저자는 자금 지원 NIH / NIDCR R00DE018212 (SPH), NIH/NIDCR-R01DE022032 (SPH), NIH / NIDCR T32 DE07306 (AJ, JDL), NIH / NCRR S10RR026645 (SPH) 및 예방과 원기를 회복시키는 치과 과학의 부서를 인정하고 구강 안면 과학, UCSF. 또한, 저자는 Xradia 친목 대학원 (AJ), Xradia 주식, 플레 즌튼, 캘리포니아를 인정합니다. 저자는 데이터의 사후 처리와 그녀의 도움 박사 캐서린 Grandfield, UCSF 감사, 박사 부부를. 스티븐 와이너와 길리 Naveh, 과학, 레호 보트 이스라엘의 Weizmann 연구소;의 현장 로딩 장치에 특정 그들의 통찰력있는 논의 박사 론 샤 하르, 예루살렘의 히브리 대학, 이스라엘. 저자는 또한 마이크로 XCT의 사용과에서 현장 로딩 장치 UCSF에서 생체 재료 및 생체 공학 MicroCT 이미징 시설을 감사드립니다.

축사와 안락사 :이 데모에 사용 된 모든 동물이 기관 동물 케어 및 사용위원회 (IACUC)와 국립 보건 연구소 (NIH)의 지침에 따라 무균 조건 하에서 보관 하였다. 표준 하드 펠렛 쥐 차우와 물 광고 리브 동물을 제공합니다. IACUC 승인 한 UCSF의 표준 프로토콜에 따라 이산화탄소 질식, 양자 개흉술의 2 단계 방법을 통해 동물을 안락사. 조직의 저하를 방지하기 위해 동물의 희생을 24 시간 이내에 생체 역학 테스트를 수행합니다. 1. 준비 및 쥐 하악골이나 상악골의 해부 <ol><li> 척골 구상 과정과 과두 과정 (그림 1) (15)를 포함하여 전체 하악골을 유지하면서 부드럽게 막 조직과 근육 조직 첨부 파일을 단절하여 쥐의 턱을 제거합니다. </li><li> 자동차로 구분 hemimandiblesefully 메스 블레이드 하악 치골의 섬유 조직을 절단. 참고 : 물리적으로 2 차 몰의 생체 역학 테스트를 방해하는 경우 관상 동맥과 과두 프로세스, 하악 하악 (그림 1)를 제거해야합니다. </li><li> 몰의 로딩을 방해하지로 펄프 챔버를 노출하지 않고 앞니를 잘라. </li></ol> 2. 현장 압축 하중에 대한 표본 준비 (그림 2) <ol><li> 이전에 현장 로딩 장치 (그림 2A)에서에 넣기로 실험 표본보다 훨씬 더 뻣뻣 재료를 사용하여 스틸 스텁에 시편을 고정시킨다. 주 : 폴리 메틸 메타 크릴 레이트 (PMMA)가있는 경우, 치과 탐색기를 사용하여 제거하고, 본 연구와 과량의 시료를 고정화 하였다. </li><li> 모두 직선을​​ 사용하여 AFM 금속 시편 디스크와 관심 평행의 몰 (들)의 교합면을 맞 춥니 다면 (즉, 근심-말단과 구강 - 언어). </li><li> 어금니 주변 둔기로 저점을 만듭니다. 참고 :이 공간은 여분의 액체를 포함하고 현장 로딩하는 동안 조직의 수분을 유지하기 위해 &quot;해자&quot;의 역할을해야한다. </li><li> 치과 합성을 사용하여 동심 (그림 2B) 또는 편심 (그림 2C) 로딩 구축 치아 표면을 준비합니다. 15 초 동안 교합면에 35 % 인산 젤로 또한 치아의 표면을 에칭. </li><li> 탈 이온수로 철저하게 에칭을 씻어​​ 공기 / 물 주사기 또는 압축 공기 용기를 사용하여 표면을 건조. 탐험가로, 얇은 층에 열려있는 교두에 접착제 한 방울을 확산. 치과 치료 빛과 복합 치료. 참고 : 복합 재료를 포함한 모든 단계는 램프에서 직접 빛없이 수행해야합니다. 이러한 조건은 바람직하지 않게 중합 공정을 가속화하고 coul 것D 합성의 적절한 배치를 방지합니다. 실내 조명이 허용됩니다. </li><li> 미세한 메스 또는 면도날 인접 치아에서 과잉 접착제를 제거합니다. </li><li> 표면의 준비 다음 표면에 유동성 치과 합성을 놓고 치과 탐색기를 사용하여 관심의 몰 (들)의 홈에 확산. </li><li> 30 초 동안 치과 치료 빛에 복합 노출. </li><li> 그 몰 (들)과 30 초 동안 빛 치료의 교합면에서, 치과 수지 조성물을 사용하여 3 ~ 4 ㎜의 교합 형성을 금형. </li><li> 직선 및 고속 핸드 피스를 사용하여 모든 표본 일관된 로딩 방식을 사용하도록 평평한 표면에 평행으로 복합 상승의 상단을 줄일 수 있습니다. 주 : 생체 역학 테스트하는 동안 다른 시료 50 ㎎ / ㎖ 페니실린과 트리스 - 인산염 완충 용액 (TBS)에 저장하고, 스트렙토 마이신 (15)되어야한다. </li></ol> 3. 로딩 장치 드리프트강성, 소재 부동산의 차별화 된 기능, 섬유 공동의 현장 로딩 중 <ol><li> 그림 (b)에 나타낸 바와 같이, 균일 한 동안의로드 단계의 모루의 복합 상승과 시편, 테스트를 고정합니다. </li><li> 동심 또는 편심 하중 (그림 2B 및 2C)를 확인하기 위해 유한 부하에 시료를로드하여 다음 복합체의 표면에 관절 용지를 넣습니다. </li><li> 표본의 수분을 보장하기 위해 표본 주위에 TBS에 젖은 킴 와이프를 놓습니다. 시편 주위에 저점을 확인하고 촬영하는 동안 수화 기관을 유지하기 위해 TBS로 채 웁니다. </li><li> hemimandible의 고정 다음 변위 속도로 원하는 피크 부하에 몰을 압축하는 데벤 (Deben) 소프트웨어에 입력 피크 하중과 변위 속도. 참고 : 재료가 부하 센서 감도 = 0.1 (시간이 지남에 압축 전형적인 판독은 반동적 부하를 포함해야N). 로드 시간과 변위 시간에서, 압축 된 자료에 대한 하중 - 변위 곡선은 16-18를 얻을 수 있어야합니다. 로드 사이클로부터 수집 된 데이터를 사용하여, 공동의 여러가지 특성도 결정될 수있다. 조인트의 강성은 선형 부 변위 곡선 19 대 부하의 적재 단계 (데이터의 마지막 약 30 %)의 기울기를 고려하여 계산되어야한다. </li></ol> 4. 포스 포 산 (PTA)와 연조직, PDL의 염색, 참고 : X-선 감쇠 대비를 강화하기 위해, PDL은 5 % PTA 솔루션 (20)으로 염색해야한다. <ol><li> 백필 PTA 염색 깨끗한 1.8 ㎖의 유리 carpule에 솔루션 및 주사기에로드 carpule를 놓습니다. </li><li> 그 몰 주변 치주 조직에 구조적 손상을 방지하기 위해 인접 치아의 PDL 공간에 솔루션 천천히 (5 분 / carpule)를 주입. 참고 : 위의 단계 ㄱ한다용액의 약 5 완전 carpules (15 ㎖)까지 반복 전자가 주입되고 주변 조직에 흐르게된다. 수험 공부를 표본은 나머지 PTA 솔루션 (8 시간)에 하룻밤 담가 할 수 있습니다. </li></ol> 5. 추천 μ-XCT 스캔 설정 다음 스캔 설정 M-XCT를 수행 <table border="1" cellpadding="0" cellspacing="0"><tbody><tr><td> 목표 확대 </td><td> 4X, 10X </td></tr><tr><td></td><td> 1800 이미지 </td></tr><tr><td> X 선관 전압 </td><td> 75 KVP (PTA 스테인드 샘플 50 KVP) </td></tr><tr><td></td><td> 8 W </td></tr><tr><td> 노출 시간 </td><td> ~ 8-25 초 * </td></tr><tr><td></td><td> ~ 4 μm의 (4X 목적), 1 ~ 2 μm의 (10X 목적) ** </td></tr></tbody></table> * 노광 시간은 기하학과 시험편과 X-선관 VO의 광학 밀도에 기초하여 변화 할 수있다ltage. * 실제 픽셀 해상도가 다소 소스, 시험편 및 검출기의 구성에 따라 다를 것이다.

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Biomech. 40, 634-644 (2007).</li><li>Lin, J. D., 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=Biomechanics+of+a+bone-periodontal+ligament-tooth+fibrous+joint.">Biomechanics of a bone-periodontal ligament-tooth fibrous joint.</a> J. Biomech. 46, 443-449 (2013).</li><li>Quintarelli, G., Zito, R., Cifonelli, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+phosphotungstic+acid+staining.">On phosphotungstic acid staining.</a> I. J. Histochem. Cytochem. 19, 641-647 (1971).</li><li>Quintarelli, G., Cifonelli, J. A., Zito, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+phosphotungstic+acid+staining.">On phosphotungstic acid staining.</a> II. J. Histochem. Cytochem. 19, 648-653 (1971).</li><li>Quintarelli, G., Bellocci, M., Geremia, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+phosphotungstic+acid+staining.+IV.+Selectivity+of+the+staining+reaction.">On phosphotungstic acid staining. IV. Selectivity of the staining reaction.</a> J. Histochem. Cytochem. 21, 155-160 (1973).</li><li>Crabtree, W. N., Murphy, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+value+of+ethanol+as+a+fixative+in+urinary+cytology.">The value of ethanol as a fixative in urinary cytology.</a> Acta Cytol. 24, 452-455 (1980).</li></ol>

저자가 공개하는 게 없다.

이 프로토콜을 수립하는 첫번째 단계는 강체를 사용하여로드 프레임의 강성을 평가 참여. 결과에 따라, 강성이 상당히 낮은 강성 값과 시험편의 추가 테스트를위한 장착 장치의 사용을 가능하게 높았다. 두 번째 단계는 강체 다른 가교 밀도 PDMS 재료 및 섬유상 조인트를 사용하여 생성 된 하역 곡선의 두 단계를 이용하여 서로 다른 강성 값을 구별하는 기기의 능력을 강조했다. 백래쉬 단계 동안?...

여러 가지 실험 방법은 diarthrodial 및 섬유 관절의 생체 역학 조사를 계속 사용. 치아 기관의 역학에 대한 구체적인 방법은 스트레인 게이지 1-3, 광탄성 방법 4, 5, 모아레 간섭 6, 7, 전자 스페 클 간섭 8, 디지털 이미지의 상관 관계 (DIC) 9-14의 사용을 포함한다. 이 연구에서 혁신적인 접근 방식은 섬유 관절의 내부 구조를 노출하는 X-선을 사용하여 (부드러운 영역으로 구성된 광물 조직과 자신의 인터페이스를하고 인대 등의 조직을 인터페이스) 생체 내 조건에 해당하는 부하에서 비 침습적 영상이 포함되어 있습니다. 마이크로 X-선 현미경에 결합 시츄 장착 장치가 사용될 것이다. 로드 시간과 하중 - 변위 곡선은 갓 수확 쥐 헤미 - 하악 내 관심의 몰로드로 수집됩니다. M1) 무부하 때로드 때 2)과 동심으로 편심로드 : 본 연구에서 제시된 방법의 아인 목표로 조건을 비교하여 치아 뼈의 3 차원 형태의 효과를 강조하고있다. 잘라 시험편에 대한 필요성을 제거하고, 습식 조건 하에서 전체 그대로 기관에 실험을 수행하여 3D 응력 상태의 최대 보존을 허용한다. 이것은 다양한로드 시나리오에서 복잡한 동적 프로세스를 이해하는 연구의 새로운 영역을 엽니 다. 본 연구에서는, 스프 라그 돌리 쥐의 손상되지 않은 섬유 관절 내에서 테스트 PDL의 생체 역학에 대한 방법은 최적의 생체 공학 모델 시스템으로 간주 관절 상세히 설명한다. 실험들은 기관 수준의 생체 역학 관련으로 관절의 세 가지 중요한 기능을 강조하기 위해 수화 된 상태에서 저작 부하의 시뮬레이션이 포함됩니다. 3 점 포함됩니다 변위 대 1) 반동을 :치아 치조 소켓 내에서 변위 및 하중에의 반동 반응, 2) 3 차원 (3D) 공간 구성 및 morphometrics : 폐포 소켓과 치아의 기하학적 관계와 수의 변동으로 인해 판독 1, 2, 3) 변경 동심의 편심 하중, 즉로드 축. 제안 된 기술의 세 가지 기본 판독 인해 기능적 요구의 변화 및 ​​/ 또는 질병의 하나 척추 관절의 적응 특성을 조사하기 위해 적용될 수있다. 상기 판독 값의 변화, 다른 로딩 속도에서 구체적으로 변위 반동 부하 사이의 상관 관계, 그리고 반동로드 시간과 하중 - 변위 곡선의 결과는 공동 역학의 전반적인 변화를 강조하기 위해 적용 할 수 있습니다. 제안 된 프로토콜의 효능은 3D morphometrics과 관절의 전반적인 생체 역학에 기계적 시험 판독 결합에 의해 평가됩니다.

<table border="0" cellpadding="0" cellspacing="0" width="652">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Bard Parker Blade</td>
			<td style="width:101px;">BD</td>
			<td style="width:123px;">MEDC-001054</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">AFM metal disk</td>
			<td style="width:101px;">Ted Pella</td>
			<td style="width:123px;">16218</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Polymethyl methacrylate&#160;</td>
			<td style="width:101px;">GC America</td>
			<td style="width:123px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Uni-Etch</td>
			<td style="width:101px;">Bisco</td>
			<td style="width:123px;">E5502EBM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Optibond Solo Plus</td>
			<td style="width:101px;">Kerr Corp</td>
			<td style="width:123px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Filtek Flow</td>
			<td style="width:101px;">3M</td>
			<td style="width:123px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Hurculite Ultra</td>
			<td style="width:101px;">Kerr</td>
			<td style="width:123px;">34346</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Tris buffer</td>
			<td style="width:101px;">Mediatech Inc.</td>
			<td style="width:123px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Articulating paper</td>
			<td style="width:101px;"></td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Phosphotungstic Acid</td>
			<td style="width:101px;">Sigma Aldrich</td>
			<td style="width:123px;">HT152</td>
			<td></td>
		</tr>
	</tbody>
</table>

in situ compressive loading and correlative noninvasive imaging of the bone-periodontal ligament-tooth fibrous joint

이 연구는 소설의 역학 시험 프로토콜을 보여줍니다. 이 프로토콜의 장점은 따라서 모의 생리적 하중 및 습식 조건 하에서 내부 구조 요소의 시각화를 가능하게 고해상 X-레이 현미경에 결합 시츄 장착 장치에서의 사용을 포함한다. 실험 표본은 그대로 뼈, 치주 인대 (PDL) - 치아 섬유 관절을 포함 할 것이다. 1) 변위 대 반동 : 치아 변위 폐포 소켓 사이 및 로딩, 2) 3 차원 (3D) 공간적 구성보기에 그 반동 응답 결과들은 장기 레벨 생체 역학에 적용 할 수있는 프로토콜의 세 가지 중요한 기능을 설명 할 것이다 및 morphometrics : 기하학적 인 폐포 소켓과 치아의 관계, 인한, 즉 편심 하중에 동심에서로드 축 변화에 판독 1, 2, 3)으로 변경됩니다. 제안 된 프로토콜의 효능 기계 테 커플 링에 의해 평가 될 것입니다3D morphometrics과 관절의 전반적인 생체 역학에 따끔 판독. 또한,이 기술은 특히 반동로드 전에 섬유상 관절 단층 촬영 취득에, 실험 조건을 평형화 필요성을 강조한다. 그것은 제안 된 프로토콜은 생체 조건에서 표본을 테스트로 제한하고, 부드러운 조직 기계적인 반응을 시각화하는 조영제의 사용은 조직과 기관 수준의 생체 역학에 대한 잘못된 결론으로 이어질 수 있음을 유의해야한다.

로딩 장치 &quot;반발&quot;일정한 부하가 &quot;푸시&quot;, 강성 및 시스템 드리프트 추정 백래쉬 : 사이클의 로딩 및 언 로딩 부분 사이에 진정한 하역 표본 멀리 가기 턱 (그림 3)에서 끌어 즉로, 개시하기 전에 모터에서 역 기어하는 동안 3 초 일시 정지가 존재한다. 이 기간은 시스템이 턱의 개구를 폐쇄로 전환하려고 할 때 시간 세그...

Watch this Scientific Journal Video about In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint at JoVE.com

In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint

본 연구에서는, 섬유 관절 생체 역학에 대한 마이크로 X-선 계산 tomography 결합 시츄 장착 장치에서의 사용이 논의 될 것이다. 관절 생체 역학의 전반적인 변화에 식별 실험 판독이 포함됩니다 : 1) 반동 변위 대 힘, 폐포 소켓 내에서, 즉 치아의 변위 및 하중에의 반동 반응을, 2) 3 차원 (3D) 공간 구성 및 morphometrics, 즉 기하학적 로딩이 축, 즉 동심 또는 편심 하중의 변화에 폐포 소켓 치아 및 판독 1, 2, 3) 변화의 관계.

현장 압축 하중과 뼈, 치주 인대 치아 섬유 공동의 상관적인 비 침습 이미징

This study demonstrates a novel biomechanics testing protocol. The advantage of this protocol includes the use of an in situ loading device coupled to a ...

in-situ-compressive-loading-correlative-noninvasive-imaging-bone

Research

JoVE Journal

Bioengineering

In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint

13.3K 조회수.  University of California San Francisco.  본 연구에서는, 섬유 관절 생체 역학에 대한 마이크로 X-선 계산 tomography 결합 시츄 장착 장치에서의 사용이 논의 될 것이다. 관절 생체 역학의 전반적인 변화에 식별 실험 판독이 포함됩니다 : 1) 반동 변위 대 힘, 폐포 소켓 내에서, 즉 치아의 변위 및 하중에의 반동 반응을, 2) 3 차원 (3D) 공간 구성 및 morphometrics, 즉 기하학적 로딩이 축, ?...

비디오: In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution. Traditionally, multiple experiments are run in parallel, with individual samples removed from the study at sequential time points for evaluation by light microscopy. Several intravital techniques have been developed, with confocal, multiphoton, and second harmonic microscopy all demonstrating their ability to be used for imaging in situ 1. With these systems, however, the required infrastructure is complex and expensive, involving scanning laser systems and complex light sources. Here we present a protocol for the design and assembly of a high-resolution microendoscope which can be built in a day using off-the-shelf components for under US$5,000. The platform offers flexibility in terms of image resolution, field-of-view, and operating wavelength, and we describe how these parameters can be easily modified to meet the specific needs of the end user.
We and others have explored the use of the high-resolution microendoscope (HRME) in in vitro cell culture 2-5, in excised 6 and living animal tissues 2,5, and in human tissues in vivo 2,7. Users have reported the use of several different fluorescent contrast agents, including proflavine 2-4, benzoporphyrin-derivative monoacid ring A (BPD-MA) 5, and fluoroscein 6,7, all of which have received full, or investigational approval from the FDA for use in human subjects. High-resolution microendoscopy, in the form described here, may appeal to a wide range of researchers working in the basic and clinical sciences. The technique offers an effective and economical approach which complements traditional benchtop microscopy, by enabling the user to perform high-resolution, longitudinal imaging in situ.

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

In many biological and clinical situations it is advantageous to study cellular processes as they evolve in their native microenvironment. Here we describe the assembly and use of a low-cost fiber-optic microscope which can provide real time imaging in cell culture, animal studies, and clinical patient studies.

고해상도 광섬유 Microendoscopy에 대한 현장에서 세포 이미징

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution.  ...

연구

생체공학

Demonstrating the Uses of the Novel Gravitational Force Spectrometer to Stretch and Measure Fibrous Proteins

The study of macromolecular structure has become critical to the elucidation of molecular mechanisms and function. There are several limited, but important bioinstruments capable of testing the force dependence of structural features in proteins. Scale has been a limiting parameter on how accurately researchers can peer into the nanomechanical world of molecules, such as nucleic acids, enzymes, and motor proteins that perform life-sustaining work. Atomic force microscopy (AFM) is well tuned to determine native structures of fibrous proteins with a distance resolution on par with electron microscopy. However, in AFM force studies, the forces are typically much higher than a single molecule might experience 1, 2. Optical traps (OT) are very good at determining the relative distance between the trapped beads and they can impart very small forces 3. However, they do not yield accurate absolute lengths of the molecules under study. Molecular simulations provide supportive information to such experiments, but are limited in the ability to handle the same large molecular sizes, long time frames, and convince some researchers in the absence of other supporting evidence2, 4.
The gravitational force spectrometer (GFS) fills a critical niche in the arsenal of an investigator by providing a unique combination of abilities. This instrument is capable of generating forces typically with 98% or better accuracy from the femtonewton range to the nanonewton range. The distance measurements currently are capable of resolving the absolute molecular length down to five nanometers, and relative bead pair separation distances with a precision similar to an optical trap. Also, the GFS can determine stretching or uncoiling where the force is near equilibrium, or provide a graded force to juxtapose against any measured structural changes. It is even possible to determine how many amino acid residues are involved in uncoiling events under physiological force loads 2. Unlike in other methods where there is extensive force calibration that must precede any assay, the GFS requires no such force calibration 5. By complementing the strengths of other methods, the GFS will bridge gaps in understanding the nanomechanics of vital proteins and other macromolecules.

This is a step-by step guide showing the purpose, operation, and representative results from the novel gravitational force spectrometer.

섬유 단백질을 늘이기 및 측정 소설 중력 포스 분석기의 사용을 보여주는

The study of macromolecular structure has become critical to the elucidation of molecular mechanisms and function. There are several limited, but ...

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

Collagen fibrils are present in the extracellular matrix of animal tissue to provide structural scaffolding and mechanical strength. These native collagen fibrils have a characteristic banding periodicity of ~67 nm and are formed in vivo through the hierarchical assembly of Type I collagen monomers, which are 300 nm in length and 1.4 nm in diameter. In vitro, by varying the conditions to which the monomer building blocks are exposed, unique structures ranging in length scales up to 50 microns can be constructed, including not only native type fibrils, but also fibrous long spacing and segmental long spacing collagen. Herein, we present procedures for forming the three different collagen structures from a common commercially available collagen monomer. Using the protocols that we and others have published in the past to make these three types typically lead to mixtures of structures. In particular, unbanded fibrils were commonly found when making native collagen, and native fibrils were often present when making fibrous long spacing collagen. These new procedures have the advantage of producing the desired collagen fibril type almost exclusively. The formation of the desired structures is verified by imaging using an atomic force microscope.

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

Simple and reproducible procedures are described for making three structurally distinct collagen assemblies from a common commercially available Type I collagen monomer. Native type, fibrous long spacing or segmental long spacing collagen can be constructed by varying the conditions to which the 300 nm long and 1.4 nm diameter monomer building block is exposed.

기본, 섬유 긴 간격 및 Segmental 긴 간격 콜라겐의 체외 합성에

Collagen fibrils are present in the  extracellular matrix of animal tissue to provide structural scaffolding and  mechanical strength. These native ...

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

조직 공학을위한 이축 기계 로딩 생물 반응기의 설계

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

FIBS-enabled Noninvasive Metabolic Profiling

In the era of computational biology, new high throughput experimental systems are necessary in order to populate and refine models so that they can be validated for predictive purposes. Ideally such systems would be low volume, which precludes sampling and destructive analyses when time course data are to be obtained. What is needed is an in situ monitoring tool which can report the necessary information in real-time and noninvasively. An interesting option is the use of fluorescent, protein-based in vivo biological sensors as reporters of intracellular concentrations. One particular class of in vivo biosensors that has found applications in metabolite quantification is based on F&#246;rster Resonance Energy Transfer (FRET) between two fluorescent proteins connected by a ligand binding domain. FRET integrated biological sensors (FIBS) are constitutively produced within the cell line, they have fast response times and their spectral characteristics change based on the concentration of metabolite within the cell. In this paper, the method for constructing Chinese hamster ovary (CHO) cell lines that constitutively express a FIBS for glucose and glutamine and calibrating the FIBS in vivo in batch cell culture in order to enable future quantification of intracellular metabolite concentration is described. Data from fed-batch CHO cell cultures demonstrates that the FIBS was able in each case to detect the resulting change in the intracellular concentration. Using the fluorescent signal from the FIBS and the previously constructed calibration curve, the intracellular concentration was accurately determined as confirmed by an independent enzymatic assay.

A description of how to calibrate F&#246;rster Resonance Energy Transfer integrated biological sensors (FIBS) for in situ metabolic profiling is presented.&#160;The FIBS can be used to measure intracellular levels of metabolites noninvasively aiding in the development of metabolic models and high throughput screening of bioprocess conditions.

비 침습 대사 프로파일 링에게 FIBS 지원

In the era of computational biology, new high throughput experimental systems are necessary in order to populate and refine models so that they can be ...

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM) of whole cells in hydrated state. Fluorescent quantum dots (QDs) were coupled to EGFR via a two-step labeling protocol, providing an efficient and specific protein labeling, while avoiding label-induced clustering of the receptor. Fluorescence microscopy provided overview images of the cellular locations of the EGFR. The scanning transmission electron microscopy (STEM) detector was used to detect the QD labels with nanoscale resolution. The resulting correlative images provide data of the cellular EGFR distribution, and the stoichiometry at the single molecular level in the natural context of the hydrated intact cell. ESEM-STEM images revealed the receptor to be present as monomer, as homodimer, and in small clusters. Labeling with two different QDs, i.e.,&#160;one emitting at 655 nm and at 800 revealed similar characteristic results.

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative light- and electron microscopy of whole cells in hydrated state. The label contained fluorescent quantum dots. The protocol can be used to study the stoichiometry of EGFR at the single molecule level.

상호 현미경을 사용하여 본래 세포의 표피 성장 인자 수용체의 화학 양론을 공부

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence ...

Optimized Setup and Protocol for Magnetic Domain Imaging with In Situ Hysteresis Measurement

This paper elaborates the sample preparation protocols required to obtain optimal domain patterns using the Bitter method, focusing on the extra steps compared to standard metallographic sample preparation procedures. The paper proposes a novel bespoke rig for dynamic domain imaging with in situ BH (magnetic hysteresis) measurements and elaborates the protocols for the sensor preparation and the use of the rig to ensure accurate BH measurement. The protocols for static and ordinary dynamic domain imaging (without in situ BH measurements) are also presented. The reported method takes advantage of the convenience and high sensitivity of the traditional Bitter method and enables in situ BH measurement without interrupting or interfering with the domain wall movement processes. This facilitates establishing a direct and quantitative link between the domain wall movement processes&#8211;microstructural feature interactions in ferritic steels with their BH loops. This method is anticipated to become a useful tool for the fundamental study of microstructure&#8211;magnetic property relationships in steels and to help interpret the electromagnetic sensor signals for non-destructive evaluation of steel microstructures.

Optimized Setup and Protocol for Magnetic Domain Imaging with In Situ Hysteresis Measurement

This paper elaborates the sample and sensor preparation procedures and the protocols for using the test rig particularly for dynamic domain imaging with in situ BH measurements in order to achieve optimal domain pattern quality and accurate BH measurements.

최적화 된 설치 및 히스테리시스 측정 현장에서 자기 도메인 이미징에 대 한 프로토콜

This paper elaborates the sample preparation protocols required to obtain optimal domain patterns using the Bitter method, focusing on the extra steps ...

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and allostasis. Moreover, dysregulated protease activity drives pathogenesis and is a functional biomarker of diseases such as cancer; therefore, the ability to detect protease activity in vivo may provide clinically relevant information for biomedical diagnostics. The goal of this protocol is to create nanosensors that probe for protease activity in vivo by producing a quantifiable signal in urine. These protease nanosensors consist of two components: a nanoparticle and substrate. The nanoparticle functions to increase circulation half-life and substrate delivery to target disease sites. The substrate is a short peptide sequence (6-8 AA), which is designed to be specific to a target protease or group of proteases. The substrate is conjugated to the surface of the nanoparticle and is terminated by a reporter, such as a fluorescent marker, for detection. As dysregulated proteases cleave the peptide substrate, the reporter is filtered into urine for quantification as a biomarker of protease activity. Herein we describe construction of a nanosensor for matrix metalloproteinase 9 (MMP9), which is associated with tumor progression and metastasis, for detection of colorectal cancer in a mouse model.

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are tightly regulated enzymes involved in fundamental biological processes, and dysregulated protease activity drives progression of complex diseases such as cancer. This method&#39;s goal is to create nanosensors that measure protease activity in vivo by producing a cleavage signal that is detectable from host urine and discriminates disease.

Nanosensors 프로 테아 제 활동에 감지 비보에 비 침 투 적인 진단에 대 한

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and ...

Correlative Light Electron Microscopy (CLEM) for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing virus-infected or transfected cells via EM are the low efficiencies of infection or transfection, hindering the examination of these cells. In order to overcome this difficulty, light microscopy (LM) can be performed first to allocate the subpopulation of infected or transfected cells. Thus, taking advantage of the use of fluorescent proteins (FPs) fused to viral proteins, LM is used here to record the positions of the "positive-transfected" cells, expressing a FP and growing on a support with an alphanumeric pattern. Subsequently, cells are further processed for EM via high pressure freezing (HPF), freeze substitution (FS) and resin embedding. The ultra-rapid freezing step ensures excellent membrane preservation of the selected cells that can then be analyzed at the ultrastructural level by transmission electron microscopy (TEM). Here, a step-by-step correlative light electron microscopy (CLEM) workflow is provided, describing sample preparation, imaging and correlation in detail. The experimental design can be also applied to address many cell biology questions.

Correlative Light Electron Microscopy CLEM for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

A correlative light electron microscopy (CLEM) method is applied to image virus-induced intracellular structures via electron microscopy (EM) in cells that are previously selected by light microscopy (LM). LM and EM are combined as a hybrid imaging approach to achieve an integrated view of virus-host interactions.

상호 빛 전자 현미경 검사 법 (클레 멘 타인) 추적 하 고 바이러스 성 단백질을 이미징 관련 Cryo 움직일 셀 구조

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing ...

Isolation, Expansion, and Differentiation of Mesenchymal Stem Cells from the Infrapatellar Fat Pad of the Goat Stifle Joint

The IFP, present in the knee joint, serves as a promising source of MSCs. The IFP is an easily accessible tissue as it is routinely resected and discarded during arthroscopic procedures and knee replacement surgeries. Additionally, its removal is associated with minimal donor site morbidity. Recent studies have demonstrated that IFP-MSCs do not lose their proliferation capacity during in vitro expansion and have age-independent osteogenic differentiation potential. IFP-MSCs possess superior chondrogenic differentiation potential compared to bone marrow-derived MSCs (BMSCs) and adipose-derived stem cells (ADSCs). Although these cells are easily obtainable from aged and diseased patients, their effectiveness is limited. Hence, using IFP-MSCs from healthy donors is important to determine their efficacy in biomedical applications. As access to a healthy human donor is challenging, animal models could be a better alternative to enable fundamental understanding. Large animals such as dogs, horses, sheep, and goats play a crucial role in translational research. Amongst these, the goat could be a preferred model since the stifle joint of the goat has the closest anatomy to the human knee joint. Moreover, goat-IFP can fulfill the higher MSC numbers needed for tissue regeneration applications. Furthermore, low cost, availability, and compliance with the 3R principles for animal research make them an attractive model. This study demonstrates a simple protocol for isolating IFP-MSCs from the stifle joint of goats and in vitro culture conditions for their expansion and differentiation. The aseptically isolated IFP from the goat was washed, minced, and digested enzymatically. After filtration and centrifugation, the collected cells were cultured. These cells were adherent, had MSCs-like morphology, and demonstrated remarkable clonogenic ability. Further, they differentiated into adipogenic, chondrogenic, and osteogenic lineages, demonstrating their multipotency. In conclusion, the study demonstrates the isolation and expansion of MSCs, which show potential in tissue engineering and regenerative medicine applications.

Infrapatellar fat pad mesenchymal&#160;stem cells (IFP-MSCs) can be isolated easily from the infrapatellar fat pad of the knee joint. They proliferate well in vitro, form CFU-F colonies, and differentiate into adipogenic, chondrogenic, and osteogenic lineages. Herein, the methodology for the isolation, expansion, and differentiation of IFP-MSCs from goat stifle joint is provided.

염소 스티플 관절의 슬개골 하 지방 패드에서 중간엽 줄기 세포의 분리, 확장 및 분화

The IFP, present in the knee joint, serves as a promising source of MSCs. The IFP is an easily accessible tissue as it is routinely resected and discarded ...