저자는 골 연골 플러그의 과정을 포장에있는 그들의 도움을 UMC 위트레흐트에서 개발 역학 그룹에서 씨 제론 반 덴 베르그 씨 Matthijs Wassink 그들의 감사의 말씀을드립니다. 이 작품은 네덜란드 관절염 재단에서 부여에 의해 지원되었다.

참고 : 여기에 제시된 프로토콜은 최근의 연구 논문 6, 8, 9의 실험과 계산 과정에서 채택된다. 이 프로토콜은도 1에 도시되어있다. 사체 자료는 위트레흐트 대학의 수의학 교수의 허가를 수집 하였다. 1. 시료와 욕제 <ol><li> 차가운 인산 완충 식염수 (PBS)를 분사하면서 과열 이후 연골의 손상을 방지하기 위해 맞춤 제작 드릴 비트 (도 1)를 이용하여 사후 말 대퇴골로부터 원통 골 연골 플러그 (8.5 mm의 직경) 밖으로 드릴. </li><li> 오버레이 욕의 측면 확산을 최소화하기 위해 슬리브를 수축 플라스틱으로 골 연골 플러그 열 수축. <ol><li> 일에서 연골과 골 연골 플러그를 장착E는 플라스틱 슬리브 내부 수축 초기 상단과 열풍을 불어 따른다. 열 관련 손상을 방지하기 위해 연골의 표면에 젖은면 조각을 추가합니다. </li></ol></li><li> 별도 부과 (예를 들어, 650 μL, 420 mm의 ioxaglate, 분자량 (Mw) = 1,269 다 충전 = -1) 및 중성 (650 μL, 420 mm의 iodixanol, MW = 1,550 다) 용질 유한 화장실을 준비한다. </li><li> 주사기를 사용하여 연골의 표면에 제조 유한 화장실로드 및 RT (도 2A)에서 실험 동안에 증발을 방지하기 위해, 랩 샘플에 코르크 마개를 배치했다. 중립 용질 수송을 연구하기 위해, iodixanol 목욕을 배치하고 연골 표면에 음전하를 띤 용질 수송 장소 ioxaglate 목욕을 연구. </li></ol> 2. 이미징 및 이미지 처리 <ol><li> 마이크로 CT의 모터 구동 스테이지에 장착 주문품 홀더에 코르크 마개로 감싸 절연 샘플을 놓는다. 장소 t그 때문에 조영제 용액 위를 향하고 덮여 그 연골 표면을 샘플링. </li><li> 마이크로 CT (40 × 40 × 40 μm의 3 복셀의 크기가 2 분의 주사 시간을 90 kV로의 관전압 180 μA의 관전류) 연골 연골 판 구성된 시야를 사용하여 검색하고,의 유한 평형 상태 (48 시간)까지 여러 시점에서의 조를 (도 2A)에 도달한다. 농도 값이 시간에 따라 변화하지 않는 경우에 평형 상태가 달성된다. </li><li> 제조업체의 소프트웨어를 사용하여 관심 (ROI)의 지역의 위치를 용이하게하기 위해 초기 이미지에 따라 서로 다른 시점에서 3D 이미지를 등록합니다 (예를 들어, 분석). </li><li> 변환 3 차원 제조 업체의 소프트웨어를 사용하여 처리하기 전에 2D 태그가 지정된 이미지 파일 형식 (TIFF) 스택에 마이크로 CT 영상을 재구성. </li><li> 글로벌 세그먼트 (ImageJ에&gt; 조정&gt; 임계치) 연골 하골 이상에서 연골소프트웨어에서 목욕을 거짓말. </li><li> 상이한 시점에서 연골의 평균 그레이 값을 구하는 이전 단계에서 생성 된 연골 마스크를 사용하여 (은 ImageJ에&gt;&gt; 측정 분석). </li><li> 연골의 초기 욕 농도 및 초기 조영제 농도 주어 용질의 실제 농도 평균 그레이 값을 변환하는 선형 보정 곡선을 사용한다. 이전의 데이터는 그레이 값 조영제의 농도를 갖는 선형 관계를 유지한다는 사실을지지한다. </li><li> 실험 시점에 비해 용질 농도를 플롯. </li></ol> 3. 전산 모델링 주 :이 문제의 확산은 실험 경계 조건을 준수 (Z 축을 따라) (1D)에서 일어나는 것으로한다. 따라서, 형상 임의로 생성 할 수 있습니다. <ol><li> 유한 목욕을 기반으로 연골 멀티 존 모델을 구축 : 1) 연골 표면 영역으로 구성<html> (전체 연골 두께의 20 %), 중간 영역 (전체 연골 두께의 50 %)과 깊은 영역 (전체 연골 두께의 30 %) 12 FEBio 13, 14 (도 2b 2) 유한 욕). </li><li> FEBio에서 연골 및 목욕의 서로 다른 영역의 기계적 및 물리적 특성을 할당합니다. 영률 (MPa의 10)이 상부로 조 작용 삼투압 레지스트 따라서 과도한 변형에서 연골을 보호하기에 충분히 높은 것으로 가정 하였다. <ol><li> 10-3 mm의 유압 투과도를 사용 4 / Ns를 상기 시뮬레이션 8 욕 0 실제 사용 용질 확산 계수의 포아송 비, 9. </li></ol></li><li> 메쉬 (8 노드 삼선 육면체 요소)를 생성하고, 경계 근처를 세분화 (도 2B)클래스 = &quot;외부 참조&quot;&gt; 8, 9. </li><li> 바이 페이 - 용질 모델 <ol><li> 이것에 대응하는 욕조 유효 압력에서 용질의 초기 농도를 적용한다. 15 9에서 유효 압력의 설명을보십시오. </li><li> 다른 연골 영역에 소정의 확산 계수에 따른 시간 곡선 대 용질의 농도를 얻기 위해 과도 모드 모델을 실행. </li></ol></li><li> 다상 모델 참고 : 욕조와 조직 사이의 전기 변동은 욕조와 조직 모두에 두 가의 반대 이온을 추가하여 우회 할 수 있습니다. <ol><li> 정상 상태 모델 : 소기 FCD 값을 증가시키면서 연골 상부 욕 동일한 유효 유체 압력 및 농도를 사용한다. </li><li> 과도 모델 : 충분히 높은 욕 중에서의 용질 확산 계수를 유지하여 잘 교반 유한 조를 만든다. 주입목욕 욕조에 공기 인터페이스에서 용질의 농도 소정의 값에 도달한다. </li><li> 과도 : 이전 단계에서 소정의 용질 농도 경계 조건을 제거하고 그것의 실제 확산 계수로 유한 욕의 확산 계수를 되돌릴. </li><li> 연골 다른 영역에 적용 FCDs 및 확산 계수에 기초하여 용질 농도 - 시간 곡선을 획득하기 위해 모델을 실행. </li></ol></li><li> FEBio - MATLAB 인터페이스 <ol><li> 자동 FEBio 플롯 농도 - 시간 곡선 (FEBio-MATLAB 인터페이스) (8, 9)에서의 시뮬레이션을 수행하는 MATLAB 코드를 개발한다. </li><li> FEBio - MATLAB 인터페이스를 사용하여 연골 영역에서 확산 계수와 FCDs을 변경합니다. FEBio에서 모델을 실행하고, 용질의 농도 - 시간 곡선 9 8 압축을 풉니 다. </li><li> 실험적으로 얻어진 용질 농도 - 시간 곡선을 비교최소 루트에 따라 다른 연골 영역의 데이터 획득 확산 계수들의 세트 FCDs 제곱 오차 (RMSE) (8, 9)를 의미한다. </li></ol></li></ol><img alt="그림 1" class="xfigimg" src="/files/ftp_upload/54984/54984fig1.jpg" /> 그림 1 : 실험 설정. A) 사용자 정의 만든 드릴 비트를 사용하여 샘플 추출 과정. B) 마이크로 CT 이미징 절차 확산 프로세스를 모니터링한다. <a href="http://ecsource.jove.com/files/ftp_upload/54984/54984fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 2" class="xfigimg" src="/files/ftp_upload/54984/54984fig2.jpg" /> 그림 2 : 도식.A) 실험 설계. 유한 목욕, 연골 및 관련 메쉬의 표면, 중간과 깊은 영역으로 구성된 B) 다중 영역 계산 모델. <a href="http://ecsource.jove.com/files/ftp_upload/54984/54984fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>

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T., 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=Computed+tomography+detects+changes+in+contrast+agent+diffusion+after+collagen+cross-linking+typical+to+natural+aging+of+articular+cartilage.">Computed tomography detects changes in contrast agent diffusion after collagen cross-linking typical to natural aging of articular cartilage.</a> Osteoarthritis and Cartilage. 19 (10), 1190-1198 (2011).</li><li>Decker, S. G., Moeini, M., Chin, H. C., Rosenzweig, D. H., Quinn, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adsorption+and+Distribution+of+Fluorescent+Solutes+near+the+Articular+Surface+of+Mechanically+Injured+Cartilage.">Adsorption and Distribution of Fluorescent Solutes near the Articular Surface of Mechanically Injured Cartilage.</a> Biophysical Journal. 105 (10), 2427-2436 (2013).</li><li>Silvast, T. S., Jurvelin, S. J., Tiitu, V., Quinn, T. M., Töyräs, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bath+Concentration+of+Anionic+Contrast+Agents+Does+Not+Affect+Their+Diffusion+and+Distribution+in+Articular+cartilage+In+Vitro.">Bath Concentration of Anionic Contrast Agents Does Not Affect Their Diffusion and Distribution in Articular cartilage In Vitro.</a> Cartilage. 4 (1), 42-51 (2013).</li><li>Silvast, T. S., Jurvelin, J. S., Lammi, M. J., T&#246;yr&#228;s, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=pQCT+study+on+diffusion+and+equilibrium+distribution+of+iodinated+anionic+contrast+agent+in+human+articular+cartilage+-+associations+to+matrix+composition+and+integrity.">pQCT study on diffusion and equilibrium distribution of iodinated anionic contrast agent in human articular cartilage - associations to matrix composition and integrity.</a> Osteoarthritis and Cartilage. 17 (1), 26-32 (2009).</li><li>Pouran, B., Arbabi, V., Villamar, J., Weinans, H., Zadpoor, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contrast+agent's+transport+across+healthy+articular+cartilage+under+various+bath+conditions.">Contrast agent's transport across healthy articular cartilage under various bath conditions.</a> , (2015).</li><li>Arbabi, V., Pouran, B., Weinans, H., Zadpoor, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+a+biphasic-solute+model+in+predicting+diffusive+properties+of+osteochondral+interface.">Application of a biphasic-solute model in predicting diffusive properties of osteochondral interface.</a> , (2016).</li></ol>

저자가 공개하는 게 없다.

우리는 관절 연골에서 중립 및 충전 용질의 확산을 연구하기 위해 유한 요소 모델링 절차와 결합 된 실험 프로토콜을 제시 하였다. 우리의 최근 연구에 따르면, 제안 된 모델은 정확하게 관절 연골 8, 9의 서로 다른 영역에서 중립 (이상성 - 용질) 모두의 전송 및 음전하 (다상) 용질을 설명 할 수있다. 널리 관절 연골이 기능적으로 같은 OA 진행 <sup class="xr...

<html> 분자 수송 관절 형 조인트의 항상성에 중요한 역할을하는 관절 치료제의 전달 연골 및 연골 조영 증강 영상 1, 2, 3. 이러한 연골 통합 intactness, 용질의 전하 및 크기뿐만 아니라 삼투압 및 전송 레이트 4, 5, 6에 영향을 미칠 수있는 연골 접촉 욕의 농도 요소. 각 영역은 다른 농도의 주요 세포 외 매트릭스 분자의 배향으로 구성되어 있기 때문에 중성 또는 충전 중 용질의 수송은, 관절 연골 영역과 상이 할 수있다, 즉, 프로테오글리칸 (PG를) 콜라겐 타입 II (1), 7, 8, 9,아가씨 = &quot;외부 참조&quot;&gt; 10, 11. 더 중요한 것은, 충전 용질의 수송은 관절 연골 (8, 9)에 걸쳐 증가 외 매트릭스 내에 고정 된 네거티브 전하를 포함 프로테오글리칸의 농도에 크게 의존 할 수있다. 골관절염 (OA)가되어 연골 걸쳐 확산 연구의 중요성을 나타내는, 진행됨에 따라 이러한 매개 변수, 특히 고정 된 전하 밀도 (FCD)는 콜라겐 원 섬유 연골 전체 수분 함량의 변화의 방향은 변화를 거칠 수있다. 현재의 연구에서, 프로토콜은 6, 8, 9, 정확하게 확산의 유한 목욕 모델에서 중립 및 충전 용질을 사용하여 다양한 경계 조건에서 확산을 조사하기 위해 제안 이전에 설정된 실험 및 전산 연구를 기반으로. 티그 방법은 연골 및 고급 이상성-용질 다상 유한 요소 모델에 의해지지되는 유한 욕조를 포함하는 시스템의 마이크로 컴퓨터 단층 촬영 영상 (마이크로 CT)으로 구성되어 제안. 이러한 모델은 관절 연골의 다양한 영역에 걸쳐 중성 및 하전 분자뿐만 아니라 FCDs의 확산 계수를 취득 할 수 있습니다. 이 모델을 사용하여, 하나는 연골 및 오버레이 유한 화장실 사이의 상호 작용을 조사하는 데 사용할 수 있습니다 확산 중립 및 충전 분자의 행동의 더 나은 이해를 얻을 수 있습니다.

<table><tbody><tr><td>Hexabrix</td><td>Guerbet</td><td>15HX005D</td><td>Negatively-charged contrast agent</td></tr><tr><td>Visipaque</td><td>GE healthcare</td><td>12570511</td><td>Nuetral contrast agent</td></tr><tr><td>PBS (Phosphate-buffered Saline)</td><td>Life technologies</td><td>10010023</td><td>Medium</td></tr><tr><td>micro-CT</td><td>Perkin Elmer</td><td></td><td>Monitoring diffusion</td></tr><tr><td>Freezing-point osmometer</td><td>Advanced instruments</td><td></td><td>Measuring solution osmolality</td></tr></tbody></table>

an experimental and finite element protocol to investigate the transport of neutral and charged solutes across articular cartilage

<html> 골관절염 (OA)은 관절 연골과 연골 하골의 변성과 연관된 쇠약하게하는 질병이다. 그것은 상당한 화학적 열화, 즉 손실 프로테오글리칸 및 콜라겐 브릴 중단을 경험으로 관절 연골의 변성이 실질적으로 그 하중 기능을 손상시킨다. OA에서 화학적 손상 메커니즘을 조사하는 한 유망한 방법은 외부 용질에 연골 표본을 노출하고, 분자의 확산을 모니터하는 것입니다. 건강한 연골에 비해 상이한 확산 특성을 생성 관절 연골을 가로 질러 이동하는 동안 연골 손상 (즉, 농도 및 고분자 필수 구성)의 정도는 외부 용질 충돌하는 에너지 손실과 관련된다. 이 연구에서 우리는 몇 가지 단계로 구성되어 있으며 이전에 개발 된 실험 마이크로-C를 기반으로하는 프로토콜을 소개14px; &quot;&gt; omputed T omography (마이크로 CT) 및 유한 요소 모델링 제 이상성-용질 확산 계수를 얻기 다상 유한 요소 모델에 적용함으로써 뒤에 마이크로 CT를 사용하여 기록되어 충전 및 비 대전 요오드 분자의 수송. 연골 영역에 걸쳐 전하 밀도를 고정.

여기에 제공된 대표적인 결과는 이전의 연구 논문 6, 8, 9, 16에서 채택된다. OA에서 관절 연골은 큰 변화 가장 중요한 GAG 손실 및 콜라겐 원 섬유 손상 17, 18, 19을</su...

Watch this Scientific Journal Video about An Experimental and Finite Element Protocol to Investigate the Transport of Neutral and Charged Solutes across Articular Cartilage at JoVE.com

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

우리는 최근에 개발 된 실험 및 수치 방법의 도움으로 관절 연골에서 충전 및 충전되지 않은 분자의 이동을 조사하기위한 프로토콜을 제안한다.

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 ...

an-experimental-finite-element-protocol-to-investigate-transport

Research

JoVE Journal

Bioengineering

6.2K 조회수.  Delft University of Technology (TU Delft).  우리는 최근에 개발 된 실험 및 수치 방법의 도움으로 관절 연골에서 충전 및 충전되지 않은 분자의 이동을 조사하기위한 프로토콜을 제안한다. 

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

Real-time Visualization and Analysis of Chondrocyte Injury Due to Mechanical Loading in Fully Intact Murine Cartilage Explants

Homeostasis of articular cartilage depends on the viability of resident cells (chondrocytes). Unfortunately, mechanical trauma can induce widespread chondrocyte death, potentially leading to irreversible breakdown of the joint and the onset of osteoarthritis. Additionally, maintenance of chondrocyte viability is important in osteochondral graft procedures for optimal surgical outcomes. We present a method to assess the spatial extent of cell injury/death on the articular surface of intact murine synovial joints after application of controlled mechanical loads or impacts. This method can be used in comparative studies to investigate the effects of different mechanical loading regimens, different environmental conditions or genetic manipulations, as well as different stages of cartilage degeneration on short- and/or long-term vulnerability of in situ articular chondrocytes. The goal of the protocol introduced in the manuscript is to assess the spatial extent of cell injury/death on the articular surface of murine synovial joints. Importantly, this method enables testing on fully intact cartilage without compromising native boundary conditions. Moreover, it allows for real-time visualization of vitally stained articular chondrocytes and single image-based analysis of cell injury induced by application of controlled static and impact loading regimens. Our representative results demonstrate that in healthy cartilage explants, the spatial extent of cell injury depends sensitively on load magnitude and impact intensity. Our method can be easily adapted to investigate the effects of different mechanical loading regimens, different environmental conditions or different genetic manipulations on the mechanical vulnerability of in situ articular chondrocytes.

We present a method to assess the spatial extent of cell injury/death on the articular surface of intact murine joints after application of controlled mechanical loads or impacts. This method can be used to investigate how osteoarthritis, genetic factors and/or different loading regimens affect the vulnerability of in situ chondrocytes.

실시간 시각화 및 Chondrocyte 부상 때문에 완전히 그대로 Murine 연골 Explants 기계적 부하의 분석

Homeostasis of articular cartilage depends on the viability of resident cells (chondrocytes). Unfortunately, mechanical trauma can induce widespread ...

연구

생체공학

A 3D System for Culturing Human Articular Chondrocytes in Synovial Fluid

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not regenerate very efficiently in vivo; and as a result, osteoarthritis leads to irreversible cartilage loss and is accompanied by chronic pain and immobility 1,2. Cartilage tissue engineering offers promising potential to regenerate and restore tissue function. This technology typically involves seeding chondrocytes into natural or synthetic scaffolds and culturing the resulting 3D construct in a balanced medium over a period of time with a goal of engineering a biochemically and biomechanically mature tissue that can be transplanted into a defect site in vivo 3-6. Achieving an optimal condition for chondrocyte growth and matrix deposition is essential for the success of cartilage tissue engineering. 
 In the native joint cavity, cartilage at the articular surface of the bone is bathed in synovial fluid. This clear and viscous fluid provides nutrients to the avascular articular cartilage and contains growth factors, cytokines and enzymes that are important for chondrocyte metabolism 7,8. Furthermore, synovial fluid facilitates low-friction movement between cartilaginous surfaces mainly through secreting two key components, hyaluronan and lubricin 9 10. In contrast, tissue engineered cartilage is most often cultured in artificial media. While these media are likely able to provide more defined conditions for studying chondrocyte metabolism, synovial fluid most accurately reflects the natural environment of which articular chondrocytes reside in.
 Indeed, synovial fluid has the advantage of being easy to obtain and store, and can often be regularly replenished by the body. Several groups have supplemented the culture medium with synovial fluid in growing human, bovine, rabbit and dog chondrocytes, but mostly used only low levels of synovial fluid (below 20&#37;) 11-25. While chicken, horse and human chondrocytes have been cultured in the medium with higher percentage of synovial fluid, these culture systems were two-dimensional 26-28. Here we present our method of culturing human articular chondrocytes in a 3D system with a high percentage of synovial fluid (up to 100&#37;) over a period of 21 days. In doing so, we overcame a major hurdle presented by the high viscosity of the synovial fluid. This system provides the possibility of studying human chondrocytes in synovial fluid in a 3D setting, which can be further combined with two other important factors (oxygen tension and mechanical loading) 29,30 that constitute the natural environment for cartilage to mimic the natural milieu for cartilage growth. Furthermore, This system may also be used for assaying synovial fluid activity on chondrocytes and provide a platform for developing cartilage regeneration technologies and therapeutic options for arthritis.

A 3D system of culturing human articular chondrocytes in high levels of synovial fluid is described. Synovial fluid reflects the most natural microenvironment for articular cartilage, and can be easily obtained and stored. This system thus can be used for studying cartilage regeneration and for screening therapeutics for treating arthritis.

활액 유체에 Culturing 인간 관절 Chondrocytes을위한 3D 시스템

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not ...

생물학

Characterization of Intra-Cartilage Transport Properties of Cationic Peptide Carriers

Several negatively charged tissues in the body, like cartilage, present a barrier to the targeted drug delivery due to their high density of negatively charged aggrecans and, therefore, require improved targeting methods to increase their therapeutic response. Because cartilage has a high negative fixed charge density, drugs can be modified with positively charged drug carriers to take advantage of electrostatic interactions, allowing for enhanced intra-cartilage drug transport. Studying the transport of drug carriers is, therefore, crucial towards predicting the efficacy of drugs in inducing a biological response. We show the design of three experiments which can quantify the equilibrium uptake, depth of penetration and non-equilibrium diffusion rate of cationic peptide carriers in cartilage explants. Equilibrium uptake experiments provide a measure of the solute concentration within the cartilage compared to its surrounding bath, which is useful for predicting the potential of a drug carrier in enhancing therapeutic concentration of drugs in cartilage. Depth of penetration studies using confocal microscopy allow for the visual representation of 1D solute diffusion from the superficial to deep zone of cartilage, which is important for assessing whether solutes reach their matrix and cellular target sites. Non-equilibrium diffusion rate studies using a custom-designed transport chamber enables the measurement of the strength of binding interactions with the tissue matrix by characterizing the diffusion rates of fluorescently labeled solutes across the tissue; this is beneficial for designing carriers of optimal binding strength with cartilage. Together, the results obtained from the three transport experiments provide a guideline for designing optimally charged drug carriers which take advantage of weak and reversible charge interactions for drug delivery applications. These experimental methods can also be applied to evaluate the transport of drugs and drug-drug carrier conjugates. Further, these methods can be adapted for the use in targeting other negatively charged tissues such as meniscus, cornea and the vitreous humor.

This protocol determines equilibrium uptake, depth of penetration and non-equilibrium diffusion rate for cationic peptide carriers in cartilage. Characterization of transport properties is critical for ensuring an effective biological response. These methods can be applied for designing an optimally charged drug carriers for targeting negatively charged tissues.

양이온 펩타이드 캐리어의 인트라-연골 수송 특성의 특성화

Several negatively charged tissues in the body, like cartilage, present a barrier to the targeted drug delivery due to their high density of negatively ...

Labeling of Extracellular Vesicles for Monitoring Migration and Uptake in Cartilage Explants

Extracellular vesicles (EVs) are used in different studies to prove their potential as a cell-free treatment due to their cargo derived from their cellular source, such as platelet lysate (PL). When used as treatment, EVs are expected to enter the target cells and effect a response from these. In this research, PL-derived EVs have been studied as a cell-free treatment for osteoarthritis (OA). Thus, a method was set up to label EVs and test their uptake on cartilage explants. PL-derived EVs are labeled with the lipophilic dye PKH26, washed twice through a column, and then tested in an in vitro inflammation-driven&#160;OA model for 5 h after particle quantification by nanoparticle tracking analysis (NTA). Hourly, cartilage explants are fixed, paraffined, cut into 6 &#181;m sections to mount on slides, and observed under a confocal microscope. This allows verification of whether EVs enter the target cells (chondrocytes) during this period and analyze their direct effect.

Here, we present a protocol to label platelet lysate-derived extracellular vesicles to monitor their migration and uptake in cartilage explants used as a model for osteoarthritis.

연골 내의 마이그레이션 및 섭취 모니터링을 위한 세포외 소포 라벨링

Extracellular vesicles (EVs) are used in different studies to prove their potential as a cell-free treatment due to their cargo derived from their ...

Mechanical Stimulation of Chondrocyte-agarose Hydrogels

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy this deficiency, several medical interventions have been developed. One such method is to resurface the damaged area with tissue-engineered cartilage; however, the engineered tissue typically lacks the biochemical properties and durability of native cartilage, questioning its long-term survivability. This limits the application of cartilage tissue engineering to the repair of small focal defects, relying on the surrounding tissue to protect the implanted material. To improve the properties of the developed tissue, mechanical stimulation is a popular method utilized to enhance the synthesis of cartilaginous extracellular matrix as well as the resultant mechanical properties of the engineered tissue. Mechanical stimulation applies forces to the tissue constructs analogous to those experienced in vivo. This is based on the premise that the mechanical environment, in part, regulates the development and maintenance of native tissue1,2. The most commonly applied form of mechanical stimulation in cartilage tissue engineering is dynamic compression at physiologic strains of approximately 5-20% at a frequency of 1 Hz1,3. Several studies have investigated the effects of dynamic compression and have shown it to have a positive effect on chondrocyte metabolism and biosynthesis, ultimately affecting the functional properties of the developed tissue4-8. In this paper, we illustrate the method to mechanically stimulate chondrocyte-agarose hydrogel constructs under dynamic compression and analyze changes in biosynthesis through biochemical and radioisotope assays. This method can also be readily modified to assess any potentially induced changes in cellular response as a result of mechanical stimuli.

The biosynthesis of cartilaginous extracellular matrix by chondrocytes can be affected by application of mechanical stimuli. This method describes the technique of applying dynamic compressive strains to chondrocytes encapsulated in 3D constructs and the evaluation of induced changes in chondrocyte metabolism.

Chondrocyte - 아가로 오스 Hydrogels의 기계적 자극

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy ...

A Friction Testing-Bioreactor Device for Study of Synovial Joint Biomechanics, Mechanobiology, and Physical Regulation

In primary osteoarthritis (OA), normal 'wear and tear' associated with aging inhibits the ability of cartilage to sustain its load-bearing and lubrication functions, fostering a deleterious physical environment. The frictional interactions of articular cartilage and synovium may influence joint homeostasis through tissue level wear and cellular mechanotransduction. To study these mechanical and mechanobiological processes, a device capable of replicating the motion of the joint is described. The friction testing device controls the delivery of reciprocal translating motion and normal load to two contacting biological counterfaces. This study adopts a synovium-on-cartilage configuration, and friction coefficient measurements are presented for tests performed in a phosphate-buffered saline (PBS) or synovial fluid (SF) bath. The testing was performed for a range of contact stresses, highlighting the lubricating properties of SF under high loads. This friction testing device can be used as a biomimetic bioreactor for studying the physical regulation of living joint tissues in response to applied physiologic loading associated with diarthrodial joint articulation.

The present protocol describes a friction testing device that applies simultaneous reciprocal sliding and normal load to two contacting biological counterfaces.

마찰 테스트 - 활막 관절 생물 역학, Mechanobiology 및 물리적 조절 연구를위한 생물 반응기 장치

In primary osteoarthritis (OA), normal 'wear and tear' associated with aging inhibits the ability of cartilage to sustain its load-bearing and lubrication ...

Addressing Practical Issues in Atomic Force Microscopy-Based Micro-Indentation on Human Articular Cartilage Explants

Without a doubt, atomic force microscopy (AFM) is currently one of the most powerful and useful techniques to assess micro and even nano-cues in the biological field. However, as with any other microscopic approach, methodological challenges can arise. In particular, the characteristics of the sample, sample preparation, type of instrument, and indentation probe can lead to unwanted artifacts. In this protocol, we exemplify these emerging issues on healthy as well as osteoarthritic articular cartilage explants. To this end, we first show via a step-by-step approach how to generate, grade, and visually classify ex vivo articular cartilage discs according to different stages of degeneration by means of large 2D mosaic fluorescence imaging of the whole tissue explants. The major strength of the ex vivo model is that it comprises aged, native, human cartilage that allows the investigation of osteoarthritis-related changes from early onset to progression. In addition, common pitfalls in tissue preparation, as well as the actual AFM procedure together with the subsequent data analysis, are also presented. We show how basic but crucial steps such as sample preparation and processing, topographic sample characteristics caused by advanced degeneration, and sample-tip interaction can impact data acquisition. We also subject to scrutiny the most common problems in AFM and describe, where possible, how to overcome them. Knowledge of these limitations is of the utmost importance for correct data acquisition, interpretation, and, ultimately, the embedding of findings into a broad scientific context.

We present a step-by-step approach to identify and address the most common problems associated with atomic force microscopy micro-indentations. We exemplify the emerging problems on native human articular cartilage explants characterized by various degrees of osteoarthritis-driven degeneration.

인간 관절 연골 외식물에 대한 원자력 현미경 기반 미세 압입의 실제 문제 해결

Without a doubt, atomic force microscopy (AFM) is currently one of the most powerful and useful techniques to assess micro and even nano-cues in the ...

Biotribological Testing and Analysis of Articular Cartilage Sliding against Metal for Implants

Osteochondral defects in middle-aged patients might be treated with focal metallic implants. First developed for defects in the knee joint, implants are now available for the shoulder, hip, ankle and the first metatarsalphalangeal joint. While providing pain reduction and clinical improvement, progressive degenerative changes of the opposing cartilage are observed in many patients. The mechanisms leading to this damage are not fully understood. This protocol describes a tribological experiment to simulate a metal-on-cartilage pairing and comprehensive analysis of the articular cartilage. Metal implant material is tested against bovine osteochondral cylinders as a model for human articular cartilage. By applying different loads and sliding speeds, physiological loading conditions can be imitated. To provide a comprehensive analysis of the effects on the articular cartilage, histology, metabolic activity and gene expression analysis are described in this protocol. The main advantage of tribological testing is that loading parameters can be adjusted freely to simulate in vivo conditions. Furthermore, different testing solutions might be used to investigate the influence of lubrication or pro-inflammatory agents. By using gene expression analysis for cartilage-specific genes and catabolic genes, early changes in the metabolism of articular chondrocytes in response to mechanical loading might be detected.

This protocol describes the preparation, biotribological testing, and analysis of osteochondral cylinders sliding against metal implant material. Outcome measures included in this protocol are metabolic activity, gene expression and histology.

임플란트용 금속에 대한 관절 연골 슬라이딩의 생체 립학 테스트 및 분석

Osteochondral defects in middle-aged patients might be treated with focal metallic implants. First developed for defects in the knee joint, implants are ...

의학

A Non-Invasive Method for Generating the Cyclic Loading-Induced Intra-Articular Cartilage Lesion Model of the Rat Knee

The pathophysiology of primary osteoarthritis (OA) remains unclear. However, a specific subclassification of OA in relatively younger age groups is likely correlated with a history of articular cartilage damage and ligament avulsion. Surgical animal models of OA of the knee play an important role in understanding the onset and progression of post-traumatic OA and aid in the development of novel therapies for this disease. However, non-surgical models have been recently considered to avoid traumatic inflammation that could affect the evaluation of the intervention. 
In this study, an intra-articular cartilage lesion rat model induced by in vivo cyclic compressive loading was developed, which allowed researchers to (1) determine the optimal magnitude, speed, and duration of load that could cause focal cartilage damage; (2) assess post-traumatic spatiotemporal pathological changes in chondrocyte vitality; and (3) evaluate the histological expression of destructive or protective molecules that are involved in the adaptation and repair mechanisms against joint compressive loads. This report describes the experimental protocol for this novel cartilage lesion in a rat model.

Here, we present the cyclic loading-induced intra-articular cartilage lesion model of the rat knee, generated by 60 cyclic compressions over 20 N, resulting in damage to the femoral condylar cartilage in rats.

래트 무릎의 순환 로딩 유도 관절내 연골 병변 모델을 생성하기 위한 비침습적 방법

The pathophysiology of primary osteoarthritis (OA) remains unclear. However, a specific subclassification of OA in relatively younger age groups is likely ...

<meta charset="utf8"></head><body>연골형성체(Chondroprogenitor and Chondrocyte Isolation from Human Articular Cartilage)

Isolation of Chondrocytes and Chondroprogenitors Using Fibronectin Adhesion and Migratory Assay

Chondroprogenitor cells (CPCs), recently identified as a distinct subpopulation, exhibit promise due to their mesenchymal properties, heightened chondrogenesis, and limited hypertrophic traits. The enrichment of progenitors is achieved through differential fibronectin adhesion and migration-based explant assays, with Fibronectin Adhesion Assay-derived Chondroprogenitors (FAA-CPs) and Migratory Chondroprogenitors (MCPs) demonstrating superior potential compared to chondrocytes. This article delves into the details of isolating resident cartilage-derived cells, namely chondrocytes and chondroprogenitors. While valuable insights from chondrocyte research contribute to our understanding of cartilage repair, ongoing efforts are directed toward the use of chondroprogenitors and exploring their potential as an alternative therapeutic approach. Additionally, this methodology article provides a detailed step-by-step protocol for isolating three specific cell types from cartilage: chondrocytes, FAA-CPs, and MCPs. By following standardized procedures, this protocol facilitates the successful extraction of these cell subtypes. Grounded in extensive research, the article focuses on the intricate techniques utilized in isolating the different subsets and the optimized culture conditions required to expand and maintain their cultures. The methodology encompasses enzymatic isolation of human articular cartilage-derived chondrocytes, differential fibronectin adhesion following sequential enzymatic digestion, and migration-based explant assays to obtain cartilage-resident cells.

<meta charset="utf8"></head><body>Fibronectin Adhesion and Migratory Assay를 사용한 연골세포와 연골형성세포의 분리

This protocol details the isolation of chondrocytes, Fibronectin Adhesion Assay-derived Chondroprogenitors (FAA-CPs), and Migratory Chondroprogenitors (MCPs) from human articular cartilage. It covers enzymatic digestion, fibronectin adhesion, and migration-based assays for isolating and characterizing these cells.