作者想表达他们在乌得勒支UMC从开发力学组感谢的Jeroen先生范登贝尔赫和Matthijs Wassink先生为他们的包裹骨软骨插头的过程中提供帮助。这项工作是由来自荷兰关节炎基金会的资助。

注意:这里介绍的协议是从最近的研究论文6，8，9，实验和计算程序通过。该协议是在图1中示出。 该尸体材料与乌得勒支大学的兽医学院的许可收集。 1.样品和浴的制备<ol><li>使用定制的钻头（ 图1），同时喷涂凉爽磷酸盐缓冲盐水（PBS），以防止过热和随后的软骨损伤钻出从尸体马股骨髁圆柱形骨软骨塞（8.5mm的直径）。 </li><li>用塑料收缩套筒以最小化重叠浴的横向扩散热缩骨软骨塞。 <ol><li>安装骨软骨插头与日软骨E中的塑料收缩套内侧顶最初和通过吹送热空气到它跟随。加入软骨，防止热相关的损害表面湿棉片。 </li></ol></li><li>分别准备电荷的（ 例如，650微升，420毫摩尔，碘克沙酸盐，分子量（Mw）= 1269道尔顿，电荷= -1）和中性（650μL，420毫米，碘克沙醇，MW = 1550道尔顿）溶质的有限浴。 </li><li>加载使用注射器将软骨的表面上制备的有限浴并放置一个软木塞上包裹样品，以防止在实验过程中的蒸发在RT（ 图2A）。为了研究中性溶质运移，将碘克沙醇浴，并研究带负电荷的溶质运移地方碘克酸浴到软骨表面。 </li></ol> 2.成像和图像处理<ol><li>将带有软木塞上附连到微CT的电动载物台定制的保持器中分离的包裹样品。地方Ť他取样所以覆盖有造影剂溶液朝上软骨表面。 </li><li>扫描使用微CT（40×40×40微米的3体素尺寸，2分钟的扫描时间，90千伏的管电压和180μA管电流）的视图，包括软骨，软骨下板的领域，并且finite-浴在几个时间点，直到平衡状态（48小时）达到（ 图2A）。当浓度值不随时间变化达到平衡状态。 </li><li>注册在基于初始图像上使用制造商的软件，方便利息（ROI）的区域定位不同时间点的3D图像（ 例如，分析）。 </li><li>转换3D使用制造商的软件处理它们之前重建微CT图像为二维标记图像文件格式（TIFF）栈。 </li><li>全局段（ImageJ的&gt;调整&gt;阈值）从软骨下骨和在软骨在软件躺在浴缸。 </li><li>获得在不同时间点软骨的平均灰度值使用所生成的软骨掩模在前面的步骤（ImageJ的&gt;分析&gt;测量）。 </li><li>鉴于该软骨的初始浴浓度和初始造影剂浓度，使用线性校准曲线的平均灰度值转换为溶质的实际浓度。以前的数据支持该灰度值保持与造影剂的浓度成线性关系这一事实。 </li><li>剧情中的溶质浓度与实验的时间点。 </li></ol> 3.计算建模注意:在这个问题的扩散假设采取1D位置（沿着z轴），其与实验边界条件的要求。因此，几何形状可以任意创建。 <ol><li>构建基于有限浴软骨多区域模式:1）由软骨浅表区<html> （总软骨厚度的20％），中间区（总软骨厚度的50％）和深区（总软骨厚度的30％）12和2）在FEBio 13，14（ 图2B有限浴）。 </li><li>分配软骨和沐浴在FEBio的不同区域的机械和物理性能。杨氏模量（10MPa的）被假定为足够高以抵抗由覆浴施加的渗透压，因此保护软骨免受过度的变形。 <ol><li>使用的10 -3毫米的液压渗透性4 / Ns和泊松在模拟8，0。使用的浴的实际溶质扩散系数比9。 </li></ol></li><li>生成网格（8节点六面体三线性元素）和完善它的边界附近（ 图2B）类="外部参照"&gt; 8,9。 </li><li>双相溶质模型<ol><li>在对应于它的浴和有效压力施加初始的溶质浓度。看的有效压力的说明在图9中 ，15。 </li><li>运行瞬态模式模型根据在不同的区域软骨规定扩散系数，以获得溶质浓度对时间的曲线。 </li></ol></li><li>多相模型 注意:浴和组织之间的电波动可以通过添加两个单价抗衡离子的浴，将组织两者来规避。 <ol><li>对于稳态模型:同时增加FCD到其期望值使用软骨和覆浴相同的有效流体压力和浓度。 </li><li>对于瞬态模型:通过保持溶质扩散系数在浴中足够高的创建充分搅拌的有限浴。注入溶质从浴 - 空气界面到浴达到其期望的浓度值。 </li><li>瞬态:除去在先前步骤中规定的溶质浓度的边界条件，并恢复有限浴的扩散系数其实际扩散系数。 </li><li>运行模型，以获得基于施加FCDS和扩散系数在不同的软骨区溶质浓度 - 时间曲线。 </li></ol></li><li> FEBio-MATLAB界面<ol><li>开发一个MATLAB代码在FEBio和情节浓度-时间曲线（FEBio-MATLAB接口）8，9自动执行仿真。 </li><li>改变扩散系数和FCDS使用FEBio-MATLAB界面软骨区。在FEBio运行模式和提取的溶质浓度-时间曲线8,9。 </li><li>与实验进行比较所获得的溶质浓度 - 时间曲线基于最小根扩散系数的数据，并获取集和FCDS在不同软骨区均方误差（RMSE）8,9。 </li></ol></li></ol><img alt="图1" class="xfigimg" src="/files/ftp_upload/54984/54984fig1.jpg" /> 图1: 实验设置。 A）使用一个特制的钻头样品提取过程。 B）显微CT成像程序以监测扩散过程。 <a href="http://ecsource.jove.com/files/ftp_upload/54984/54984fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" class="xfigimg" src="/files/ftp_upload/54984/54984fig2.jpg" /> 图2: 示意图。A）实验设计。 B）多区域计算模型包括有限浴，软骨以及相关联的网格的浅，中，深区。 <a href="http://ecsource.jove.com/files/ftp_upload/54984/54984fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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</ol>

作者什么都没有透露。

我们提出了一个实验方案用有限元建模程序组合来研究跨关节软骨中性和带电溶质的扩散。根据我们最近的研究，所提出的模型可以准确地描述这两种中性（双相溶质）的跨关节软骨8,9的不同区域的运输和带负电荷（多相）溶质。人们普遍认为，关节软骨变得通过其重要部件的损耗功能的限制，诸如OA进展21，22，23，24中带负电荷的大分子GAG以及胶原纤维。使用在本研究中提出的技术，可以潜在地检查关节软骨的健全性。中性溶质的运输可能在OA PRIMAR来扩充<html> ILY由于胶原的溶质之间的较低的相互作用以及聚糖和。在另一方面，带负电荷的溶质运输可以帮助获得相对于蛋白聚糖的固定收费浓度的信息，从而给OA发展水平的一些指示。 该双相溶质和多相模型基础上根据以前的研究有限浴概念的出现是被开发可以作为由可以提供关节软骨的纬向性的精确估计的平台。用造影剂，大浴相关的限制即可能射束硬化伪影，和分配单扩散系数关节软骨7，20，25，26，27，28，姑娘="外部参照"&gt; 29，30开车到开发当前研究的动机。在未来OA的研究，我们开发的车型可能会潜在地发现早期OA诊断中的应用。 有两个实验和计算机模拟所需的一些关键步骤。为了在实验过程中保持软骨完整性，一个将需要加入足够量的蛋白酶抑制剂，以防止后续的酶活性。在使用一个样本一个以上的实验的情况下，洗出时间为平衡后的渗透溶质约为48小时。洗出效率需要使用微CT进行检查。当使用我们的设置采用马软骨，上覆浴相当于无限浴的最小体积计算比软骨量的5倍。此外，为了进行扩散的有限元建模，重要的是应用实际溶质扩散coefficient以来从偏离洗澡可能会影响结果。 在底层的有限元模型，溶质尺寸效应并没有实现，因此无法进行研究。我们提出的有限浴模型提供了一定的优势，在铰接接头更恰当地理解，即扩散过程，并减少束硬化伪影。当应用带正电的造影剂我们所提出的技术相结合的实验和计算模型能使审议软骨的扩散属性。在洗澡的实际溶质扩散系数的知识似乎是关键，因为这可能影响显著跨关节软骨所获得的扩散系数的准确性。这将需要获得任一浴的扩散系数实验或容易地使用文献值。 总之，我们提出了包括实验的通用协议，计算研究带电和不带电的溶质的跨关节软骨的运输。使用协议，可以成功地获得扩散系数和固定带电密度在不同软骨层。

<html>分子运输在铰接接头的内稳态中起着至关重要的作用，治疗剂递送至关节软骨和对比度增强软骨成像1，2，3。因素，如软骨整合和完整性，溶质电荷和大小以及重量渗透摩尔浓度，并与软骨可能影响传输速率4，5，6接触浴的浓度。溶质，无论是中性的或带电荷的传输，可以是关节软骨区域之间不同，因为每个区由不同浓度的和主要的细胞外基质分子的取向，即蛋白聚糖素（PGs）和II型胶原蛋白1，7，8，9，小姑娘="外部参照"&gt; 10,11。更重要的是，带电荷的溶质运输可以是高度依赖于包括横跨关节软骨8,9增加了的细胞外基质内负的固定电荷的蛋白聚糖的浓度。这些参数尤其是固定电荷密度（FCD），胶原原纤维和穿过软骨含水量变化的取向可经历改变为骨关节炎（OA）的进行，从而表示跨越软骨研究扩散的重要性。 在目前的研究中，协议基于先前建立的实验和计算研究6，8，9，提出了准确调查使用中性和带电溶质扩散的有限浴模型中的各种边界条件下扩散。 Ť他提出的方法是由包括软骨和由先进的双相 - 溶质和多相有限元模型支持的有限浴的系统的微型计算机断层扫描成像（微CT）的。这些模型使跨关节软骨的不同区域获得中性和带电分子以及FCDS的扩散系数。利用这些模型，可以更好地了解可用于研究软骨和覆盖有限浴之间的相互作用的扩散中性和带电分子的行为。

<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>骨关节炎（OA）是与关节软骨和软骨下骨的退化相关联的令人衰弱的疾病。关节软骨的退化基本上损害其承载功能，因为它经历了巨大的化学降解， 即 ，蛋白多糖的损失和胶原纤维的破坏。 OA期间，调查化学损伤机制中的一种有前途的方法是将试样软骨暴露到外部溶质和监控分子的扩散。软骨损伤（ 即浓度和基本大分子的构型）的程度与外部溶质的碰撞能量损失，同时跨越关节软骨移动相比健康软骨产生不同的扩散特性相关联。在这项研究中，我们引入一个协议，该协议包括几个步骤，并且基于先前开发的试验微-C14px的;"&gt; omputedŤomography（微CT）和有限元建模使用微CT，随后是通过施加双相-溶质和多相有限元模型，以获得扩散系数首先被记录带电和不带电碘化分子的运输。固定跨软骨区的电荷密度。

这里提供的代表性的结果是从先前的研究论文6，8，9，16日通过。 在OA中，关节软骨发生显著变化最重要的GAG损失，以及胶原纤维17的损坏，18个 ，19。这些变化可通过关节软骨20,21影响溶质的扩散行为。我们研究了两种碘化造影剂的轴向扩散， 即 ，碘克沙醇（电荷= 0）和碘克沙酸盐（电荷= -1），在使用显微CT尸体马骨软骨塞。为了定量的中性溶质（碘克沙醇），双相-溶质模型和带电SOLU的扩散过程<html> TE（碘克酸）多相模型中在FEBio所考虑软骨的纬向结构发达。将两相-溶质和多相模型可以预测碘克沙醇的扩散和整个关节软骨碘克沙酸盐（ 图3）。启用这些模型在不同的软骨区8获得的碘克沙醇（双相-溶质）和扩散系数的扩散系数以及FCD（碘克沙酸盐），9。 <img alt="图3" class="xfigimg" src="/files/ftp_upload/54984/54984fig3.jpg" /> 图3: 计算上曲线拟合数据。 A）多区域双相-溶质（虚线）与实验数据和B）多相模型拟合（虚线）与实验数据（符号）8,9。://ecsource.jove.com/files/ftp_upload/54984/54984fig3large.jpg"目标=‘_空白’&gt;点击此处查看该图的放大版本。

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

我们提出了一个协议，以调查带电和不带电分子的横跨关节软骨与最近开发的实验和数值方法的帮助下运输。

实验和有限元议定书调查中性和带电溶质的跨关节软骨运输

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

Nanyang Technological University

Research

JoVE Journal

Bioengineering

6.2K 次观看.  Delft University of Technology (TU Delft). 我们提出了一个协议，以调查带电和不带电分子的横跨关节软骨与最近开发的实验和数值方法的帮助下运输。 

视频: 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.

全完整型小鼠软骨外种机械负荷对软骨细胞损伤的实时可视化与分析

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

科研

生物工程

Application of Atomic Force Microscopy to Detect Early Osteoarthritis

Biomechanical properties of cells and tissues not only regulate their shape and function but are also crucial for maintaining their vitality. Changes in elasticity can propagate or trigger the onset of major diseases like cancer or osteoarthritis (OA). Atomic force microscopy (AFM) has emerged as a strong tool to qualitatively and quantitatively characterize the biomechanical properties of specific biological target structures on a microscopic scale, measuring forces in a range from as small as the piconewton to the micronewton. Biomechanical properties are of special importance in musculoskeletal tissues, which are subjected to high levels of strain. OA as a degenerative disease of the cartilage results in the disruption of the pericellular matrix (PCM) and the spatial rearrangement of the chondrocytes embedded in their extracellular matrix (ECM). Disruption in PCM and ECM has been associated with changes in the biomechanical properties of cartilage. In the present study we used AFM to quantify these changes in relation to the specific spatial pattern changes of the chondrocytes. With each pattern change, significant changes in elasticity were observed for both the PCM and ECM. Measuring the local elasticity thus allows for drawing direct conclusions about the degree of local tissue degeneration in OA.

We present a method to investigate early osteoarthritic changes at the cellular level in articular cartilage by using atomic force microscopy (AFM).

原子力显微镜在早期骨关节炎检测中的应用

Biomechanical properties of cells and tissues not only regulate their shape and function but are also crucial for maintaining their vitality. Changes in ...

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.

一个培养人关节软骨细胞的三维系统在滑液

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

生物学

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.

阳性肽载体软骨内运输特性的特征

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.

标记细胞外囊泡以监测软骨外植体的迁移和摄取

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

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.

一种摩擦试验-用于研究滑膜关节生物力学、机械生物学和物理调节的生物反应器装置

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.

基于原子力显微镜的人关节软骨外植体微压痕研究的实际问题

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 ...

A Probing Device for Quantitatively Measuring the Mechanical Properties of Soft Tissues during Arthroscopy

Probing in arthroscopic surgery is performed by pulling or pushing the soft tissue, which provides feedback for understanding the condition of the soft tissue. However, the output is only qualitative based on the &#8220;surgeon&#8217;s feeling&#8221;. Herein is described a probing device developed to address this issue by measuring the resistance of soft tissues quantitatively with a tri-axial force sensor. Under both conditions (i.e., pull- and push-probing certain tissues mimicking the acetabular labrum and cartilage), this probing device is found to be useful for measuring some mechanical properties in joints during arthroscopy.

Probing during arthroscopy surgery is normally done to assess the condition of the soft tissue, but this approach has always been subjective and qualitative. This report describes a probing device that can measure the resistance of the soft tissue quantitatively with a tri-axial force sensor during arthroscopy.

定量测量关节镜期间软组织机械性能的探测装置

Probing in arthroscopic surgery is performed by pulling or pushing the soft tissue, which provides feedback for understanding the condition of the soft ...

医学

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.

植入物关节软骨滑向金属的生物摩擦学测试与分析

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

Synovial Fluid Analysis to Identify Osteoarthritis

Synovial fluid (SF) analysis is important in diagnosing osteoarthritis (OA). Macroscopic and microscopic features, including total and differential white blood cell (WBC) count, help define the non-inflammatory nature of SF, which is a hallmark of OA. In patients with OA, WBC in SF samples usually does not exceed 2000 cells per microliter, and the percentage of inflammatory cells, such as neutrophils, is very low or absent. Calcium crystals are frequent in SF collected from OA patients. Although their role in the pathogenesis of OA remains unclear, they have been associated with a mild inflammatory process and a more severe disease progression. Recently, calcium crystals have been described in both the early and late stages of OA, indicating that they may play a vital role in diagnosing different clinical subsets of OA and pharmacological treatment. The overall goal of SF analysis in OA is two-fold: to ascertain the non-inflammatory degree of SF and to highlight the presence of calcium crystals.

Synovial fluid analysis under transmitted, polarized, and compensated light microscopy is used to evaluate the inflammatory or non-inflammatory nature of a sample through simple steps. It is particularly useful in osteoarthritis to detect calcium crystals and identify a more severe subset of osteoarthritis.

滑液分析以识别骨关节炎

Synovial fluid (SF) analysis is important in diagnosing osteoarthritis (OA). Macroscopic and microscopic features, including total and differential white ...