우리는 닥터 롭 노박을 포함하여 수년에 걸쳐 CT-FIRE 및 CurveAlign에 많은 기여자와 사용자에게 감사드립니다, 캐롤린 펠케 박사, 제레미 브레드펠트 박사, 구네트 메타, 앤드류 라이히트 박사, 아디브 케이호스라비 박사, 맷 콘클린 박사, 제인 다람쥐, 파올로 프로방스자노 박사, 브렌다 오글 박사, 패트리샤 킬리 박사, 조셉 셀체프스키 박사, 스앤 스니크 박사 이 작품은 반도체 연구 회사, 모드리지 연구소, NIH보조금 R01CA199996, R01CA181385 및 U54CA210190을 K.W.E에 지원했습니다.

저자는 공개 할 것이 없습니다.

이 프로토콜은 피브릴라 콜라겐 정량화를 위해 CT-FIRE 및 CurveAlign의 사용을 설명하고 콜라겐 섬유 또는 CT-FIRE 또는 CurveAlign에 의해 분석에 적합한 다른 라인 과 같은 또는 섬유와 같은 길쭉한 구조를 가진 모든 이미지에 적용 될 수 있습니다. 예를 들어 엘라스틴 또는 탄성 섬유는 이 플랫폼에서 유사한 방식으로 처리될 수 있습니다. 우리는 계산 생성 합성 섬유(21)에두 도구를 모?...

피브릴라 콜라겐은 눈에 띄는 구조ECM 구성 요소입니다. 이들의 조직 변화는 조직 기능에 영향을 미치고 골발생불완전성 1,심장 기능 장애2,상처 치유3에서 유방4,5,6,난소7,8,신장9,췌장암10을포함한 다양한 유형의 암에 이르는 많은 질병의 진행과 연관될 가능성이 있다. 많은 확립된 이미징 양식은 제2 고조파 생성 현미경검사법(11),밝은 필드 또는 형광 현미경 또는 편광 광 현미경 검사법과 함께 피브릴라 콜라겐을 시각화하는 데 사용할 수 있으며, 액정 계 편광 현미경 검사법(LC-PolScope) 13, 전자 현미경검사법14. 피브릴라 콜라겐 조직의 중요성이 명확해지고 이러한 방법의 사용이 증가함에 따라 콜라겐 섬유 분석 접근 방식의 개선필요성도 커지고 있습니다.
피브릴라 콜라겐의 자동화된 측정을 위한 계산 방법을 개발하기 위한 많은 노력이 있었습니다. 자유롭게 사용할 수 있는 소프트웨어 도구는 주로 이미지타일(17)에대한 제1유도체 또는 구조 텐서 또는 포리에 변환 기반 스펙트럼 분석을 채택하여 섬유 정렬 또는 방향의 계산에 초점을 맞추고 있다. 이러한 모든 도구는 수동 단계의 요구 사항, 시끄러운 배경에서 섬유 모서리 감지의 부정확성 또는 지역화 된 기능 특성화의 부족과 같은 제한사항이 적용됩니다.
자유롭게 사용할 수 있는 다른 오픈 소스 무료 소프트웨어 도구와 비교하여 이 프로토콜에 설명된 메서드는 최적의 다중 규모의 방향성 이미지 표현 방법인 CT를 사용하여 피브릴라 콜라겐 이미지에서 노이즈를 제거하고 섬유 가장자리를 향상시키거나 추적합니다. 위치 및 방향에 대한 정보는 간접 픽셀 또는 창 별 정보를 사용하여 섬유 조직의 메트릭을 추론하는 것이 아니라 섬유에서 직접 제공될 수 있습니다. 이 CT 기반프레임워크(18,19,20,21)는 주로 두 개의 분리된 것을 통해 글로벌, ROI 또는 섬유 기초에 섬유 조직을 정량화할 수 있지만, "CT-FIRE"18,21 및"CurveAlign"19,21이라는이름의 패키지가 있습니다. 소프트웨어 구현과 관련하여 CT-FIRE에서 여러 스케일의 CT 계수를 사용하여 가장자리를 향상시키고 노이즈를 줄이는 이미지를 재구성할 수 있습니다. 그런 다음, 개별 섬유 추출 알고리즘이 CT 재구성 된 이미지에 적용되어 대표 중심 점을 찾고, 중앙 점에서 섬유 분기를 확장하고, 섬유 가지를 연결하여 섬유 네트워크를 형성하기 위한 섬유를 추적합니다. CurveAlign에서 사용자 지정 규모의 CT 계수를 사용하여 곡선의 방향과 위치를 추출하고 그룹화하여 해당 위치에서 섬유 방향을 추정하는 로컬 섬유 방향을 추적할 수 있습니다. 이러한 결과 정량화 프레임워크는 10년 이상 개발되었으며 기능, 사용자 인터페이스 및 모듈성 과 같은 여러 측면에서 크게 발전해 왔습니다. 예를 들어, 이 도구는 로컬 섬유 방향을 시각화할 수 있으며 길이, 각도, 너비 및 직선성과 같은 개별 섬유 특성과 밀도 및 정렬과 같은 벌크 측정을 포함하여 최대 30개의 섬유 기능을 측정할 수 있습니다. 또한, 사용자는 수동으로 또는 자동으로 분할된 경계에 비해 섬유각을 측정할 수 있으며, 이는 예를 들어유방암(22)과 췌장암연구(10)에서이미지 기반 바이오마커 개발에 중요한 역할을 한다. 이 플랫폼은 ROI 분석, 자동 경계 생성 및 후처리를 위한 기능을 포함한 여러 기능 모듈을 제공합니다. ROI 모듈은 다양한 형태의 ROI에 알리고 해당 ROI 분석을 수행하는 데 사용할 수 있습니다. 응용 사례로서, 자동 경계 생성 모듈은 제2 고조파 생성(SHG) 이미지를 가진 헤마톡시린 및 에오신(H&E) 밝은 필드 영상을 등록하고 등록된 H&E 심상으로부터 종양 경계의 이미지 마스크를 생성하는 데 사용될 수 있다. 후처리 모듈은 가능한 통계 분석을 위해 개별 이미지에서 출력 데이터 파일을 처리하고 통합하는 데 도움이 될 수 있습니다.
이 정량화 플랫폼은 프로그래밍 또는 이미지 처리에 대한 사전 경험이 필요하지 않으며 수백 또는 수천 개의 이미지를 포함한 대규모 데이터 집합을 처리할 수 있으므로 생물학적 또는 생체 의학 응용 프로그램을 위한 콜라겐 조직의 효율적인 정량화를 가능하게 합니다. 그것은 널리 전 세계 많은 연구자에 의해 다른 연구 분야에서 사용 되었습니다., 자신을 포함 하 여. CT-FIRE 및 CurveAlign18,19,20,21에4 개의 주요 간행물이 있으며, 그 중 처음 세 개는 272 번 인용되었습니다 (Google 학자에 따르면 2020-05-04 현재). 이 플랫폼 (CT-FIRE 또는 CurveAlign)을 인용 한 출판물의 검토는 약 35 개의 출판물이 우리 그룹과 협력하고 다른 그룹 (~ 75)이 다른 그룹에 의해 작성된 분석을 위해 직접 사용하는 약 110 개의 저널 논문이 있음을 나타냅니다. 예를 들어, 이 플랫폼은 유방암22,23,24,췌장암10,25,신장암9,26,상처치유3,27,28,29,30,난소암8,31,7,자궁 대장대인32,저혈당 상종33,기저세포암(34) 저산소육종(35) , 연골조직36,심장기능장애37개,뉴런38,교모세포종39,림프수축40,섬유성 카펠드41,위암42,미세투벌레43,및 광섬유증44. 도 1은 SHG 이미지로부터 유방암19의 종양 관련 콜라겐 시그니처를 찾기 위해 CurveAlign의 암 이미징 응용을 입증한다. 그림 2는 이 플랫폼의 일반적인 회로도 워크플로우를 설명합니다. 이러한 도구는 기술적으로18,19,21을 검토하고 CurveAlign과의 정렬 분석을 위한 정규프로토콜(20)도 사용할 수 있지만 모든 필수 기능을 보여주는 시각적 프로토콜도 유용할 수 있습니다. 여기에 제시된 바와 같이 시각화된 프로토콜은 이 플랫폼을 사용하는 학습 프로세스를 용이하게 할 뿐만 아니라 사용자가 가질 수 있는 우려 사항과 질문을 보다 효율적으로 해결할 수 있습니다.

<table>
	<tbody>
		<tr>
			<td>CT-FIRE</td>
			<td>Univerity of Wisconsin-Madison</td>
			<td>N/A</td>
			<td>open source software available from https://eliceirilab.org/software/ctfire/</td>
		</tr>
		<tr>
			<td>CurveAlign</td>
			<td>University of Wisconsin-Madison</td>
			<td>N/A</td>
			<td>open source software available from https://eliceirilab.org/software/curvealign/</td>
		</tr>
	</tbody>
</table>

quantifying fibrillar collagen organization with curvelet transform-based tools

피브릴라 콜라겐은 눈에 띄는 세포 외 매트릭스 (ECM) 구성 요소이며, 그들의 토폴로지 변화는 유방, 난소, 신장 및 췌장암을 포함한 광범위한 질병의 진행과 관련이 있는 것으로 나타났습니다. 자유롭게 사용할 수 있는 섬유 정량화 소프트웨어 도구는 주로 섬유 정렬 또는 방향계산에 중점을 두고 있으며, 수동 단계의 요구 사항, 시끄러운 배경에서 섬유 에지 감지의 부정확성 또는 국소화 된 기능 특성화의 부족과 같은 제한 사항이 적용됩니다. 이 프로토콜에 설명된 콜라겐 섬유 수량 도구는 커브릿 변환(CT)에 의해 활성화된 최적의 다중 스케일 이미지 표현을 사용하는 것이 특징입니다. 이러한 알고리즘 접근 방식을 통해 피브릴라 콜라겐 이미지에서 노이즈를 제거하고 섬유 가장자리가 향상되어 다른 도구에서 얻은 간접 픽셀 또는 창 별 정보를 사용하는 대신 섬유에서 직접 위치 및 방향 정보를 제공할 수 있습니다. 이 CT 기반 프레임워크에는 글로벌, 관심 영역(ROI) 또는 개별 섬유 기준으로 섬유 조직을 정량화할 수 있는 "CT-FIRE" 및 "CurveAlign"이라는 두 개의 별도 패키지가 포함되어 있습니다. 이 정량화 프레임워크는 10년 이상 개발되었으며 이제 포괄적인 사용자 중심의 콜라겐 정량화 플랫폼으로 발전했습니다. 이 플랫폼을 사용하면 길이, 각도, 너비 및 직선과 같은 개별 섬유 특성과 밀도 및 정렬과 같은 벌크 측정을 포함하여 최대 30개의 섬유 기능을 측정할 수 있습니다. 또한 사용자는 수동으로 또는 자동으로 분할된 경계를 기준으로 광섬유 각도를 측정할 수 있습니다. 또한 이 플랫폼은 ROI 분석, 자동 경계 생성 및 후처리를 위한 모듈을 포함한 몇 가지 추가 모듈을 제공합니다. 이 플랫폼을 사용하면 프로그래밍 또는 이미지 처리에 대한 사전 경험이 필요하지 않으며 수백 또는 수천 개의 이미지를 포함한 대규모 데이터 집합을 처리할 수 있으므로 생물학적 또는 생체 의학 응용 을 위한 콜라겐 섬유 조직의 효율적인 정량화를 가능하게 합니다.

이러한 방법은 수많은 연구에서 성공적으로 적용되었습니다. 몇몇 전형적인 응용 프로그램은 다음을 포함합니다: 1) Conklin 외.22 종양 관련 콜라겐 서명을 계산하기 위하여 CurveAlign를 이용하고, 콜라겐 섬유가 더 빈번하게 그(DCIS) 병변에 있는 덕트 암에 있는 덕트 둘레에 수직으로 정렬되었다는 것을 것을을 발견했습니다; 2) Drifka외. 10 은 커브얼에서 CT-FIRE 모드?...

Watch this Scientific Journal Video about Quantifying Fibrillar Collagen Organization with Curvelet Transform-Based Tools at JoVE.com

Quantifying Fibrillar Collagen Organization with Curvelet Transform-Based Tools

여기서는 정상 및 병이 있는 조직의 세포외 매트릭스에서 세브릴라 콜라겐 조직을 정량화하기 위한 커브릿 변환 기반 오픈 소스 MATLAB 소프트웨어 도구를 사용하는 프로토콜을 제시합니다. 이 도구는 콜라겐 섬유 또는 다른 유형의 라인 과 같은 구조를 가진 이미지에 적용 할 수 있습니다.

곡선 변환 기반 도구를 사용하여 피브릴라 콜라겐 조직을 정량화

Fibrillar collagens are prominent extracellular matrix (ECM) components, and their topology changes have been shown to be associated with the progression ...

quantifying-fibrillar-collagen-organization-with-curvelet-transform

Research

JoVE Journal

Bioengineering

5.9K 조회수.  University of Wisconsin-Madison. 여기서는 정상 및 병이 있는 조직의 세포외 매트릭스에서 세브릴라 콜라겐 조직을 정량화하기 위한 커브릿 변환 기반 오픈 소스 MATLAB 소프트웨어 도구를 사용하는 프로토콜을 제시합니다. 이 도구는 콜라겐 섬유 또는 다른 유형의 라인 과 같은 구조를 가진 이미지에 적용 할 수 있습니다.

비디오: Quantifying Fibrillar Collagen Organization with Curvelet Transform-Based Tools

Quantifying the Mechanical Properties of the Endothelial Glycocalyx with Atomic Force Microscopy

Our understanding of the interaction of leukocytes and the vessel wall during leukocyte capture is limited by an incomplete understanding of the mechanical properties of the endothelial surface layer. It is known that adhesion molecules on leukocytes are distributed non-uniformly relative to surface topography 3, that topography limits adhesive bond formation with other surfaces 9, and that physiological contact forces (&asymp; 5.0 &minus; 10.0 pN per microvillus) can compress the microvilli to as little as a third of their resting length, increasing the accessibility of molecules to the opposing surface 3, 7. We consider the endothelium as a two-layered structure, the relatively rigid cell body, plus the glycocalyx, a soft protective sugar coating on the luminal surface 6. It has been shown that the glycocalyx can act as a barrier to reduce adhesion of leukocytes to the endothelial surface 4. In this report we begin to address the deformability of endothelial surfaces to understand how the endothelial mechanical stiffness might affect bond formation. Endothelial cells grown in static culture do not express a robust glycocalyx, but cells grown under physiological flow conditions begin to approximate the glycocalyx observed in vivo 2. The modulus of the endothelial cell body has been measured using atomic force microscopy (AFM) to be approximately 5 to 20 kPa 5. The thickness and structure of the glycocalyx have been studied using electron microscopy 8, and the modulus of the glycocalyx has been approximated using indirect methods, but to our knowledge, there have been no published reports of a direct measurement of the glycocalyx modulus in living cells. In this study, we present indentation experiments made with a novel AFM probe on cells that have been cultured in conditions to maximize their glycocalyx expression to make direct measurements of the modulus and thickness of the endothelial glycocalyx.

The mechanical characteristics of endothelial glycocalyx were measured by indentation using micron sized spheres on AFM cantilevers. Endothelial cells were cultured in a custom chamber under physiological flow conditions to induce glycocalyx expression. Data were analyzed using a thin film model to determine the glycocalyx thickness and modulus.

원자 힘 현미경과 내피 Glycocalyx의 기계적 성질을 정량화

Our  understanding of the interaction of leukocytes and the vessel wall during  leukocyte capture is limited by an incomplete understanding of the ...

연구

생체공학

Observing and Quantifying Fibroblast-mediated Fibrin Gel Compaction

Cells embedded in collagen and fibrin gels attach and exert traction forces on the fibers of the gel. These forces can lead to local and global reorganization and realignment of the gel microstructure. This process proceeds in a complex manner that is dependent in part on the interplay between the location of the cells, the geometry of the gel, and the mechanical constraints on the gel. To better understand how these variables produce global fiber alignment patterns, we use time-lapse differential interference contrast (DIC) microscopy coupled with an environmentally controlled bioreactor to observe the compaction process between geometrically spaced explants (clusters of fibroblasts). The images are then analyzed with a custom image processing algorithm to obtain maps of the strain. The information obtained from this technique can be used to probe the mechanobiology of various cell-matrix interactions, which has important implications for understanding processes in wound healing, disease development, and tissue engineering applications.

Time-lapse microscopy and image processing techniques were used to observe and analyze fibroblast-mediated gel compaction and fibrin fiber realignment in an environmentally controlled bioreactor over a 48 hr&#160;period.

섬유아세포 매개 피브린 겔 다짐 관찰 및 정량화

Cells embedded in collagen and fibrin gels attach and exert traction forces on the fibers of the gel. These forces can lead to local and global ...

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an integral part of normal tissue renewal, excessive amount of remodeling activity is involved in tumors, arthritis, and many other pathological conditions. During collagen remodeling, the triple helical structure of collagen molecules is disrupted by proteases in the extracellular environment. In addition, collagens present in many histological tissue samples are partially denatured by the fixation and preservation processes. Therefore, these denatured collagen strands can serve as effective targets for biological imaging. We previously developed a caged collagen mimetic peptide (CMP) that can be photo-triggered to hybridize with denatured collagen strands by forming triple helical structure, which is unique to collagens. The overall goals of this procedure are i)&#160;to image denatured collagen strands resulting from normal remodeling activities in vivo, and ii) to visualize collagens in ex vivo tissue sections using the photo-triggered caged CMPs. To achieve effective hybridization and successful in vivo and ex vivo imaging, fluorescently labeled caged CMPs are either photo-activated immediately before intravenous injection, or are directly activated on tissue sections. Normal skeletal collagen remolding in nude mice and collagens in prefixed mouse cornea tissue sections are imaged in this procedure. The imaging method based on the CMP-collagen hybridization technology presented here could lead to deeper understanding of the tissue remodeling process, as well as allow development of new diagnostics for diseases associated with high collagen remodeling activity.

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

This procedure demonstrates in vivo near IR fluorescence imaging of collagen remodeling activities in mice as well as ex vivo staining of collagens in tissue sections using caged collagen mimetic peptides that can be photo-triggered to hybridize with denatured collagen strands.

변성 콜라겐 물가 이미징<em&gt; 생체 내</em&gt;와<em&gt; 예 생체 내</em&gt; 리테이너 타입 콜라겐 미메시스 펩티드의 사진 트리거 하이브리드를 통해

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an ...

Helical Organization of Blood Coagulation Factor VIII on Lipid Nanotubes

Cryo-electron microscopy (Cryo-EM)1 is a powerful approach to investigate the functional structure of proteins and complexes in a hydrated state and membrane environment2.
Coagulation Factor VIII (FVIII)3 is a multi-domain blood plasma glycoprotein. Defect or deficiency of FVIII is the cause for Hemophilia type A&#160;-&#160;a severe bleeding disorder. Upon proteolytic activation, FVIII binds to the serine protease Factor IXa on the negatively charged platelet membrane, which is critical for normal blood clotting4. Despite the pivotal role FVIII plays in coagulation, structural information for its membrane-bound state is incomplete5. Recombinant FVIII concentrate is the most effective drug against Hemophilia type A and commercially available FVIII can be expressed as human or porcine, both forming functional complexes with human Factor IXa6,7.
In this study we present a combination of Cryo-electron microscopy (Cryo-EM), lipid nanotechnology and structure analysis applied to resolve the membrane-bound structure of two highly homologous FVIII forms: human and porcine. The methodology developed in our laboratory to helically organize the two functional recombinant FVIII forms on negatively charged lipid nanotubes (LNT) is described.&#160;The representative results demonstrate that our approach is sufficiently sensitive to define the differences in the helical organization between the two highly homologous in sequence (86% sequence identity) proteins. Detailed protocols for the helical organization, Cryo-EM and electron tomography (ET) data acquisition are given. The two-dimensional (2D) and three-dimensional (3D) structure analysis applied to obtain the 3D reconstructions of human and porcine FVIII-LNT is discussed. The presented human and porcine FVIII-LNT structures show the potential of the proposed methodology to calculate the functional, membrane-bound organization of blood coagulation Factor VIII at high resolution.

We present a combination of Cryo-electron microscopy, lipid nanotechnology, and structure analysis applied to resolve the membrane-bound structure of two highly homologous FVIII forms: human and porcine. The methodology developed in our laboratory&#160;to helically organize the two functional recombinant FVIII forms on negatively charged lipid nanotubes (LNT) is described.

지질 나노 튜브에 혈액 응고 인자 VIII의 나선형 조직

Cryo-electron microscopy (Cryo-EM)1 is a powerful approach to investigate the functional structure of proteins and complexes in a hydrated state and ...

Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible applications in controlling cell morphology/orientation. Nanofiber organization is controlled with a new fabrication apparatus that enables the gradual decrease of fiber organization in a scaffold. Changing the alignment of fibers is achieved through decreasing deposition time of random electrospun fibers on a uniaxially aligned fiber mat. By covering the collector with a moving barrier/mask, along the same axis as fiber deposition, the organizational structure is easily controlled. For tissue engineering purposes, adipose-derived stem cells can be seeded to these scaffolds. Stem cells undergo morphological changes as a result of their position on the varied organizational structure, and can potentially differentiate into different cell types depending on their locations. Additionally, the graded organization of fibers enhances the biomimicry of nanofiber scaffolds so they more closely resemble the natural orientations of collagen nanofibers at tendon-to-bone insertion site compared to traditional scaffolds. Through nanoencapsulation, the gradated fibers also afford the possibility to construct chemical gradients in fiber scaffolds, and thereby further strengthen their potential applications in fast screening of cell-materials interaction and interfacial tissue regeneration. This technique enables the production of continuous gradient scaffolds, but it also can potentially produce fibers in discrete steps by controlling the movement of the moving barrier/mask in a discrete fashion.

Here, we present a protocol to fabricate electrospun nanofiber scaffolds with gradated organization of fibers and explore their applications in regulating cell morphology/orientation. Gradients with regard to physical and chemical properties of the nanofiber scaffolds offer a wide variety of applications in the biomedical field.

섬유 조직의 그라데이션으로 전기 방사 나노 섬유 공사장 공중 발판

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

혈관 조직 재생을위한 엔지니어링 3D Cellularized 콜라겐 젤

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a deeper comprehension of the neural system scientists aim to simplistically reconstruct the tissue by assembling it in vitro from basic building blocks using a tissue engineering approach. Our group developed a tissue-engineered silk and collagen-based 3D brain-like model resembling the white and gray matter of the cortex. The model consists of silk porous sponge, which is pre-seeded with rat brain-derived neurons, immersed in soft collagen matrix. Polarized neuronal outgrowth and network formation is observed with separate axonal and cell body localization. This compartmental architecture allows for the unique development of niches mimicking native neural tissue, thus enabling research on neuronal network assembly, axonal guidance, cell-cell and cell-matrix interactions and electrical functions.

Insight into the complex actions of the brain requires advanced research tools. Here we demonstrate a novel silk-collagen-based 3D engineered model of neural tissue resembling brain-like architecture. The model can be used to study neuronal network assembly, axonal guidance, cell-cell interactions and electrical activity.

편광 신경 조직의 엔지니어링 3D 실크 콜라겐 기반 모델

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a ...

A Novel Tenorrhaphy Suture Technique with Tissue Engineered Collagen Graft to Repair Large Tendon Defects

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily identified and used. Currently, this gap is filled with tendon auto-, allo-, xeno-, or artificial grafts, but clinical methods to secure them are not necessarily translatable to animals because of the scale. In order to evaluate new biomaterials or study a tendon graft made up of collagen type 1, we have developed a modified suture technique to help maintain the engineered tendon in alignment with the tendon ends. Mechanical properties of these grafts are inferior to the native tendon. To incorporate engineered tendon into clinically relevant models of loaded repair, a strategy was adopted to offload the tissue engineered tendon graft and allow for the maturation and integration of the engineered tendon in vivo until a mechanically sound neo-tendon was formed. We describe this technique using incorporation of the collagen type 1 tissue engineered tendon construct.

In this paper, we present an in vitro and in situ protocol to repair a tendon gap of up to 1.5 cm by filling it with engineered collagen graft. This was performed by developing a modified suture technique to take the mechanical load until the graft matures into the host tissue.

큰 힘줄 결함을 복구하는 조직 엔지니어링 콜라겐 이식과 새로운 테노르래피 봉합사 기술

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily ...

Quantifying Intermembrane Distances with Serial Image Dilations

A recently-described extracellular nanodomain, termed the perinexus, has been implicated in ephaptic coupling, which is an alternative mechanism for electrical conduction between cardiomyocytes. The current method for quantifying this space by manual segmentation is slow and has low spatial resolution.We developed an algorithm that uses serial image dilations of a binary outline to count the number of pixels between two opposing 2 dimensional edges.This algorithm requires fewer man hours and has a higher spatial resolution than the manual method while preserving the reproducibility of the manual process.In fact, experienced and novice investigators were able to recapitulate the results of a previous study with this new algorithm.The algorithm is limited by the human input needed to manually outline the perinexus and computational power mainly encumbered by a pre-existing pathfinding algorithm.However, the algorithm&#39;s high-throughput capabilities, high spatial resolution and reproducibility make it a versatile and robust measurement tool for use across a variety of applications requiring the measurement of the distance between any 2-dimensional (2D) edges.

The purpose of this algorithm is to continuously measure the distance between two 2-dimensional edges using serial image dilations and pathfinding. This algorithm can be applied to a variety of fields such as cardiac structural biology, vascular biology, and civil engineering.

직렬 이미지 Dilations Intermembrane 거리 측정

A recently-described extracellular nanodomain, termed the perinexus, has been implicated in ephaptic coupling, which is an alternative mechanism for ...

Biomimetic Replication of Root Surface Microstructure using Alteration of Soft Lithography

Biomimetics is the use of chemistry and material sciences to mimic biological systems, specifically biological structures, to better humankind. Recently, biomimetic surfaces mimicking the microstructure of leaf surface, were used to study the effects of leaf microstructure on leaf-environment interactions. However, no such tool exists for roots. We developed a tool allowing the synthetic mimicry of the root surface microstructure into an artificial surface. We relied on the soft lithography method, known for leaf surface microstructure replication, using a two-step process. The first step is the more challenging one as it involves the biological tissue. Here, we used a different polymer and curing strategy, relying on the strong, rigid, polyurethane, cured by UV for the root molding. This allowed us to achieve a reliable negative image of the root surface microstructure including the delicate, challenging features such as root hairs. We then used this negative image as a template to achieve the root surface microstructure replication using both the well-established polydimethyl siloxane (PDMS) as well as a cellulose derivative, ethyl cellulose, which represents a closer mimic of the root and which can also be degraded by cellulase enzymes secreted by microorganisms. This newly formed platform can be used to study the microstructural effects of the surface in root-microorganism interactions in a similar manner to what has previously been shown in leaves. Additionally, the system enables us to track the microorganism&#8217;s locations, relative to surface features, and in the future its activity, in the form of cellulase secretion.

Biomimetics has been previously used as a tool to study leaf-microorganism interactions. However, no such tool exists for roots. Here, we develop a protocol to form synthetic surfaces mimicking root surface microstructure for the study of root-environment interactions.

소프트 리소그래피의 변경을 사용하여 루트 표면 미세 구조의 생물 적 복제

Biomimetics is the use of chemistry and material sciences to mimic biological systems, specifically biological structures, to better humankind. Recently, ...