium Iodide</td>
			<td style="width:155px;">Sigma-Aldrich</td>
			<td>383112-2.5KG&#160;</td>
			<td style="width:208px;">Crystalline</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glycerol</td>
			<td style="width:155px;">Sigma-Aldrich</td>
			<td>G5516-1L</td>
			<td style="width:208px;">Liquid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Deionized Water</td>
			<td style="width:155px;">-</td>
			<td style="width:119px;">-</td>
			<td style="width:208px;">Liquid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium thiosulfate anhydrous</td>
			<td style="width:155px;">Sigma-Aldrich</td>
			<td>72049-250G</td>
			<td style="width:208px;">Powder</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PIV Recording medium</td>
			<td style="width:155px;">&#160;LaVision&#160;</td>
			<td style="width:119px;">Imager Intense 10Hz</td>
			<td style="width:208px;">PIV Image acquisition CCD camera</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">PIV Illumination source</td>
			<td style="width:155px;">New Wave Research</td>
			<td style="width:119px;">Solo III-15</td>
			<td style="width:208px;">PIV Laser source, Nd:YAG laser, 532 nm, dual pulse 70 mJ/pulse</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PIV Imaging software</td>
			<td style="width:155px;">&#160;LaVision&#160;</td>
			<td style="width:119px;">DaVis 7.2&#160;</td>
			<td style="width:208px;">PIV data acquisition and instrument control&#160;</td>
		</tr>
		<tr height="211">
			<td height="211" style="height:211px;width:216px;">PIV Seeding material</td>
			<td style="width:155px;">Thermo-scientific&#160;&#160;</td>
			<td style="width:119px;">Flouro-Max</td>
			<td style="width:208px;">Red fluorescent polymer microspheres (&#8776; 7 &#181;m); Dry dyed polystyrene (DVB) fluorescent microspheres emit bright and distinct colors when illuminated by the light of shorter&#160; wavelengths than the emission wavelength.&#160;</td>
		</tr>
	</tbody>
</table>

experimental investigation of secondary flow structures downstream of a model type iv stent failure in a 180&#176; curved artery test section

인간의 혈관의 동맥 네트워크는 복잡한 형상 (지점, 곡률과 비틀림)에 보편적으로 존재하는 혈관 구성되어 있습니다. 보조 유동 구조에 의한 원심력, 역 압력 구배 유입 특성의 결합 작용으로 만곡 동맥에 발생하는 선회 흐름 패턴이다. 이러한 흐름 모폴로지 크게 박동 및 생리 학적 유입 조건의 여러 고조파의 영향과 1 흐르는 비 생리적 (정상 및 진동)에 비해 크기 강도 형 특성이 크게 변화된다 - (7). 보조 유동 구조 궁극적 죽상 경화증, 재 협착, 혈소판 감작 및 혈전증 (4)의 진행 방향으로 혈액 유래 입자 벽 전단 응력 및 노출 시간에 영향을 미칠 수 - 6, 8-13 따라서, 실험에 따라 이러한 구조를 검출하고 특성화하는 능력. -controlled 조건은 precurs입니다임상 연구를 촉진합니다. 죽상 동맥 경화증에 대한 일반적인 수술 적 치료는 탁 트인 혈액 흐름에 대한 협착 동맥을 열어 스텐트 시술이다. 그러나 때문에 스텐트 설치에 수반 흐름 교란 멀티 규모의 이차 흐름 모폴로지 (4) 결과 -. 6 이러한 일관성의 비대칭 손실로 점진적으로 더 높은 차수의 복잡성은 마주 교란 흐름 5 세 미만 계속되는 스텐트 장애에 의해 유발 될 수있다. 이러한 스텐트 실패는 실패의 고려 사항 및 임상 적 중증도 (14)에 따라 &quot;유형 I-에-IV&quot;로 분류되고있다. 본 연구는 만곡 동맥 스텐트 모델 횡 골절 및 골절 부의 선형 변위 ( &quot;타입 IV&quot;)을 완료하기 때문에 복잡한 보조 유동 구조의 실험 연구를위한 프로토콜을 제공한다. 실험 방법은 입자 화상 속도계의 구현을 포함한다 (도 2C-2전형적인 경동맥 유입 파형 D PIV)는 기술, 굴절률 위상 평균 측정치 (15)에 대한 작동 유체 혈액 아날로그 일치 -. 18 차 유동 구조의 양적 식별이 흐름 물리학의 개념, 중요한 점 이론과 소설을 사용하여 달성되었다 26 - PIV 실험 데이터 5, 6, 19에 적용되는 알고리즘을 웨이블릿 변환.

도 7A-D에 제시된 결과 (참조 :도 5, 6)도 3a에 도시 2C-2D PIV 시스템으로부터 취득한 후 처리 이차 유속 데이터 후에 발생 하였다. 이상화 된 &quot;타입 IV&quot;스텐트 골절 만곡 동맥 시험 부에 공급되는 유입 상태는도 4b에 도시 된 경동맥 파형이다. 우리의 이전 연구 Womersley 번호의 특정 범위 내에 유입 맥동 파형?...

Watch this Scientific Journal Video about Experimental Investigation of Secondary Flow Structures Downstream of a Model Type IV Stent Failure in a 180Â° Curved Artery Test Section at JoVE.com

Experimental Investigation of Secondary Flow Structures Downstream of a Model Type IV Stent Failure in a 180&#176; Curved Artery Test Section

Stent implants in stenosed arterial curvatures are prone to "Type IV" failures involving the complete transverse fracture of stents and linear displacement of the fractured parts. We present a protocol for detection of secondary flow (vortical) structures in a curved artery model, downstream of clinically relevant "Type IV" stent failures.

180 ° 곡선 동맥 테스트 섹션에서 모델 유형 IV 스텐트 실패의 다운 스트림 보조 흐름 구조의 실험 조사

The arterial network in the human vasculature comprises of ubiquitously present blood vessels with complex geometries (branches, curvatures and ...

experimental-investigation-secondary-flow-structures-downstream-model

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

11.5K 조회수.  The George Washington University. Stent implants in stenosed arterial curvatures are prone to "Type IV" failures involving the complete transverse fracture of stents and linear displacement of the fractured parts. We present a protocol for detection of secondary flow (vortical) structures in a curved artery model, downstream of clinically relevant "Type IV" stent failures.

비디오: Experimental Investigation of Secondary Flow Structures Downstream of a Model Type IV Stent Failure in a 180° Curved Artery Test Section

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

Parallel-plate Flow Chamber and Continuous Flow Circuit to Evaluate Endothelial Progenitor Cells under Laminar Flow Shear Stress

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well quantifiable fluid shear stresses1.
Our flow chamber design and flow circuit (Fig. 1) contains a transparent viewing region that enables testing of cell adhesion and imaging of cell morphology immediately before flow (Fig. 11A, B), at various time points during flow (Fig. 11C), and after flow (Fig. 11D). These experiments are illustrated with human umbilical cord blood-derived endothelial progenitor cells (EPCs) and porcine EPCs2,3.
This method is also applicable to other adherent cell types, e.g. smooth muscle cells (SMCs) or fibroblasts.
The chamber and all parts of the circuit are easily sterilized with steam autoclaving. In contrast to other chambers, e.g. microfluidic chambers, large numbers of cells (&gt; 1 million depending on cell size) can be recovered after the flow experiment under sterile conditions for cell culture or other experiments, e.g. DNA or RNA extraction, or immunohistochemistry (Fig. 11E), or scanning electron microscopy5. The shear stress can be adjusted by varying the flow rate of the perfusate, the fluid viscosity, or the channel height and width. The latter can reduce fluid volume or cell needs while ensuring that one-dimensional flow is maintained. It is not necessary to measure chamber height between experiments, since the chamber height does not depend on the use of gaskets, which greatly increases the ease of multiple experiments. Furthermore, the circuit design easily enables the collection of perfusate samples for analysis and/or quantification of metabolites secreted by cells under fluid shear stress exposure, e.g. nitric oxide (Fig. 12)6.

We are describing a method to subject adherent cells to laminar flow shear stress in a sterile continuous flow circuit. The cells' adhesion, morphology can be studied through the transparent chamber, samples obtained from the circuit for metabolite analysis and cells harvested after shear exposure for future experiments or culture.

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well ...

Monitoring the Wall Mechanics During Stent Deployment in a Vessel

Clinical trials have reported different restenosis rates for various stent designs1. It is speculated that stent-induced strain concentrations on the arterial wall lead to tissue injury, which initiates restenosis2-7. This hypothesis needs further investigations including better quantifications of non-uniform strain distribution on the artery following stent implantation. A non-contact surface strain measurement method for the stented artery is presented in this work. ARAMIS stereo optical surface strain measurement system uses two optical high speed cameras to capture the motion of each reference point, and resolve three dimensional strains over the deforming surface8,9. As a mesh stent is deployed into a latex vessel with a random contrasting pattern sprayed or drawn on its outer surface, the surface strain is recorded at every instant of the deformation. The calculated strain distributions can then be used to understand the local lesion response, validate the computational models, and formulate hypotheses for further in vivo study.

Stent-induced arterial strain distributions are characterized using an optical surface strain measurement system. This visualization technique is used to gain insights into the impact of stent implantation on the host vessel.

선박의 스텐 트를 배포하는 동안 벽 역학 모니터링

Clinical trials have reported different restenosis rates for various stent designs1. It is speculated that stent-induced strain concentrations on the ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

인간의 3 차원의 조직 공학<em&gt; 시험관</em&gt; 종양 테스트 시스템

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

An Improved Method for the Preparation of Type I Collagen From Skin

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. Clinically, this protein is widely used for cosmetic surgery, dermal injections, bone grafting, and reconstructive surgery. The sources of COL1 for these procedures are commonly nonhuman, which increases the potential for inflammation and rejection as well as xenobiotic disease transmission. In view of this, a method to efficiently and quickly purify COL1 from limited quantities of autologously-derived tissues would circumvent many of these issues; however, standard isolation protocols are lengthy and often require large quantities of collagenous tissues. Here, we demonstrate an efficient COL1 extraction method that reduces the time needed to isolate and purify this protein from about 10 days to less than 3 hr. We chose the dermis as our tissue source because of its availability during many surgical procedures. This method uses traditional extraction buffers combined with forceful agitation and centrifugal filtration to obtain highly-pure, soluble COL1 from small amounts of corium. Briefly, dermal biopsies are washed thoroughly in ice-cold dH2O after removing fat, connective tissue, and hair. The skin samples are stripped of noncollagenous proteins and polysaccharides using 0.5 M sodium acetate and a high speed bench-top homogenizer. Collagen from residual solids is subsequently extracted with a 0.075 M sodium citrate buffer using the homogenizer. These extracts are purified using 100,000 MW cut-off centrifugal filters that yield COL1 preparations of comparable or superior quality to commercial products or those obtained using traditional procedures. We anticipate this method will facilitate the utilization of autologously-derived COL1 for a multitude of research and clinical applications.

Traditional procedures for the isolation of soluble type 1 collagen (COL1) require about 10 days from start to finish because of lengthy buffer incubations and laborious resuspensions of fibrils. Here, we describe a means to purify COL1 from small dermal biopsies in less than 3 hr.

피부에서 타입 I 콜라겐의 제조를위한 개선 된 방법

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. ...

Characterization Of Multi-layered Fish Scales (Atractosteus spatula) Using Nanoindentation, X-ray CT, FTIR, and SEM

The hierarchical architecture of protective biological materials such as mineralized fish scales, gastropod shells, ram&#8217;s horn, antlers, and turtle shells provides unique design principles with potentials for guiding the design of protective materials and systems in the future. Understanding the structure-property relationships for these material systems at the microscale and nanoscale where failure initiates is essential. Currently, experimental techniques such as nanoindentation, X-ray CT, and SEM provide researchers with a way to correlate the mechanical behavior with hierarchical microstructures of these material systems1-6. However, a well-defined standard procedure for specimen preparation of mineralized biomaterials is not currently available. In this study, the methods for probing spatially correlated chemical, structural, and mechanical properties of the multilayered scale of A. spatula using nanoindentation, FTIR, SEM, with energy-dispersive X-ray (EDX) microanalysis, and X-ray CT are presented.

Characterization Of Multi-layered Fish Scales Atractosteus spatula Using Nanoindentation, X-ray CT, FTIR, and SEM

This paper presents the methods used for probing spatially correlated chemical, structural, and mechanical properties of the multilayered scale of Atractosteus spatula (A. spatula) using nanoindentation, Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), and X-ray computed tomography (X-ray CT). The experimental results have been used to investigate the design principles of protective biological materials.

다층 물고기 비늘 (의 특성<em&gt; Atractosteus 주걱</em&gt;) 나노 인 덴 테이션, X-선 CT, FTIR, 및 SEM을 사용하여

The hierarchical architecture of protective biological materials such as mineralized fish scales, gastropod shells, ram&#8217;s horn, antlers, and turtle ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

복원 된 인간의 각막 모양의 상피 (RHCE) 조직 모델을 사용하여 눈에 자극성 화학 물질의 위험 식별을위한 눈 자극 시험 (EIT)

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in every tissue to be identified and characterized. Our laboratory is actively working to make technological advances in X-ray micro-computed tomography (micro-CT) that will bring the diagnostic power of histology to the study of full tissue volumes at cellular resolution (i.e., an X-ray Histo-tomography modality). Toward this end, we have made targeted improvements to the sample preparation pipeline. One key optimization, and the focus of the present work, is a straightforward method for rigid embedding of fixed and stained millimeter-scale samples. Many of the published methods for sample immobilization and correlative micro-CT imaging rely on placing the samples in paraffin wax, agarose, or liquids such as alcohol. Our approach extends this work with custom procedures and the design of a 3-dimensional printable apparatus to embed the samples in an acrylic resin directly into polyimide tubing, which is relatively transparent to X-rays. Herein, sample preparation procedures are described for the samples from 0.5 to 10 mm in diameter, which would be suitable for whole zebrafish larvae and juveniles, or other animals and tissue samples of similar dimensions. As proof of concept, we have embedded the specimens from Danio, Drosophila, Daphnia, and a mouse embryo; representative images from 3-dimensional scans for three of these samples are shown. Importantly, our methodology leads to multiple benefits including rigid immobilization, long-term preservation of laboriously-created resources, and the ability to re-interrogate samples.

We developed protocols and designed a custom apparatus to enable embedding of millimeter-scale specimens. We present sample preparation procedures with an emphasis on embedding in acrylic resin and polyimide tubing to achieve rigid immobilization and long-term storage of specimens for the interrogation of tissue architecture and cell morphology by micro-CT.

엄밀한 포함의 고정 및 스테인드, 전체, 마이크로 단층에 의해 섹션-무료 3D 조직학에 대 한 밀리미터 단위 표본

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in ...

Gene Expression Analysis of Endothelial Cells Exposed to Shear Stress Using Multiple Parallel-plate Flow Chambers

We describe a workflow for the analysis of gene expression from endothelial cells subject to a steady laminar flow using multiple monitored parallel-plate flow chambers. Endothelial cells form the inner cellular lining of blood vessels and are chronically exposed to the frictional force of blood flow called shear stress. Under physiological conditions, endothelial cells function in the presence of various shear stress conditions. Thus, the application of shear stress conditions in in vitro models can provide greater insight into endothelial responses in vivo. The parallel-plate flow chamber previously published by Lane et al.9 is adapted to study endothelial gene regulation in the presence and absence of steady (non-pulsatile) laminar flow. Key adaptations in the set-up for laminar flow as presented here include a large, dedicated environment to house concurrent flow circuits, the monitoring of flow rates in real-time, and the inclusion of an exogenous reference RNA for the normalization of quantitative real-time PCR data. To assess multiple treatments/conditions with the application of shear stress, multiple flow circuits and pumps are used simultaneously within the same heated and humidified incubator. The flow rate of each flow circuit is measured continuously in real-time to standardize shear stress conditions throughout the experiments. Because these experiments have multiple conditions, we also use an exogenous reference RNA that is spiked-in at the time of RNA extraction for the normalization of RNA extraction and first-strand cDNA synthesis efficiencies. These steps minimize the variability between samples. This strategy is employed in our pipeline for the gene expression analysis with shear stress experiments using the parallel-plate flow chamber, but parts of this strategy, such as the exogenous reference RNA spike-in, can easily and cost-effectively be used for other applications.

Here, a workflow for the culture and gene expression analysis of endothelial cells under fluid shear stress is presented. Included is a physical arrangement for simultaneously housing and monitoring multiple flow chambers in a controlled environment and the use of an exogenous reference RNA for quantitative PCR.

가 여러 명의 병렬 접시 흐름 챔버를 사용 하 여 스트레스에 노출 된 내 피 세포의 유전자 표정 분석

We describe a workflow for the analysis of gene expression from endothelial cells subject to a steady laminar flow using multiple monitored parallel-plate ...

An Automated Method to Perform The In Vitro Micronucleus Assay using Multispectral Imaging Flow Cytometry

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and introduces uncertainty in results due to variability between scorers. To remedy this, automated slide-scanning microscopy as well as conventional flow cytometry methods have been introduced in an attempt to remove scorer bias and improve throughput. However, these methods have their own inherent limitations such as inability to visualize the cytoplasm of the cell and the lack of visual MN verification or image data storage with flow cytometry. Multispectral Imaging Flow Cytometry (MIFC) has the potential to overcome these limitations. MIFC combines the high resolution fluorescent imagery of microscopy with the statistical robustness and speed of conventional flow cytometry. In addition, all collected imagery can be stored in dose-specific files. This paper describes the protocol developed to perform a fully automated version of the MN assay on MIFC. Human lymphoblastoid TK6 cells were enlarged using a hypotonic solution (75 mM KCl), fixed with 4% formalin and the nuclear content was stained with Hoechst 33342. All samples were run in suspension on the MIFC, permitting acquisition of high resolution images of all key events required for the assay (e.g. binucleated cells with and without MN as well as mononucleated and polynucleated cells). Images were automatically identified, categorized and enumerated in the MIFC data analysis software, allowing for automated scoring of both cytotoxicity and genotoxicity. Results demonstrate that using MIFC to perform the in vitro MN assay allows statistically significant increases in MN frequency to be detected at several different levels of cytotoxicity when compared to solvent controls following exposure of TK6 cells to Mitomycin C and Colchicine, and that no significant increases in MN frequency are observed following exposure to Mannitol.

The in vitro micronucleus assay is a well-established method for evaluating genotoxicity and cytotoxicity but scoring the assay using manual microscopy is laborious and suffers from subjectivity and inter-scorer variability. This paper describes the protocol developed to perform a fully automated version of the assay using multispectral imaging flow cytometry.

다중 스펙트럼 화상 진찰 교류 세포측정을 사용하여 시험관 내 미세핵 분석기를 능력을 발휘하는 자동화된 방법

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and ...

180 ° 곡선 동맥 테스트 섹션에서 모델 유형 IV 스텐트 실패의 다운 스트림 보조 흐름 구조의 실험 조사

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