Name: biomechanical testing of murine tendons
Uploaded: 2019-10-15T14:30:00
Description: Watch this Scientific Journal Video about Biomechanical Testing of Murine Tendons at JoVE.com

이 연구는 NIH/NIAMS(R01 AR055580, R01 AR057836)에 의해 지원되었다.

동물 연구는 컬럼비아 대학 기관 동물 관리 및 사용 위원회에 의해 승인 되었다. 이 연구에서 사용된 마우스는 C57BL/6J 배경이었으며 잭슨 연구소(미국 바 하버)에서 구입했습니다. 그(것)들은 병원균 자유로운 방벽 조건에 보관되고 음식과 물 광고 libitum를 제공되었습니다.
1. 그립 뼈를 위한 맞춤형 3D 인쇄 설비 개발
<ol><li>뼈 이미지 수집 및 3D 뼈 모델 구성<ol><li>3D 모델 생성 ...

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뮤린 동물 모델은 일반적으로 힘줄 장애를 연구하는 데 사용되지만, 기계적 특성의 특성화는 문헌에서 도전적이고 드문 일입니다. 이 프로토콜의 목적은 뮤린 힘줄의 인장 시험을 위한 시간 효율적이고 재현 가능한 방법을 설명하는 것이다. 새로운 방법은 샘플을 테스트하는 데 필요한 시간을 몇 시간에서 몇 분으로 단축하고 이전 방법에서 일반적인 문제였던 주요 파지 아티팩트를 제거했습니?...

힘줄 장애는 노화, 운동 및 활성 인구 중 일반적이고 매우 널리 퍼져1,2,3. 미국에서는, 16.4 백만 결합 조직 상해는 해마다 보고되고모든 상해 관련 의사 사무실 방문의 30%를차지합니다3,5,6,7, 8. 가장 일반적으로 영향을받는 사이트는 회전근, 아킬레스 힘줄, 슬개골 힘줄9을포함한다. 항염증제, 재활, 외과적 수리 등 다양한 비수술적 및 수술적 치료법이 탐구되었지만, 결과는 여전히 좋지 않고, 기능 회복과 높은실패율로인해5, 6. 이 가난한 임상 결과는 건병을 이해하고 새로운 처리 접근을 개발하기 위하여 노력하는 기본적인 및 번역학 연구 동기를 부여했습니다.
인장 생체 역학 적 특성은 힘줄 기능을 정의하는 주요 정량적 결과입니다. 따라서, 힘줄 병증 및 치료 효능의 실험실 특성은 힘줄 인장 특성의 엄격한 테스트를 포함해야합니다. 수많은 연구는 쥐, 양, 개 및 토끼10,11,12와같은 동물 모델로부터 힘줄의 생체 역학적 특성을 결정하는 방법을 기술하였다. 그러나, 몇몇 연구 결과는 뮤린 힘줄의 생체 역학 적 특성을 테스트했습니다, 주로 인장 테스트를 위한 작은 조직을 붙잡는 어려움 때문에. 뮤린 모델은 유전자 조작, 광범위한 시약 옵션 및 저렴한 비용을 포함하여 기계적으로 건병증을 연구하는 데 많은 이점이 있기 때문에 생체 역학적으로 뮤린 조직을 테스트하는 정확하고 효율적인 방법의 개발이 필요합니다.
힘줄의 기계적 특성을 적절하게 테스트하기 위해서는 조직이 그립 인터페이스에서 미끄러지거나 관절이 찢어지거나 성장 판의 골절없이 효과적으로 그립해야합니다. 많은 경우에, 특히 짧은 힘줄에 대 한, 뼈는 한쪽 끝에 잡고 하 고 힘 줄은 다른 쪽 끝에 그립. 뼈는 전형적으로 에폭시수지(13) 및 폴리메틸메타크릴레이트(14,15)와같은 물질에 이들을 포함시킴으로써 확보된다. 힘줄은 종종 시안아크라일레이트와 접착, 사포의 두 층 사이에 배치하고, 압축 클램프를 사용하여 고정 (단면이 평평한 경우) 또는 냉동 매체 (단면이 큰 경우)15,16,17 . 이러한 방법은 생체 역학적으로 뮤린 힘줄에 적용되었지만, 시편의 작은 크기와 성장 판의 준수로 인해 문제가 발생하며, 이는 결코18을ossifies하지 않습니다. 예를 들어, 뮤린 상완골 머리의 직경은 불과 몇 밀리미터이므로 뼈의 파지가 어려워집니다. 특히, 뮤린 상부 힘줄 - 뼈 샘플의 인장 테스트는 종종 힘줄이나 힘줄 enthesis보다는 성장 판에 실패를 초래. 마찬가지로, 아킬레스 건의 생체 역학 테스트는 도전이다. 아킬레스 건은 다른 뮤린 힘줄보다 크지만, 종골은 작아서이 뼈를 잡기가 어렵습니다. 뼈를 제거 할 수 있습니다, 두 힘줄 끝을 잡고 다음; 그러나, 이것은 힘줄 -뼈 부착의 테스트를 배제. 다른 그룹은 맞춤형 비품19,20,클램프(21)에의한 고정, 자체 경화 플라스틱 시멘트(22) 또는 원추형 모양 슬롯(22)을사용하여 칼카누뼈를 잡는 것을보고하지만, 아직이들은 이전 방법은 낮은 재현성, 높은 파지 실패율 및 지루한 준비 요구 사항에 의해 제한됩니다.
현재 연구의 목적은 뮤린 힘줄의 인장 생체 역학 적 테스트를위한 정확하고 효율적인 방법을 개발하는 것이었습니다, 예로 상피와 아킬레스 건에 초점을 맞추고. 네이티브 뼈 해부학, 고체 모델링 및 적첨가 제법에서 3D 재구성의 조합을 사용하여 뼈를 잡는 새로운 방법이 개발되었습니다. 이러한 설비는 효과적으로 뼈를 고정, 성장 판 실패를 방지, 감소 시편 준비 시간, 증가 테스트 재현성. 새로운 방법은 쥐와 다른 동물의 힘줄뿐만 아니라 다른 뮤린 힘줄을 테스트하기 위해 쉽게 적응할 수 있습니다.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >Agarose</td>
			<td >Fisher Scientific</td>
			<td >BP160-100</td>
			<td >Dissovle 1g in 100 ml ultrapure water to make 1% agarose&#160;</td>
		</tr>
		<tr >
			<td >Bruker microCT&#160;</td>
			<td >Bruker BioSpin Corp</td>
			<td >Skyscan 1272&#160;</td>
			<td>Used by authors</td>
		</tr>
		<tr >
			<td >ElectroForce&#160;</td>
			<td >TA Instruments</td>
			<td >3200</td>
			<td>Testing platform</td>
		</tr>
		<tr >
			<td >Ethanol 200 Proof</td>
			<td >Fisher Scientific</td>
			<td >A4094</td>
			<td>Dilute to 70% and use as suggested in protocol</td>
		</tr>
		<tr >
			<td >Fixture to attach grips</td>
			<td >Custom made</td>
			<td ></td>
			<td>Used by authors</td>
		</tr>
		<tr >
			<td >Kimwipes</td>
			<td >Kimberly-Clark</td>
			<td >&#160;S-8115</td>
			<td>As suggested in protocol</td>
		</tr>
		<tr >
			<td >MicroCT CT-Analyser (Ctan)</td>
			<td >Bruker BioSpin Corp</td>
			<td ></td>
			<td>Used by authors for visualizing and analyzing micro-CT scans&#160;</td>
		</tr>
		<tr >
			<td >MilliQ water (Ultrapure water)</td>
			<td>Millipore Sigma</td>
			<td>QGARD00R1 (or related purifier)</td>
			<td>100 ml&#160;</td>
		</tr>
		<tr >
			<td >Meshmixer</td>
			<td >Autodesk</td>
			<td >http://www.meshmixer.com/</td>
			<td >Free engineering software used by authors to refine mesh</td>
		</tr>
		<tr >
			<td >Objet EDEN 260VS&#160;</td>
			<td >Stratasys LTD</td>
			<td ></td>
			<td>Precision Prototyping</td>
		</tr>
		<tr >
			<td >Objet Studio</td>
			<td >Stratasys LTD</td>
			<td ></td>
			<td>Used by authors with 3D printer</td>
		</tr>
		<tr >
			<td >PBS - Phosphate-Buffered Saline</td>
			<td >ThermoFisher Scientific</td>
			<td >10010031</td>
			<td >2.5 L of 10% PBS&#160;</td>
		</tr>
		<tr >
			<td >S&#38;T Forceps</td>
			<td >Fine Science Tools</td>
			<td >00108-11</td>
			<td>Used by authors</td>
		</tr>
		<tr >
			<td >Scalpel Blade - #11</td>
			<td >Fine Science Tools</td>
			<td >10011-00</td>
			<td>Used by authors</td>
		</tr>
		<tr >
			<td >Scalpel Handle - #3</td>
			<td >Fine Science Tools</td>
			<td >10003-12</td>
			<td>Used by authors</td>
		</tr>
		<tr >
			<td >SkyScan 1272</td>
			<td >Bruker BioSpin Corp</td>
			<td ></td>
			<td>Used by authors for visualizing and analyzing micro-CT scans&#160;</td>
		</tr>
		<tr >
			<td >Skyscan CT-Vox</td>
			<td >Bruker BioSpin Corp</td>
			<td ></td>
			<td>Used by authors for visualizing and analyzing micro-CT scans&#160;</td>
		</tr>
		<tr >
			<td >SkyScan NRecon</td>
			<td >Bruker BioSpin Corp</td>
			<td ></td>
			<td>Used by authors for visualizing and analyzing micro-CT scans&#160;</td>
		</tr>
		<tr >
			<td >SolidWorks CAD</td>
			<td >Dassault Syst&#232;mes</td>
			<td >SolidWorks Research Subsription</td>
			<td>Solid modeling computer-aided design used by authors</td>
		</tr>
		<tr >
			<td >SuperGlue</td>
			<td >Loctite</td>
			<td >234790</td>
			<td>As suggested in protocol</td>
		</tr>
		<tr >
			<td >Testing bath</td>
			<td >Custom made</td>
			<td ></td>
			<td>Used by authors</td>
		</tr>
		<tr >
			<td >Thin film grips&#160;</td>
			<td >Custom made</td>
			<td ></td>
			<td>Used by authors</td>
		</tr>
		<tr >
			<td >VeroWhitePlus</td>
			<td >Stratasys LTD</td>
			<td >NA</td>
			<td>3D printing material used by authors</td>
		</tr>
		<tr >
			<td >WinTest&#160;</td>
			<td >WinTest Software</td>
			<td ></td>
			<td>Used by authors to collect data</td>
		</tr>
	</tbody>
</table>

biomechanical testing of murine tendons

힘줄 장애는 일반적이며 모든 연령대의 사람들에게 영향을 미치며 종종 쇠약해합니다. 항염증제, 재활 및 외과 적 수리와 같은 표준 치료는 종종 실패합니다. 힘줄 기능을 정의하고 새로운 치료법의 효능을 입증하기 위해서는 동물 모델의 힘줄의 기계적 특성을 정확하게 결정해야 합니다. 뮤린 동물 모델은 지금 널리 힘줄 장애를 연구하고 건선 에 대한 새로운 치료를 평가하는 데 사용됩니다; 그러나 마우스 힘줄의 기계적 특성을 결정하는 것은 어려운 일이었습니다. 이 연구에서는 상완골과 칼카누의 해부학과 정확히 일치하는 3D 인쇄 설비를 포함하는 힘줄 기계 적 테스트를 위해 새로운 시스템이 개발되어 상완골과 아킬레스 건을 기계적으로 테스트합니다. 이러한 설비는 네이티브 뼈 해부학, 고체 모델링 및 적색 제법의 3D 재구성을 사용하여 개발되었습니다. 새로운 접근법은 예술적 파지 실패(예: 힘줄이 아닌 성장 판 실패)를 제거하고 전체 테스트 시간을 줄였으며 재현성을 증가시켰습니다. 또한,이 새로운 방법은 다른 동물에서 다른 뮤린 힘줄과 힘줄을 테스트하기 위해 쉽게 적응할 수 있습니다.

3D 프린팅 비품은 8주 된 뮤린 수명과 아킬레스 건을 테스트하는 데 사용되었습니다. 기계적으로 테스트된 모든 샘플은 인장 검사 후 microCT 스캔, 육안 검사 및 비디오 분석을 특징으로 하는 enthesis에서 실패했습니다. 우리의 실험실에서 상부 척추 힘줄 테스트에 대한 이전 및 현재 방법의 일대일 비교는 그림 3에도시되어 있습니다. 이전 방법28,<sup cl...

Watch this Scientific Journal Video about Biomechanical Testing of Murine Tendons at JoVE.com

Biomechanical Testing of Murine Tendons

이 프로토콜은 맞춤형 맞춤 3D 인쇄 설비를 사용하여 뮤린 힘줄에 대한 효율적이고 재현 가능한 인장 생체 역학 테스트 방법을 설명합니다.

뮤린 힘줄의 생체 역학 테스트

Tendon disorders are common, affect people of all ages, and are often debilitating. Standard treatments, such as anti-inflammatory drugs, rehabilitation, ...

이 프로토콜은 재사용 가능한 맞춤형 3D 인쇄 설비를 통해 마우스 힘줄의 생체 역학 적 테스트를위한 효율적이고 재현 가능한 방법을 설명합니다. 새로운 메서드는 샘플을 몇 시간에서 분으로 테스트하는 데 필요한 시간을 줄이고 이전 메서드에서 일반적인 문제였던 주요 파지 아티팩트를 제거합니다. 이 방법은 초라피나투스 및 아킬레스 건에 국한되지 않습니다.그것은 쉽게 큰 동물에서 다른 마우스 힘줄과 힘줄을 테스트에 적응 할 수 있습니다. 접근 방식의 재현성과 효율성을 보장하기 위해 여기에 설명되지 않은 비품에 대해 여러 주기의 설계 프로토타이핑 및 유효성 검사가 필요할 수 있습니다. 시작하려면 아가로즈 젤에서 전체 뼈의 미세 계산 단층 촬영 검사를 수행하십시오.단면 이미지로 재구성한 후 파일 열기 명령을 선택하여 파일 데이터 집합을 엽니다. 대화 상자 파일 환경 설정을 열고 고급 탭을 선택합니다. 적응형 렌더링 알고리즘을 사용하여 표면 세부 정보를 더 부드럽게 제공하는 3D 모델을 구성합니다.개체 테두리를 찾는 데 사용되는 이웃 점까지 픽셀의 거리를 정의하는 지역성 매개변수로 10을 사용합니다. 0.1로 허용 오차를 최소화하여 파일 크기를 줄입니다. 관심 볼륨을 지정하려면 선택한 VOI 범위의 위쪽과 아래쪽으로 설정하도록 두 개의 이미지를 수동으로 선택한 다음 관심 영역의 두 번째 페이지로 이동합니다.단일 단면 이미지에서 관심 영역을 수동으로 선택합니다. 10~15개의 단면 이미지마다 ROI를 반복적으로 선택합니다. 다음으로 세 번째 페이지인 바이너리 선택으로 이동합니다.히스토그램 메뉴에서 데이터 집합을 클릭하여 데이터 집합의 모든 이미지에서 밝기의 히스토그램 분포를 표시합니다. 또한 히스토그램 메뉴에서 3D 모델 파일 만들기 메뉴를 클릭합니다. 골격의 3D 모델을 STL 파일 형식으로 저장합니다.Meshmixer 소프트웨어에서 메시를 가져오고 편집할 모든 것을 선택합니다. 도구 세트 편집에서 생성을 선택한 다음 도구 세트 에서 삼각형 예산을 선택합니다. 트리카운트를 줄이고 변경 내용을 수락합니다.수출 As.To 설계 를 선택하여 STL 형식으로 새로 감소 된 파일을 다시 저장, 상완골 그립 기구의 사용자 정의 적합 모델을 만들기 위해 솔리드 모델링 컴퓨터 지원 설계 프로그램을 사용합니다. 솔리드 모델링 프로그램에서 상완골 골격의 STL 형식 파일을 열고 SLDPRT 형식의 부품 파일로 저장합니다. 그런 다음 Part 파일을 열어 해부학적으로 관련된 세 비행기(예: 처검, 관상 및 횡방향)를 수동으로 만듭니다.새 파일 파일을 클릭하여 솔리드 블록 구성 요소 부분을 만듭니다. 파일 New를 클릭하여 솔리드 블록과 오른쪽 또는 왼쪽 상완골 골격의 두 가지 구성 요소로 어셈블리 모델을 만듭니다. 블록 내의 골격 방향을 정의하여 힘줄과 뼈 사이의 각도가 180도되도록 합니다.전체 골격 볼륨이 블록 내부에 맞는지 확인합니다. 어셈블리 창에서 블록을 선택하고 어셈블리 도구 모음에서 구성 요소 편집을 클릭합니다. 삽입, 기능, 캐비티를 클릭합니다.균일 한 배율 조정을 선택하고 모든 방향으로 확장할 값으로 0 %를 입력합니다. 골격 부분을 억제하고 어셈블리를 일부로 저장합니다. 캐비티 부품으로 실린더를 엽니다.스케치를 만들고 상완골 머리 위에 0.5 밀리미터만 남도록 하십시오. 피처에서 압출된 컷을 선택합니다. 적중 평면을 따라 어셈블리를 잘라 뼈에 앞쪽과 후방에 맞는 두 개의 대칭 성분을 만듭니다.적중 평면을 따라 어셈블리 부품을 잘라 뼈에 앞쪽과 후방에 맞는 두 개의 대칭 성분을 만듭니다. 상완골 헤드가 성장 판 고장을 방지하고 테스트 중에 모델에서 상완골이 분리되는 것을 방지하는 엄격한 간격을 정의하는 것이 중요합니다. 원고에 설명된 대로 진행하여 후방 구성 요소 및 전방 구성 요소를 완료합니다.두 구성 요소를 별도의 부품 파일로 저장합니다. 이제 삽입 패턴, 미러 및 미러를 눌러 반대 쪽 사지에 대한 고정물의 각 구성 요소에 대해 3D 미러 모델을 만듭니다. 앞면을 미러 페이스 플레인으로 선택합니다.미러할 본문으로 부품을 선택합니다. 미러 평면을 선택하고 모든 재질을 포함하는 스케치를 만듭니다. 피처에서 압출된 컷을 선택하여 원래 부품을 제거하고 미러된 부품만 유지합니다.부품의 바닥을 클릭하고 스케치를 만들기 위해 평면으로 사용합니다. 스케치 텍스트를 클릭하고 L 또는 R.이 왼쪽과 오른쪽을 구별하는 비품의 하단에 에칭을 추가합니다. 3D 프린팅을 준비하기 위해 STL 표준 파일 형식으로 모든 비품 부품을 저장합니다.상완골 근육 힘줄, 상완골 표본을 해부하기 위해, 먼저 안락사 마우스를 경향이 있는 위치에 배치하고 앞치마의 팔꿈치 위에서 어깨쪽으로 피부에 절개를 합니다. 어깨의 근육이 보이도록 조심스럽게 무딘 해부로 피부를 제거하기 위해 집게를 사용합니다. 뼈가 노출될 때까지 상완골을 둘러싼 조직을 제거합니다.상완골을 집게로 잡고 망상과 사다리꼴 근육을 조심스럽게 제거하여 코라코아코미알 아치를 노출시하십시오. AC 조인트를 식별하고 메스 블레이드로 아크로미온에서 쇄골을 조심스럽게 분리합니다. 수퍼피나투스 힘줄과 뼈 부착을 손상시키지 않도록 주의하고 메스 블레이드를 사용하여 견갑질의 부착물에서 근육을 제거하십시오.상완골 머리를 글레노이드에서 분리하고 조인트 캡슐과 인프라피나투스 카피큘러리스와 사소한 힘줄을 레이스로 장식합니다. 팔꿈치 관절을 분리하여 상완골을 척골과 반경으로부터 분리합니다. 상완골-수프라피나투스 힘줄 근육 표본을 분리하고 상완골과 상완골 머리에 과도한 연조직을 제거합니다.아킬레스 건, 칼카뉴뼈를 해부하려면 안락사 된 마우스를 경향이있는 위치에 배치합니다. 아킬레스 건과 뼈 부착을 손상시키지 않도록주의하고, 발목과 무릎 관절 주위의 근육이 노출되도록 무딘 해부로 피부를 제거합니다. 아킬레스 건-칼카뉴 부착에서 시작하여 메스 블레이드를 사용하여 위상염 근육을 근접 부착부에서 조심스럽게 분리합니다.다양한 인접한 뼈에서 칼카네를 조심스럽게 분리하고 아킬레스 건-칼카네스 표본을 분리하고 과도한 연조직을 제거합니다. 힘줄의 단면 영역을 결정하기 위해 뼈를 거꾸로 삽입하여 아가로즈 젤과 바깥쪽 근육의 뼈가 있는 아가로즈 젤로 채워진 극저온튜브의 시편을 중단한다. 마이크로 컴퓨터 단층 촬영을 사용하여 스캔 한 후 메스 블레이드를 사용하여 힘줄에서 근육을 부드럽게 제거하십시오.골격을 3D 인쇄 설비에 삽입하여 테스트 그리드에 부착합니다. 접힌 얇은 티슈 페이퍼 사이에 힘줄을 삽입하고 붙이고 박막 그립을 사용하여 고정합니다. 샘플과 그립을 섭씨 37도에서 PBS의 테스트 욕조에 삽입하고 기계적 테스트를 수행합니다.이 연구에서는 재사용 가능한 3D 인쇄 설비를 사용하여 뼈를 잡으며 시편 준비 시간이 몇 시간에서 분으로 단축되었습니다. 현재 방법을 사용하여 초프라피나투스 힘줄의 인장 테스트를 위한 대표적인 하중 변형 곡선은 성장판 고장과 같은 주요 그립 아티팩트를 제거하였다. 수프라피나투스와 아킬레스 건의 기계적 특성은 0.05 미만의 P 값을 가진 페어링되지 않은 T 테스트를 기반으로 섹스의 중요한 효과를 보여줍니다.마이크로 CT는 아킬레스 건의 길이를 따라 초라피나투스 힘줄의 길이를 따라 단면 영역을 성공적으로 측정했습니다. 각 해부학 사이트에는 효과적인 그립에 필요한 특정 설계 기준이 있음을 기억하는 것이 중요합니다. 따라서 각 설비에 대한 설계는 그에 따라 조정되어야 합니다.고장에 대한 인장 테스트 외에도 이러한 설비는 힘줄 피로 및 점성 특성에 대한 정보를 제공하는 순환 적재 테스트에 사용할 수 있습니다. 뮤린 힘줄의 기계적 특성을 특성화하는 것은 이러한 작은 조직을 잡는 데 어려움, 지루하고 시간이 많이 소요되는 표본 준비 방법 및 성장 판 골절을 포함하여 여러 가지 이유로 인해 문헌에서 흔하지 않습니다. 이 프로토콜은 관절 의 그립 실패를 제거하고 하루에 테스트 할 수있는 표본의 수를 세 배로 한 마우스 힘줄을 테스트하기위한 시간 효율적이고 재현 가능한 방법을 제공합니다.

Research

JoVE Journal

Bioengineering

Directed Cellular Self-Assembly to Fabricate Cell-Derived Tissue Rings for Biomechanical Analysis and Tissue Engineering

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do not have suitable autologous donor vessels due to disease progression or previous harvest. The aim of vascular tissue engineering is to develop a suitable alternative source for these bypass grafts. In addition, engineered vascular tissue may prove valuable as living vascular models to study cardiovascular diseases. Several promising approaches to engineering blood vessels have been explored, with many recent studies focusing on development and analysis of cell-based methods.2-5 Herein, we present a method to rapidly self-assemble cells into 3D tissue rings that can be used in vitro to model vascular tissues.
To do this, suspensions of smooth muscle cells are seeded into round-bottomed annular agarose wells. The non-adhesive properties of the agarose allow the cells to settle, aggregate and contract around a post at the center of the well to form a cohesive tissue ring.6,7 These rings can be cultured for several days prior to harvesting for mechanical, physiological, biochemical, or histological analysis. We have shown that these cell-derived tissue rings yield at 100-500 kPa ultimate tensile strength8 which exceeds the value reported for other tissue engineered vascular constructs cultured for similar durations (&lt;30 kPa).9,10 Our results demonstrate that robust cell-derived vascular tissue ring generation can be achieved within a short time period, and offers the opportunity for direct and quantitative assessment of the contributions of cells and cell-derived matrix (CDM) to vascular tissue structure and function.

This article outlines a versatile method to create cell-derived tissue rings by cellular self-assembly. Smooth muscle cells seeded into ring-shaped agarose wells aggregate and contract to form robust three-dimensional (3D) tissues within 7 days. Millimeter-scale tissue rings are conducive to mechanical testing and serve as building blocks for tissue assembly.

자기 조립 셀룰러이 Biomechanical 분석 및 조직 공학을위한 셀 유래 조직 반지를 조작하는 감독

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do ...

연구

생체공학

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

태반의 맞은 편에 생체이 물과 나노 물질의 전송 속도의 결정을 사용하여<em&gt; 생체</em&gt; 인간 태반 관류 모델

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Biomechanical Characterization of Human Soft Tissues Using Indentation and Tensile Testing

Regenerative medicine aims to engineer materials to replace or restore damaged or diseased organs. The mechanical properties of such materials should mimic the human tissues they are aiming to replace; to provide the required anatomical shape, the materials must be able to sustain the mechanical forces they will experience when implanted at the defect site. Although the mechanical properties of tissue-engineered scaffolds are of great importance, many human tissues that undergo restoration with engineered materials have not been fully biomechanically characterized. Several compressive and tensile protocols are reported for evaluating materials, but with large variability it is difficult to compare results between studies. Further complicating the studies is the often destructive nature of mechanical testing. Whilst an understanding of tissue failure is important, it is also important to have knowledge of the elastic and viscoelastic properties under more physiological loading conditions.
This report aims to provide a minimally destructive protocol to evaluate the compressive and tensile properties of human soft tissues. As examples of this technique, the tensile testing of skin and the compressive testing of cartilage are described. These protocols can also be directly applied to synthetic materials to ensure that the mechanical properties are similar to the native tissue. Protocols to assess the mechanical properties of human native tissue will allow a benchmark by which to create suitable tissue-engineered substitutes.

Tissue biomechanics is important for maintaining cell shape and function and for determining phenotype. This report demonstrates non-destructive mechanical protocols for characterizing elastic and viscoelastic properties of human soft tissues, which can be directly applied to tissue-engineered substrates to allow a close matching of engineered materials to native tissue.

들여 쓰기 및 인장 시험을 사용하여 인간의 부드러운 조직의 생체 역학적 특성

Regenerative medicine aims to engineer materials to replace or restore damaged or diseased organs. The mechanical properties of such materials should ...

An Experimental and Finite Element Protocol to Investigate the Transport of Neutral and Charged Solutes across Articular Cartilage

Osteoarthritis (OA) is a debilitating disease that is associated with degeneration of articular cartilage and subchondral bone. Degeneration of articular cartilage impairs its load-bearing function substantially as it experiences tremendous chemical degradation, i.e. proteoglycan loss and collagen fibril disruption. One promising way to investigate chemical damage mechanisms during OA is to expose the cartilage specimens to an external solute and monitor the diffusion of the molecules. The degree of cartilage damage (i.e. concentration and configuration of essential macromolecules) is associated with collisional energy loss of external solutes while moving across articular cartilage creates different diffusion characteristics compared to healthy cartilage. In this study, we introduce a protocol, which consists of several steps and is based on previously developed experimental micro-Computed Tomography (micro-CT) and finite element modeling. The transport of charged and uncharged iodinated molecules is first recorded using micro-CT, which is followed by applying biphasic-solute and multiphasic finite element models to obtain diffusion coefficients and fixed charge densities across cartilage zones.

We propose a protocol to investigate the transport of charged and uncharged molecules across articular cartilage with the aid of recently developed experimental and numerical methods.

실험적 및 유한 요소 의정서는 관절 연골에서 중립 및 충전 용질의 운송을 조사하기 위해

Osteoarthritis (OA) is a debilitating disease that is associated with degeneration of articular cartilage and subchondral bone. Degeneration of articular ...

A Test Bed to Examine Helmet Fit and Retention and Biomechanical Measures of Head and Neck Injury in Simulated Impact

Conventional wisdom and the language in international helmet testing and certification standards suggest that appropriate helmet fit and retention during an impact are important factors in protecting the helmet wearer from impact-induced injury. This manuscript aims to investigate impact-induced injury mechanisms in different helmet fit scenarios through analysis of simulated helmeted impacts with an anthropometric test device (ATD), an array of headform acceleration transducers and neck force/moment transducers, a dual high speed camera system, and helmet-fit force sensors developed in our research group based on Bragg gratings in optical fiber. To simulate impacts, an instrumented headform and flexible neck fall along a linear guide rail onto an anvil. The test bed allows simulation of head impact at speeds up to 8.3 m/s, onto impact surfaces that are both flat and angled. The headform is fit with a crash helmet and several fit scenarios can be simulated by making context specific adjustments to the helmet position index and/or helmet size. To quantify helmet retention, the movement of the helmet on the head is quantified using post-hoc image analysis. To quantify head and neck injury potential, biomechanical measures based on headform acceleration and neck force/moment are measured. These biomechanical measures, through comparison with established human tolerance curves, can estimate the risk of severe life threatening and/or mild diffuse brain injury and osteoligamentous neck injury. To our knowledge, the presented test-bed is the first developed specifically to assess biomechanical effects on head and neck injury relative to helmet fit and retention.

Using an anthropometric head and neck, optical fiber-based fit force transducers, an array of head acceleration and neck force/moment transducers, and a dual high speed camera system, we present a test bed to study helmet retention and effects on biomechanical measures of head and neck injury secondary to head impact.

테스트 침대 헬멧 적합 및 보존 및 머리와 목 부상 시뮬레이션된 영향의 Biomechanical 측정 검사

Conventional wisdom and the language in international helmet testing and certification standards suggest that appropriate helmet fit and retention during ...

Methods for In Vivo Biomechanical Testing on Brachial Plexus in Neonatal Piglets

Neonatal brachial plexus palsy (NBPP) is a stretch injury that occurs during the birthing process in nerve complexes located in the neck and shoulder regions, collectively referred to as the brachial plexus (BP). Despite recent advances in obstetrical care, the problem of NBPP continues to be a global health burden with an incidence of 1.5 cases per 1,000 live births. More severe types of this injury can cause permanent paralysis of the arm from the shoulder down. Prevention and treatment of NBPP warrants an understanding of the biomechanical and physiological responses of newborn BP nerves when subjected to stretch. Current knowledge of the newborn BP is extrapolated from adult animal or cadaveric BP tissue instead of in vivo neonatal BP tissue. This study describes an in vivo mechanical testing device and procedure to conduct in vivo biomechanical testing in neonatal piglets. The device consists of a clamp, actuator, load cell, and camera system that apply and monitor in vivo strains and loads until failure. The camera system also allows monitoring of the failure location during rupture. Overall, the presented method allows for a detailed biomechanical characterization of neonatal BP when subjected to stretch.

Methods for In Vivo Biomechanical Testing on Brachial Plexus in Neonatal Piglets

Presented here are methods to perform in vivo biomechanical testing on brachial plexus in a neonatal piglet model.

신생아 돼지의 상수 신경총에 생체 역학 테스트 방법

Neonatal brachial plexus palsy (NBPP) is a stretch injury that occurs during the birthing process in nerve complexes located in the neck and shoulder ...

Biaxial Basal Tone and Passive Testing of the Murine Reproductive System Using a Pressure Myograph

The female reproductive organs, specifically the vagina and cervix, are composed of various cellular components and a unique extracellular matrix (ECM). Smooth muscle cells exhibit a contractile function within the vaginal and cervical walls. Depending on the biochemical environment and the mechanical distension of the organ walls, the smooth muscle cells alter the contractile conditions. The contribution of the smooth muscle cells under baseline physiological conditions is classified as a basal tone. More specifically, a basal tone is the baseline partial constriction of smooth muscle cells in the absence of hormonal and neural stimulation. Furthermore, the ECM provides structural support for the organ walls and functions as a reservoir for biochemical cues. These biochemical cues are vital to various organ functions, such as inciting growth and maintaining homeostasis. The ECM of each organ is composed primarily of collagen fibers (mostly collagen types I, III, and V), elastic fibers, and glycosaminoglycans/proteoglycans. The composition and organization of the ECM dictate the mechanical properties of each organ. A change in ECM composition may lead to the development of reproductive pathologies, such as pelvic organ prolapse or premature cervical remodeling. Furthermore, changes in ECM microstructure and stiffness may alter smooth muscle cell activity and phenotype, thus resulting in the loss of the contractile force.
In this work, the reported protocols are used to assess the basal tone and passive mechanical properties of the nonpregnant murine vagina and cervix at 4-6 months of age in estrus. The organs were mounted in a commercially available pressure myograph and both pressure-diameter and force-length tests were performed. Sample data and data analysis techniques for the mechanical characterization of the reproductive organs are included. Such information may be useful for constructing mathematical models and rationally designing therapeutic interventions for women&#8217;s health pathologies.

This protocol utilized a commercially available pressure myograph system to perform pressure myograph testing on the murine vagina and cervix. Utilizing media with and without calcium, the contributions of the smooth muscle cells (SMC) basal tone and passive extracellular matrix (ECM) were isolated for the organs under estimated physiological conditions.

압력 근엽을 이용한 뮤린 생식 계통의 양축 기저톤 및 수동 테스트

The female reproductive organs, specifically the vagina and cervix, are composed of various cellular components and a unique extracellular matrix (ECM). ...

The Quantification of Injectability by Mechanical Testing

Injectable biomaterials are becoming increasingly popular for the minimally invasive delivery of drugs and cells. These materials are typically more viscous than traditional aqueous injections and may be semi-solid, therefore, their injectability cannot be assumed. This protocol describes a method to objectively assess the injectability of these materials using a standard mechanical tester. The syringe plunger is compressed by the crosshead at a set rate, and the force is measured. The maximum or plateau force value can then be used for comparison between samples, or to an absolute force limit. This protocol can be used with any material, and any syringe and needle size or geometry. The results obtained may be used to make decisions about formulations, syringe and needle sizes early in the translational process. Further, the effects of altering formulations on injectability may be quantified, and the optimum time to inject temporally changing materials determined. This method is also suitable as a reproducible way to examine the effects of injection on a material, to study phenomena such as self-healing and filter pressing or study the effects of injection on cells. This protocol is faster and more directly applicable to injectability than rotational rheology, and requires minimal post processing to obtain key values for direct comparisons.

Presented here is a protocol for quantitatively evaluating the injectability of a material through a syringe-needle system using a standard mechanical testing rig.

기계적 테스트를 통해 주사용 의 정량화

Injectable biomaterials are becoming increasingly popular for the minimally invasive delivery of drugs and cells. These materials are typically more ...

Optical Coherence Tomography Based Biomechanical Fluid-Structure Interaction Analysis of Coronary Atherosclerosis Progression

In this paper, we present a complete workflow for the biomechanical analysis of atherosclerotic plaque in the coronary vasculature. With atherosclerosis as one of the leading causes of global death, morbidity and economic burden, novel ways of analyzing and predicting its progression are needed. One such computational method is the use of fluid-structure interaction (FSI) to analyze the interaction between the blood flow and artery/plaque domains. Coupled with in vivo imaging, this approach could be tailored to each patient, assisting in differentiating between stable and unstable plaques. We outline the three-dimensional reconstruction process, making use of intravascular Optical Coherence Tomography (OCT) and invasive coronary angiography (ICA). The extraction of boundary conditions for the simulation, including replicating the three-dimensional motion of the artery, is discussed before the setup and analysis is conducted in a commercial finite element solver. The procedure for describing the highly nonlinear hyperelastic properties of the artery wall and the pulsatile blood velocity/pressure is outlined along with setting up the system coupling between the two domains. We demonstrate the procedure by analyzing a non-culprit, mildly stenotic, lipid-rich plaque in a patient following myocardial infarction. Established and emerging markers related to atherosclerotic plaque progression, such as wall shear stress and local normalized helicity, respectively, are discussed and related to the structural response in the artery wall and plaque. Finally, we translate the results to potential clinical relevance, discuss limitations, and outline areas for further development. The method described in this paper shows promise for aiding in the determination of sites at risk of atherosclerotic progression and, hence, could assist in managing the significant death, morbidity, and economic burden of atherosclerosis.

There is a need to determine which atherosclerotic lesions will progress in the coronary vasculature to guide intervention before myocardial infarction occurs. This article outlines the biomechanical modeling of arteries from Optical Coherence Tomography using fluid-structure interaction techniques in a commercial finite element solver to help predict this progression.

In this paper, we present a complete workflow for the biomechanical analysis of atherosclerotic plaque in the coronary vasculature. With atherosclerosis ...

Construction of a Human Aorta Smooth Muscle Cell Organ-On-A-Chip Model for Recapitulating Biomechanical Strain in the Aortic Wall

Conventional two-dimensional cell culture techniques and animal models have been used in the study of human thoracic aortic aneurysm and dissection (TAAD). However, human TAAD sometimes cannot be characterized by animal models. There is an apparent species gap between clinical human studies and animal experiments that may hinder the discovery of therapeutic drugs. In contrast, the conventional cell culture model is unable to simulate in vivo biomechanical stimuli. To this end, microfabrication and microfluidic techniques have developed greatly in recent years, providing novel techniques for establishing organoids-on-a-chip models that replicate the biomechanical microenvironment. In this study, a human aorta smooth muscle cell organ-on-a-chip (HASMC-OOC) model was developed to simulate the pathophysiological parameters of aortic biomechanics, including the amplitude and frequency of cyclic strain experienced by human aortic smooth muscle cells (HASMCs) that play a vital role in TAAD. In this model, the morphology of HASMCs became elongated in shape, aligned perpendicularly to the strain direction, and presented a more contractile phenotype under strain conditions than under static conventional conditions. This was consistent with the cell orientation and phenotype in native human aortic walls. Additionally, using bicuspid aortic valve-related TAAD (BAV-TAAD) and tricuspid aortic valve-related TAAD (TAV-TAAD) patient-derived primary HASMCs, we established BAV-TAAD and TAV-TAAD disease models, which replicate HASMC characteristics in TAAD. The HASMC-OOC model provides a novel in vitro platform that is complementary to animal models for further exploring the pathogenesis of TAAD and discovering therapeutic targets.

Here, we developed a human aorta smooth muscle cell organ-on-a-chip model to replicate the in vivo biomechanical strain of smooth muscle cells in the human aortic wall.

대동맥 벽의 생체 역학적 변형을 요약하기 위한 인간 대동맥 평활근 세포 장기 온 칩 모델 구축

Conventional two-dimensional cell culture techniques and animal models have been used in the study of human thoracic aortic aneurysm and dissection ...