Name: biomechanical characterization of human soft tissues using indentation and tensile testing
Uploaded: 2016-12-13T14:00:00.000+00:00
Duration: 7 min 7 s
Description: Watch this Scientific Journal Video about Biomechanical Characterization of Human Soft Tissues Using Indentation and Tensile Testing at JoVE.com

We would like to thank the funding from Medical Research Council and Action Medical Research, which provided MG with a clinical fellowship, GN 2339, to conduct this work.

该协议遵循的关于禁止使用，储存和处置人体组织我们机构的人类研究伦理委员会的指导方针道德准则。人体组织样品可以从已同意为研究目的与相关的伦理批准尸体被切除。样品还可以从接受手术治疗的，具有相关的伦理批准同意患者丢弃的组织。 1.皮肤的制备<ol><li>通过手动解剖掉脂肪组织并使用手术刀刀片和镊子真皮深层的薄层制作标本。这个步骤是重要的，以确保样品14之间的一致性。 </li><li>所得刃厚皮片材切成标准化的样本大小（如 1厘米*5厘米样本）。确定基于所述检测装置的尺寸的试样尺寸。如果组织工程构建物也被测试，该specimeÑ尺寸应该适合感兴趣14的材料。手术刀刀片在适当的尖锐件箱处理。 </li><li>以使力学计算完成后，测量皮肤的厚度之前和机械测试之后使用电子卡尺进行测试。 </li></ol> 2.拉伸测试注意:所有的材料试验机应根据制造商的测试之前的准则进行校准。 <ol><li>试验皮肤样品在单轴拉伸，在室温（22℃）14使用材料试验机（ 图2A）。 </li><li>定向在所有样品（ 例如，垂直或在符合兰格线（绘制在地图上的人体的拓扑线和参照在真皮胶原纤维）的自然取向）14相同的方向上的皮肤样品。 </li><li>固定两个夹具之间的试样（共mmercial夹具），一个固定在98.07 N加载细胞，另一个不可移动基板14。在单轴拉伸测试夹具之间所产生的面积应为1厘米×4厘米（ 图2）。 注:商业夹具用来避免不均匀的把持和损坏测试前的样品。样品被固定在"用手指拧紧"密封性。 </li><li>盖两边用凡士林样品面积（在装置放置后），以防止样品脱水。 </li><li>程序中的拉伸负荷和放松的检验制度进入软件的操作列表如下:空载|零位|查找联系人（拉伸载荷）|等待（放松）。 </li><li>先从软件程序测试。荷载作用下的张力样品29.42ñ1毫米/秒。用率和负载不会引起皮肤的失败（ 例如，29.42Ñ在1毫米/秒）。 </li><li>达到29.42 N-二负载后，允许组织放松1.5小时，一个时间点在其中有在松弛行为变化最小，由计算机软件14控制。 注意:位移在松弛阶段，而不是负载保持恒定。 </li><li>计算弹性和粘弹性性质按上述分析部的准则。调查了机械性能将代表裂层皮肤成分（表皮和真皮）14的平均性质。 注意:没有定义的皮重负载的，因为它是从当变形发生时，因此，仅包含这些数据点的原始数据清楚。 </li></ol> 3.软骨的制备<ol><li>删除使用手术刀刀片和钳子15，16中的软骨样本的皮肤和筋膜。 </li><li>划分软骨标本成使用解剖刀刀片和镊子的标准化样品的大小（ 例如，1.5厘米的块）。对于所有的样品，使用semicircu拉尔形压头（ 图2B），其具有的直径与厚度比软骨样本的大小大至少8倍。这个比率可确保压头不会受到从标本制备15的任何边缘效应。手术刀刀片在适当的尖锐件箱处理。 </li><li>以使力学计算完成后，测量之前和之后的机械试验15，16利用电子卡尺加载的软骨的厚度。 </li></ol> 4.压缩压痕测试<ol><li>压缩在室温水合环境中使用的材料试验机的软骨样本。覆盖之前和压缩测试期间的磷酸盐缓冲盐水（PBS）的软骨样本以确保样品水合。 注:PBS不完全的生理环境相匹配，但它允许两种材料和组织是玉米同样相比15，16。 </li><li>定向的软骨样品所以表面是垂直于所述压头。这允许压缩是单轴，并限制任何剪切载荷15。 </li><li>程序中的压缩载荷和放松的检验制度进入软件的操作列表如下:空载|零位|查找联系人（压缩载荷）|等待（放松）。 </li><li>使用的软件程序开始测试。负载下压缩到2.94 n个样品以1毫米/秒15,16。 注意:这被确定为一个非破坏性的负荷是足够灵敏的，以确定软骨15的弹性和粘弹性。 </li><li>达到2.94-N限制后，使软骨到15分钟放松，一个时间点在其中有在松弛行为变化最小，使用计算机软件15,16。 注: 图图2C-D显示一组典型的为人体组织标本的压缩和拉伸试验。然后相同的协议可应用到合成生物材料的生物力学特性匹配被分析的天然组织。例如， 图2E-F展示了紧密匹配的合成材料的生物力学特性的人体组织的压缩和拉伸试验。 </li></ol> 5.计算杨氏弹性模量为压痕和拉伸试验<ol><li>收集的原始数据，包括从材料试验设备14-16时间（s），位移（毫米）和负荷（N）。 </li><li>计算使用图3中所示的式的应力（兆帕）和应变（％）。 注意:如果压缩测试期间使用的半球形压头，由截面积除以力可以提供标称（平均）应力，但不是峰值应力。 </li><li>使用线性散点图绘制应力兆帕（y轴）对应变（x轴）。确定线性曲线拟合。线性曲线拟合是等于y = mx + b中与各自的R值。 注:包括所有的数据点，实现最低R值&gt; 0.98。 m值是斜率，其对应于应力的过度应变模量，这表明在兆帕压电阻或耐张力（ 即，杨氏模量）。如果R值不&gt; 0.98，那么表征线性粘弹行为的假设是无效的。 </li><li>以识别在其中因暴露于变形的流体流动达到平衡，应力随时间过去200的机械试验，并在试验结束时的最终应力水平的比例来计算的粘弹性性质。 注:随着时间，紧张程度会降低（放松），为流体流动达到平衡17,18。快速应力松弛反应indicat上课，这是困难的样品17，18内保持高的应力。 </li></ol> 6.放松属性<ol><li>在兆帕（y轴）对上一个线性散点图以s（x轴）的时间曲线图的应力。 </li><li>确定的线性曲线拟合来计算松弛率。线性曲线拟合等于Y = mx + b中与最后200秒的相应数值。 m值是松弛的速率。 </li><li>包括所有的数据点，以获得最小R值&gt; 0.98。在用于皮肤1.5小时，对于软骨15分钟的最终应力（兆帕）是最后的绝对松弛值。 </li></ol>

<ol>
	<li>Chan, B. P., Leong, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scaffolding+in+tissue+engineering:+general+approaches+and+tissue-specific+considerations.">Scaffolding in tissue engineering: general approaches and tissue-specific considerations.</a> Eur Spine J. 17, 467-479 (2008).</li><li>Nimeskern, L., van Osch, G. J., M&#252;ller, R., Stok, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+evaluation+of+mechanical+properties+in+tissue-engineered+auricular+cartilage.">Quantitative evaluation of mechanical properties in tissue-engineered auricular cartilage.</a> Tissue Eng Part B Rev. 20, 17-27 (2014).</li><li>Shaida, A. M., Kenyon, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nasal+valves:+changes+in+anatomy+and+physiology+in+normal+subjects.">The nasal valves: changes in anatomy and physiology in normal subjects.</a> Rhinology. 38, 7-12 (2000).</li><li>N&#237; Annaidh, A., Bruy&#232;re, K., Destrade, M., Gilchrist, M. D., Ott&#233;nio, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+anisotropic+mechanical+properties+of+excised+human+skin.">Characterization of the anisotropic mechanical properties of excised human skin.</a> J Mech Behav Biomed Mater. 5, 139-148 (2012).</li><li>Ottenio, M., Tran, D., N&#237; Annaidh, A., Gilchrist, M. D., Bruy&#232;re, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain+rate+and+anisotropy+effects+on+the+tensile+failure+characteristics+of+human+skin.">Strain rate and anisotropy effects on the tensile failure characteristics of human skin.</a> J Mech Behav Biomed Mater. 41, 241-250 (2015).</li><li>Silver, F. H., Freeman, J. W., DeVore, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viscoelastic+properties+of+human+skin+and+processed+dermis.">Viscoelastic properties of human skin and processed dermis.</a> Skin Res Technol. 7, 18-23 (2001).</li><li>Karimi, A., Navidbakhsh, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+the+uniaxial+mechanical+properties+of+rat+skin+using+different+stress-strain+definitions.">Measurement of the uniaxial mechanical properties of rat skin using different stress-strain definitions.</a> Skin Res Technol. 21, 149-157 (2015).</li><li>Wilkes, G. L., Brown, I. A., Wildnauer, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biomechanical+properties+of+skin.">The biomechanical properties of skin.</a> CRC Crit Rev Bioeng. 1, 453-495 (1973).</li><li>Hussain, S. H., Limthongkul, B., Humphreys, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biomechanical+properties+of+the+skin.">The biomechanical properties of the skin.</a> Dermatol Surg. 39, 193-203 (2013).</li><li>Smith, C. D., Masouros, S., Hill, A. M., Wallace, A. L., Amis, A. A., Bull, M. 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=Mechanical+testing+of+intra-articular+tissues.+Relating+experiments+to+physiological+function.">Mechanical testing of intra-articular tissues. Relating experiments to physiological function.</a> Current orthopaedics. 22, 341-348 (2008).</li><li>Korhonen, R. K., S, S. a. a. r. a. k. k. a. l. a., Klika, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biomechanics and Modeling of Skeletal Soft Tissues, Theoretical Biomechanics. , (2011).</li><li>Lu, X. L., Mow, V. C. <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+articular+cartilage+and+determination+of+material+properties.">Biomechanics of articular cartilage and determination of material properties.</a> Med Sci Sports Exerc. 40, 193-199 (2008).</li><li>Xia, Y., Zheng, S., Szarko, M., Lee, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anisotropic+Properties+of+Bovine+Nasal+Cartilage.">Anisotropic Properties of Bovine Nasal Cartilage.</a> Micros Res Tech. 75, 300-306 (2012).</li><li>Wood, J. M., Soldin, M., Shaw, T. J., Szarko, 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+biomechanical+and+histological+sequelae+of+common+skin+banking+methods.">The biomechanical and histological sequelae of common skin banking methods.</a> J Biomech. 47, 1215-1219 (2014).</li><li>Griffin, M. F., Premakumar, Y., Seifalian, A. M., Szarko, M., Butler, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanical+characterisation+of+the+human+nasal+cartilages;+implications+for+tissue+engineering.">Biomechanical characterisation of the human nasal cartilages; implications for tissue engineering.</a> J Mater Sci Mater Med. 27, 11 (2016).</li><li>Griffin, M. F., Premakumar, Y., Seifalian, A. M., Szarko, M., Butler, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanical+chacterisation+of+human+auricular+cartilages;+implications+for+tissue+engineering.">Biomechanical chacterisation of human auricular cartilages; implications for tissue engineering.</a> Annals of biomedical Engineering. , (2016).</li><li>Shrive, N. G., Frank, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Articular+Cartilage.">Articular Cartilage.</a> Biomechanics of the Musculo-Skeletal System. , 86-106 (1999).</li><li>Fung, Y. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biomechanics: Mechanical Properties of Living Tissues. 568, (1993).</li><li>Hultkrantz, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Ueber die Spaltrichtungen der Gelenkknorpel. Verhandlungen der Anatomischen Gesellschaft. , (1898).</li><li>Cheng, S., Clarke, E. C., Bilston, L. E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+preconditioning+strain+on+measured+tissue+properties.">The effects of preconditioning strain on measured tissue properties.</a> Biomech. 42, 1360-1362 (2009).</li><li>Mow, V. C., Ratcliffe, A., Mow, V. C., Hayes, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+Function+of+Articular+Cartilage+and+Meniscus.">Structure and Function of Articular Cartilage and Meniscus.</a> Basic Orthopaedic Biomechanics. , 113-178 (1997).</li><li>Tavakol, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Proteoglycan &amp; Collagen degrading activities of neural proteases from fresh and cryopreserved articular cartilage explants and the chondrocytes. An in vitro biochemical study. , (1989).</li><li>Smeathers, J. E., Vincent, J. F. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cartilage+and+Joints.">Cartilage and Joints.</a> Biomechanics: Materials. , 99-131 (1992).</li><li>Smith, C. L., Mansour, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Indentation+of+an+Osteochondral+Repair:+Sensitivity+to+Experimental+Variables+and+Boundary+Conditions.">Indentation of an Osteochondral Repair: Sensitivity to Experimental Variables and Boundary Conditions.</a> J Biomech. 33, 1507-1511 (2000).</li><li>Niederauer, G. G., Niederauer, G. M., Cullen, L. C., Athanasiou, K. A., Thomas, J. B., Niederauer, M. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+of+Cartilage+Stiffness+to+Thickness+and+Level+of+Degeneration+Using+a+Handheld+Indentation+Probe.">Correlation of Cartilage Stiffness to Thickness and Level of Degeneration Using a Handheld Indentation Probe.</a> Ann Biomed Eng. 32, 352-359 (2004).</li><li>Ball, S. T., Amiel, A. D., Willaims, S. K., Tontz, W., Chen, A. C., Sah, R. L., Bugbee, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Effects+of+Storage+on+Fresh+Human+Osteochondral+Allografts.">The Effects of Storage on Fresh Human Osteochondral Allografts.</a> Clin Orthop Relat Res. 418, 246-252 (2004).</li><li>Park, S., Hung, C. T., Ateshian, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+response+of+bovine+articular+cartilage+under+dynamic+unconfined+compression+loading+at+physiological+stress+levels.">Mechanical response of bovine articular cartilage under dynamic unconfined compression loading at physiological stress levels.</a> Osteoarthritis Cartilage. 12, 65-73 (2004).</li></ol>

The authors have nothing to declare.

一些拉伸和压痕协议已经公布的表征人体软组织。我们已经提供了另一种方法，其目的是更诊断和非破坏性。在这个协议中进行机械测试的样品通过负载，而不是通过位移的限制，因为换能器是比位移加载更加敏感。因此，该实验的复制品可以横跨组织和合成材料更精确。使用这种技术，我们已经证明了皮肤组织和软骨组织分析的压痕协议评估拉伸协议。这两个协议都容易和简单的实现，并可能被视为人体软组织和组织工程结构的表征。 一项所述的方法的重要步骤，以获得适合于分析的应力松弛曲线是确保样品不测试期间打滑。充分固定是requir版，但这必须要针对在试样上产生的任何应力，并确保压头垂直于表面，以防止任何剪切负荷平衡。至关重要的是，该组织的组合物，以及尺寸和形状的样品相似。对于软骨，至关重要的是使用重复的解剖协议和样品尺寸。对于皮肤样品，至关重要的是要除去所有的皮下组织，以便获得可重复的样品。同样重要的是，以确保所有样品中，样品的条件是相同的，包括水合，室温，解冻过程中，如果合适的。 有所呈现的协议一定的局限性。有研究表明，皮肤和软骨变形特性取决于试样取向13。皮肤被认为是各向异性的，早在19 世纪 ，随着兰格证明于1861年，皮肤具有天然的线张力的称为兰格线4。因此，表征皮肤样品时，它向定向的所有样品平行或垂直于朗格行，以避免引入的方法偏压4是重要的。软骨还显示各向异性特性，并包含Hultkrantz线，这是相当于兰格线，所以软骨可以根据在它被加载12,19的方向变形不同。因此，重要的是要增加样本大小，以允许软骨在不同方向的检测。作为组织的生物力学性能还与年龄和性别不同，研究应该具有代表性患者队列中进行，以保持有效性临床设置。此外，一些机械协议主张预处理，其中所述组织经受循环加载，以确保组织是在随后的机械测试20的稳定状态。然而，p的确切机制再生是不清楚，以产生一致的和可重复的响应所需要的循环的确切数目在不同的研究20而变化。研究者应该考虑是否要包括评估执行特定的生物力学测试20的原因后预处理。 皮肤是一个复杂的，多层次的材料，分为三个主要层:表皮，真皮，和皮下组织4。 皮肤组织的机械性质最近已使用体内评估4评价。 然而，拉伸试验的协议可以利用来理解切除皮肤4的皮肤生物力学。这样的测试可以提供信息，以应力-应变关系进行建模，由于边界条件可以定义4。通常， 在体外试验制度使用高应变的材料表征故障，而在体内系统中使用低应变范围4。当张力切除皮肤的生物力学比较值，有不同的研究之间有很大的变化，从2.9-150兆帕4。主体之间的巨大差异，预计由于自然变异生物，但在协议制度的差异也可以加剧这种自然的生物学差异。例如，在协议之间加载率的差异会导致变型中，作为更大的负荷率造成的流体流出时间较少，造成了更高的刚度。皮肤组织的制备中，切除和处理协议也将导致机械性能4的差异。这个协议证明测试皮肤为研究人员提供以表征皮肤组织的另一种方法。它提供了一些优点，包括识别一个机械测试皮肤组织的弹性和粘弹性性质的能力，允许的皮肤更好地理解在很短的时间量。此外，相同的测试可应用于组织工程替代来制造具有类似生物力学性质如天然皮肤构造。 压痕试验相比，了解软骨21的生物力学密闭压缩测试提供了一个有吸引力的选择。压痕具有保护软骨的生理结构的能力，从而提供了模仿那些临床环境的值。使用压痕，它也有可能测试软骨同时仍然附连到下面的骨骼。压痕也允许作为体内软骨的生理测试。当两个软骨表面彼此接近时，由于接触面积下的水周围接触"隆起"的区域中的边缘被压缩变形发生17,21后横向移位。软骨缩进必须与在被进行具有比软骨样本更小的半径D输入，以允许类似鼓胀。压头的大小也应至少8倍的样本大小，以确保就好像它是一个不确定的样品22的部分的软骨反应。使用压头比样品直径的半径消除存在于试样创建任何边缘效应小得多。另外，压痕避免了通过测试由样品提取损坏软骨缺损可能的实验误差。压痕也不涉及深样品制备，如密闭压缩，允许小的，薄件软骨待测试17,21。另外，压痕的非破坏性方法意味着它在临床环境的潜在应用为确认和验证的研究后的诊断工具已被执行。 有齿痕，用户必须确保approp关键假设riate结果。在缩进装载一个关键边界条件要求压头和软骨表面之间的恒定接触（ 即，该表面不会变形从压头远）23，24。压痕载荷还包括假定的边界条件，即软骨表面与压头之间的接触是无损（ 即，压头是在与表面接触，但通过表面不走;软骨表面不应下失败压头）25 - 26。有研究表明，这个边界条件，可以通过使用印油墨，当施加到软骨表面25，26，这将弄脏损坏的区域进行验证。进一步的边界条件假定压头压缩垂直于样品表面的软骨。压缩的垂直方向是一个重要的边界COND银行足球比赛，因为在一个角度压缩，特别是如果使用循环加载，可能引起滑移，这可能导致剪切元件和改变机械加载。这个条件可以保证通过仔细的测试设备设置。 汇总协议已经针对感兴趣的软组织被优化后，为研究人员寻找到感兴趣的组织的动态测试将是有用的。标本适当的循环载荷应模仿正常的生理极限和行为，如模仿行走或其他重复动作27。总之，本报告展示了简单的机械测试协议，以评估人体组织。实施这些协议将提供有关组织的生物力学特性的关键信息，使组织工程结构，以更好地模仿天然组织。

患者越来越等待各种器官移植治疗失败或受伤的器官。然而，与合适的供体器官的短缺，再生医学的目标是创建患者终末期器官衰竭替代解决方案。再生医学的目的是满足这种临床需要通过工程材料充当组织代用品，包括软组织，例如软骨和皮肤。要创造一个成功的材料，以恢复受损的组织，更换材料应模仿它要更换1-2本地组织的属性。一旦手术植入，该材料将需要提供解剖学形状的组织缺陷，因此，该材料的机械性能是至关重要的1。例如，在更换耳廓软骨的材料应当具有适当的机械性能，以防止压缩由覆盖皮肤2。同样，材料以取代鼻车tilage将需要有足够的机械性能，以防止呼吸3期间塌陷。然而，尽管制造用于注入材料时，机械性能的重要性，没有证据集中于表征不同人体组织的机械性能。 机械测试机制可用于建立压力，拉力，弯曲，或一个组织的剪切性能。皮肤是一个高度各向异性，粘弹性，几乎不可压缩材料4-9。通常切皮肤使用单轴拉伸的方法，其中皮肤的一个适当形状的条带在两端夹住并拉伸而负荷和延伸记录4-9的测试。 由于所有的软组织的主要成分为间质水，软骨的机械响应强烈相关流体穿过组织10-11的流动。软组织，如软骨ħAVE使用压缩试验传统上进行测试。在压缩测试的方法是相当多样，以密闭，潜水和缩进是最普遍的（ 图1）。内密闭压缩，软骨样品放置在一个不透流体填充井并装载通过多孔板。由于阱是无孔的，流过的软骨是在垂直方向12-13。在无侧限压缩，软骨，使用的非多孔板到非多孔室加载，迫使流体流为主要径向12-13。缩进是用于评价软骨12-13的生物力学特性的最常用的方法。它由一个压头，比试样表面较小被测试的，即放倒到样品。压痕有超过压缩的其他方法，包括事实可以原位执行的压痕，enabli许多优点纳克测试更加生理（ 图1）12-13。 理解的组织的压缩和拉伸性能，杨氏弹性模量，通常通过分析应力-应变曲线的线性部分，指示压缩或拉伸的弹性阻力计算，而不管试件尺寸12。两个拉伸和压缩试验制度可以根据负载或变形施加并且两个这样的参数的速率而变化。目前，有许多不同的测试协议，以评估组织力学，这使得它非常难以解释或从不同研究6-13比较结果。此外，许多机械方法目前集中在通过检体检测到破坏表征的组织的机械性能。 我们的目标是展示一个压痕和拉伸协议，为人类提供直接的，非破坏性的比较软组织和组织工程化构建体。 我们表明，限制了机械测试压力，但仍获得一个年轻的公司在压缩和拉伸弹性模量的方法。样品被强调无论是在拉伸或压缩到一定值，并且一旦选择了应力值已达到，样品被允许放松，同时所有的数据被记录。此方法同时捕捉相同的测试中的组织，其可以直接应用到合成材料的粘弹性和松弛性能。我们已经使用了缩进协议 评估人体软组织，包括皮肤和软骨14-16。软骨是用压痕试验评估，皮肤是用拉伸试验14-16评估。研究人员旨在工程师具有相似属性的人的软组织材料，可以考虑实施这些协议。

<table><tbody><tr><td>Digitial Vernier Calipers</td><td>Machine Mart</td><td>40218046</td><td>Digitial vernier caliper is used to measure sample thickness.&amp;nbsp;</td></tr><tr><td>Water Bath&amp;nbsp;</td><td>Cole Parmer</td><td>UY-12504-94</td><td>StableTemp Digital Water Bath Flask Holder used to defrost tissues samples if they are frozen.&amp;nbsp;</td></tr><tr><td>Mach-1 Material Testing Machine</td><td>Biomomentum&amp;nbsp;</td><td>V500c</td><td>Mechanical Testing Machine used to test the mechancial properties of the tissues.&amp;nbsp;</td></tr><tr><td>Scalpel Blade&amp;nbsp;</td><td>VWR</td><td>233-5335</td><td>Scalpel blades using to cut and dissect the tissues.&amp;nbsp;</td></tr><tr><td>Forceps&amp;nbsp;</td><td>VWR</td><td>470007-554</td><td>Forceps used to dissect the tissues.&amp;nbsp;</td></tr><tr><td>Phosphate Buffered Saline (PBS) pH 7.2</td><td>Life Technologies&amp;nbsp;</td><td>20012019</td><td>PBS is used to hydate the tissue samples&amp;nbsp;</td></tr></tbody></table>

biomechanical characterization of human soft tissues using indentation and tensile testing

再生医学的目的是工程师的材料来代替或恢复受损或患病的器官。这样的材料应该模仿他们的目标，以取代人体组织的机械性质;以提供所需的解剖学形状，材料必须能够承受的机械力时在缺损部位植入他们将遇到。虽然组织工程支架的机械性能是非常重要的，即经历恢复与工程材料许多人类组织没有得到充分的生物力学特征。几个压缩和拉伸协议报告用于评估材料，但有大的变化是困难的比较研究之间的结果。研究进一步复杂化的是机械测试的往往破坏性质。虽然组织失败的理解是重要的，同样重要的是具有下更生理学弹性和粘弹性特性的知识ogical负载条件。 此报告的目的是提供一种微创破坏性协议来评价人体软组织的压缩和拉伸性能。作为这种技术的例子中，皮肤的拉力试验和软骨的压缩测试中描述。这些协议也可以直接应用到合成材料，以确保机械性质类似于天然组织。协议评估人类天然组织的机械性能将允许一个基准由创建合适的组织工程化的替代品。

图4和5提供通过压痕和拉伸试验得到的数据的例子。 图4展示了人体软骨压痕试验后得到的典型值。 图4A是压痕试验之后获得的典型菌株抗应力曲线图的一个例子。以获得杨氏模量，所有的值都包括直到行曲线拟合具有0.98（ 图4B）的最小R值。 m值是杨氏模量在兆帕的指示符;例如，在该数据中，软骨具有1.76兆帕的模量。 图4C示出了应力对时间的典型曲线图，以评估软骨的松弛性能。松弛的速率从最后一个200秒计算的。同样，要获得松弛的速率，使用了线曲线拟合以MPa的m值的。例如，在该数据中，软骨具有的速度8.78×10 -6兆帕/秒（ 图4D）的放松。松弛的绝对最后一级是应力的以MPa最终点。例如，在该数据集，松弛的绝对最终水平将是0.028兆帕（ 图4D）。 图5显示了如何拉力试验评估后，皮肤组织的粘弹性。该分析作为每个压缩试验进行。 图5A展示了从拉伸试验协议获得的典型菌株抗应力图解。以获得杨氏拉伸模量，所有值都包含直到行曲线拟合具有0.98（ 图5B）的最小R值。 m值是杨氏模量在兆帕的指示符;例如，在该数据，对皮肤有0.62兆帕的模量。 图5C示出了相对于时间的压力的典型曲线图Ò评价松弛性能˚F皮肤。松弛的速率从最后一个200秒计算的。同样，要获得松弛的速率，使用了线曲线拟合以MPa的m值的。例如，在该数据，对皮肤有3.1×10 -5兆帕/秒（ 图5D）松弛的速率。松弛的绝对最后一级是应力的以MPa最终点。例如，在该数据组中，电平将是0.64兆帕（ 图5D）。然后在同样的分析可用于分析在压缩和拉伸测试生物材料到其生物力学特性匹配天然组织。 <img alt="图1" src="/files/ftp_upload/54872/54872fig1.jpg" /> 图1: 示意图来说明不同的压缩方法。 A.压痕测试。负载被施加到使用非多孔压头软骨的一个小区域。 B.限制ð压缩。软骨样本被放置在一个不透流体填充好。软骨然后通过多孔板加载。由于阱是不透水，流过软骨是仅在垂直方向上。 C.无侧限抗压强度。软骨是使用非多孔板到非多孔室加载，迫使流体流是主要径向的。 <img alt="图2" src="/files/ftp_upload/54872/54872fig2.jpg" /> 图 2: 力学试验机的设置。 A.试验机的插图。 B点 。用于压缩试验分析压头的插图。使用压缩压痕试验正在分析C.软骨。 D.皮肤组织被下拉力试验分析。合成生物材料E.拉伸试验。 F。 </stronG&gt; A合成生物材料的压缩试验。 <img alt="图3" src="/files/ftp_upload/54872/54872fig3.jpg" /> 图3: 用于计算的组织或组织工程构建物的压缩和拉伸的机械性能公式。 （N），用于计算力式中，应力（兆帕），和应变（％）。 <img alt="图4" src="/files/ftp_upload/54872/54872fig4.jpg" /> 图4: 人软骨的压缩分析的实施例。 A.应力与应变分析。 B.行曲线拟合方程的m个值是杨氏弹性模量在兆帕。 C.应力-时间分析来证明松弛性能。 D.行曲线拟合方程的m个值表示松弛率。最后一个bsolute速率是图表上的最后一个点。 <img alt="图5" src="/files/ftp_upload/54872/54872fig5.jpg" /> 图5: 人 的 皮肤拉伸分析的例子。 A.应力与应变分析。 B.行曲线拟合方程的m个值是杨氏弹性模量在兆帕。 C.应力-时间分析来证明松弛性能。行曲线拟合方程的D的m值的等同于松弛率。最终的绝对速率是图表上的最后一个点。

Watch this Scientific Journal Video about Biomechanical Characterization of Human Soft Tissues Using Indentation and Tensile Testing at JoVE.com

Biomechanical Characterization of Human Soft Tissues Using Indentation and Tensile Testing

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

人体软组织的生物力学特性采用压痕和拉伸试验

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

biomechanical-characterization-human-soft-tissues-using-indentation

Research

JoVE Journal

Bioengineering

31.3K 次观看.  University College London (UCL). Tissue biomechanics is important for maintaining cell shape and function and for determining phenotype. This report demonstrates non-destructive mechanical protocols for characterizing elastic and viscoelastic properties of human soft tissues, which can be directly applied to tissue-engineered substrates to allow a close matching of engineered materials to native tissue.

视频: Biomechanical Characterization of Human Soft Tissues Using Indentation and Tensile Testing

Characterization Of Multi-layered Fish Scales (Atractosteus spatula) Using Nanoindentation, X-ray CT, FTIR, and SEM

The hierarchical architecture of protective biological materials such as mineralized fish scales, gastropod shells, ram&#8217;s horn, antlers, and turtle shells provides unique design principles with potentials for guiding the design of protective materials and systems in the future. Understanding the structure-property relationships for these material systems at the microscale and nanoscale where failure initiates is essential. Currently, experimental techniques such as nanoindentation, X-ray CT, and SEM provide researchers with a way to correlate the mechanical behavior with hierarchical microstructures of these material systems1-6. However, a well-defined standard procedure for specimen preparation of mineralized biomaterials is not currently available. In this study, the methods for probing spatially correlated chemical, structural, and mechanical properties of the multilayered scale of A. spatula using nanoindentation, FTIR, SEM, with energy-dispersive X-ray (EDX) microanalysis, and X-ray CT are presented.

Characterization Of Multi-layered Fish Scales Atractosteus spatula Using Nanoindentation, X-ray CT, FTIR, and SEM

This paper presents the methods used for probing spatially correlated chemical, structural, and mechanical properties of the multilayered scale of Atractosteus spatula (A. spatula) using nanoindentation, Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), and X-ray computed tomography (X-ray CT). The experimental results have been used to investigate the design principles of protective biological materials.

表征多层鱼鳞（中<em&gt; Atractosteus锅铲</em&gt;）基于纳米压痕技术，X射线CT，红外光谱和SEM

The hierarchical architecture of protective biological materials such as mineralized fish scales, gastropod shells, ram&#8217;s horn, antlers, and turtle ...

科研

生物工程

A Coupled Experiment-finite Element Modeling Methodology for Assessing High Strain Rate Mechanical Response of Soft Biomaterials

This study offers a combined experimental and finite element (FE) simulation approach for examining the mechanical behavior of soft biomaterials (e.g. brain, liver, tendon, fat, etc.) when exposed to high strain rates. This study utilized a Split-Hopkinson Pressure Bar (SHPB) to generate strain rates of 100-1,500 sec-1. The SHPB employed a striker bar consisting of a viscoelastic material (polycarbonate). A sample of the biomaterial was obtained shortly postmortem and prepared for SHPB testing. The specimen was interposed between the incident and transmitted bars, and the pneumatic components of the SHPB were activated to drive the striker bar toward the incident bar. The resulting impact generated a compressive stress wave (i.e. incident wave) that traveled through the incident bar. When the compressive stress wave reached the end of the incident bar, a portion continued forward through the sample and transmitted bar (i.e. transmitted wave) while another portion reversed through the incident bar as a tensile wave (i.e. reflected wave). These waves were measured using strain gages mounted on the incident and transmitted bars. The true stress-strain behavior of the sample was determined from equations based on wave propagation and dynamic force equilibrium. The experimental stress-strain response was three dimensional in nature because the specimen bulged. As such, the hydrostatic stress (first invariant) was used to generate the stress-strain response. In order to extract the uniaxial (one-dimensional) mechanical response of the tissue, an iterative coupled optimization was performed using experimental results and Finite Element Analysis (FEA), which contained an Internal State Variable (ISV) material model used for the tissue. The ISV material model used in the FE simulations of the experimental setup was iteratively calibrated (i.e. optimized) to the experimental data such that the experiment and FEA strain gage values and first invariant of stresses were in good agreement.

The current study prescribes a coupled experiment-finite element simulation methodology to obtain the uniaxial dynamic mechanical response of soft biomaterials (brain, liver, tendon, fat, etc.). The multiaxial experimental results that arose because of specimen bulging obtained from Split-Hopkinson Pressure Bar testing were rendered to a uniaxial true stress-strain behavior when simulated through iterative optimization of the finite element analysis of the biomaterial.

一个耦合试验，有限元建模方法以评估生物材料软的高应变率力学响应

This study offers a combined experimental and finite element (FE) simulation approach for examining the mechanical behavior of soft biomaterials (e.g. ...

Atomic Force Microscopy Cantilever-Based Nanoindentation: Mechanical Property Measurements at the Nanoscale in Air and Fluid

An atomic force microscope (AFM) fundamentally measures the interaction between a nanoscale AFM probe tip and the sample surface. If the force applied by the probe tip and its contact area with the sample can be quantified, it is possible to determine the nanoscale mechanical properties (e.g., elastic or Young&#39;s modulus) of the surface being probed. A detailed procedure for performing quantitative AFM cantilever-based nanoindentation experiments is provided here, with representative examples of how the technique can be applied to determine the elastic moduli of a wide variety of sample types, ranging from kPa to GPa. These include live mesenchymal stem cells (MSCs) and nuclei in physiological buffer, resin-embedded dehydrated loblolly pine cross-sections, and Bakken shales of varying composition.
Additionally, AFM cantilever-based nanoindentation is used to probe the rupture strength (i.e., breakthrough force) of phospholipid bilayers. Important practical considerations such as method choice and development, probe selection and calibration, region of interest identification, sample heterogeneity, feature size and aspect ratio, tip wear, surface roughness, and data analysis and measurement statistics are discussed to aid proper implementation of the technique. Finally, co-localization of AFM-derived nanomechanical maps with electron microscopy techniques that provide additional information regarding elemental composition is demonstrated.

Quantifying the contact area and force applied by an atomic force microscope (AFM) probe tip to a sample surface enables nanoscale mechanical property determination. Best practices to implement AFM cantilever-based nanoindentation in air or fluid on soft and hard samples to measure elastic modulus or other nanomechanical properties are discussed.

原子力显微镜悬臂式纳米压痕:空气和流体中纳米级机械性能测量

An atomic force microscope (AFM) fundamentally measures the interaction between a nanoscale AFM probe tip and the sample surface. If the force applied by ...

工程学

Characterizing Multiscale Mechanical Properties of Brain Tissue Using Atomic Force Microscopy, Impact Indentation, and Rheometry

To design and engineer materials inspired by the properties of the brain, whether for mechanical simulants or for tissue regeneration studies, the brain tissue itself must be well characterized at various length and time scales. Like many biological tissues, brain tissue exhibits a complex, hierarchical structure. However, in contrast to most other tissues, brain is of very low mechanical stiffness, with Young&#39;s elastic moduli E on the order of 100s of Pa. This low stiffness can present challenges to experimental characterization of key mechanical properties. Here, we demonstrate several mechanical characterization techniques that have been adapted to measure the elastic and viscoelastic properties of hydrated, compliant biological materials such as brain tissue, at different length scales and loading rates. At the microscale, we conduct creep-compliance and force relaxation experiments using atomic force microscope-enabled indentation. At the mesoscale, we perform impact indentation experiments using a pendulum-based instrumented indenter. At the macroscale, we conduct parallel plate rheometry to quantify the frequency dependent shear elastic moduli. We also discuss the challenges and limitations associated with each method. Together these techniques enable an in-depth mechanical characterization of brain tissue that can be used to better understand the structure of brain and to engineer bio-inspired materials.

We present a set of techniques to characterize the viscoelastic mechanical properties of brain at the micro-, meso-, and macro-scales.

表征使用原子力显微镜，冲击压痕和流变测定脑组织的多尺度力学性能

To design and engineer materials inspired by the properties of the brain, whether for mechanical simulants or for tissue regeneration studies, the brain ...

神经科学

Micro-Mechanical Characterization of Lung Tissue Using Atomic Force Microscopy

Matrix stiffness strongly influences growth, differentiation and function of adherent cells1-3. On the macro scale the stiffness of tissues and organs within the human body span several orders of magnitude4. Much less is known about how stiffness varies spatially within tissues, and what the scope and spatial scale of stiffness changes are in disease processes that result in tissue remodeling. To better understand how changes in matrix stiffness contribute to cellular physiology in health and disease, measurements of tissue stiffness obtained at a spatial scale relevant to resident cells are needed. This is particularly true for the lung, a highly compliant and elastic tissue in which matrix remodeling is a prominent feature in diseases such as asthma, emphysema, hypertension and fibrosis. To characterize the local mechanical environment of lung parenchyma at a spatial scale relevant to resident cells, we have developed methods to directly measure the local elastic properties of fresh murine lung tissue using atomic force microscopy (AFM) microindentation. With appropriate choice of AFM indentor, cantilever, and indentation depth, these methods allow measurements of local tissue shear modulus in parallel with phase contrast and fluorescence imaging of the region of interest. Systematic sampling of tissue strips provides maps of tissue mechanical properties that reveal local spatial variations in shear modulus. Correlations between mechanical properties and underlying anatomical and pathological features illustrate how stiffness varies with matrix deposition in fibrosis. These methods can be extended to other soft tissues and disease processes to reveal how local tissue mechanical properties vary across space and disease progression.

The stiffness of the extracellular matrix strongly influences multiple behaviors of adherent cells. Matrix stiffness varies spatially throughout a tissue, and undergoes modification in various disease conditions. Here we develop methods to characterize spatial variations in stiffness in normal and fibrotic mouse lung tissue using atomic force microscopy microindentation.

微型机械的肺组织利用原子力显微镜表征

Matrix stiffness strongly influences growth, differentiation and function of adherent cells1-3.  On the macro scale the stiffness of tissues and organs ...

生物学

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 ...

Experimental and Data Analysis Workflow for Soft Matter Nanoindentation

Nanoindentation refers to a class of experimental techniques where a micrometric force probe is used to quantify the local mechanical properties of soft biomaterials and cells. This approach has gained a central role in the fields of mechanobiology, biomaterials design&#160;and tissue engineering, to obtain a proper mechanical characterization of soft materials with a resolution comparable to the size of single cells (&#956;m). The most popular strategy to acquire such experimental data is to employ an atomic force microscope (AFM); while this instrument offers an unprecedented resolution in force (down to pN) and space (sub-nm), its usability is often limited by its complexity that prevents routine measurements of integral indicators of mechanical properties, such as Young&#39;s Modulus (E). A new generation of nanoindenters, such as those based on optical fiber sensing technology, has recently gained popularity for its ease of integration while allowing to apply sub-nN forces with &#181;m spatial resolution, therefore being suitable to probe local mechanical properties of hydrogels and cells.
In this protocol, a step-by-step guide detailing the experimental procedure to acquire nanoindentation data on hydrogels and cells using a commercially available ferrule-top optical fiber sensing nanoindenter is presented. Whereas some steps are specific to the instrument used herein, the proposed protocol can be taken as a guide for other nanoindentation devices, granted some steps are adapted according to the manufacturer&#39;s guidelines. Further, a new open-source Python software equipped with a user-friendly graphical user interface for the analysis of nanoindentation data is presented, which allows for screening of incorrectly acquired curves, data filtering, computation of the contact point through different numerical procedures, the conventional computation of E, as well as a more advanced analysis particularly suited for single-cell nanoindentation data.

The protocol presents a complete workflow for soft material nanoindentation experiments, including hydrogels and cells. First, the experimental steps to acquire force spectroscopy data are detailed; then, the analysis of such data is detailed through a newly developed open-source Python software, which is free to download from GitHub.

软物质纳米压痕的实验和数据分析工作流程

Nanoindentation refers to a class of experimental techniques where a micrometric force probe is used to quantify the local mechanical properties of soft ...

Viscoelastic Characterization of Soft Tissue-Mimicking Gelatin Phantoms using Indentation and Magnetic Resonance Elastography

Characterization of biomechanical properties of soft biological tissues is important to understand the tissue mechanics and explore the biomechanics-related mechanisms of disease, injury, and development. The mechanical testing method is the most straightforward way for tissue characterization and is considered as verification for in vivo measurement. Among the many ex vivo mechanical testing techniques, the indentation test provides a reliable way, especially for samples that are small, hard to fix, and viscoelastic such as brain tissue. Magnetic resonance elastography (MRE) is a clinically used method to measure the biomechanical properties of soft tissues. Based on shear wave propagation in soft tissues recorded using MRE, viscoelastic properties of soft tissues can be estimated in vivo based on wave equation. Here, the viscoelastic properties of gelatin phantoms with two different concentrations were measured by MRE and indentation. The protocols of phantom fabrication, testing, and modulus estimation have been presented.

This article presents a demonstration and summary of protocols of making gelatin phantoms that mimic soft tissues, and the corresponding viscoelastic characterization using indentation and magnetic resonance elastography.

使用压痕和磁共振弹性成像对软组织模拟明胶模型进行粘弹性表征

Characterization of biomechanical properties of soft biological tissues is important to understand the tissue mechanics and explore the ...

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 ...

<meta charset="utf8"></head><body>使用外植体模型对骨形成进行多模态分析

Comprehensive Characterization of Tissue Mineralization in an Ex Vivo Model

The extensive characterization of tissue mineralization in the context of bone regeneration represents a significant challenge, given the numerous modalities that are currently available for analysis. Here, we propose a workflow for a comprehensive evaluation of new bone formation using a relevant large animal osseous ex vivo explant. A bone defect (diameter = 3.75 mm; depth = 5.0 mm) is created in an explanted sheep femoral head and injected with a macroporous bone substitute loaded with a pro-osteogenic growth factor (bone morphogenetic protein 2 - BMP2). Subsequently, the explant is maintained in culture for a 28-day period, allowing cellular colonization and subsequent bone formation. To evaluate the quality and structure of newly mineralized tissue, the following successive methods are set up: (i) Characterization and high-resolution 3D images of the entire explant using micro-CT, followed by deep learning image analyses to enhance the discrimination of mineralized tissues; (ii) Nano-indentation to determine the mechanical properties of the newly formed tissue; (iii) Histological examinations, such as Hematoxylin/Eosin/Saffron (HES), Goldner's trichrome, and Movat's pentachrome to provide a qualitative assessment of mineralized tissue, particularly with regard to the visualization of the osteoid barrier and the presence of bone cells; (iv) Back-scattering scanning electron microscopy (SEM) mapping with internal reference to quantify the degree of mineralization and provide detailed insights into surface morphology, mineral composition, and bone-biomaterial interface; (v) Raman spectroscopy to characterize the molecular composition of the mineralized matrix and to provide insights into the persistence of BMP2 within the cement through the detection of peptide bonds. This multimodal analysis will provide an effective assessment of newly formed bone and comprehensive qualitative and quantitative insights into mineralized tissues. Through the standardization of these protocols, we aim to facilitate interstudy comparisons and improve the validity and reliability of research findings.

<meta charset="utf8"></head><body>作者聚焦:在骨再生研究中表征组织矿化的高级技术

The proposed protocol entails a global approach to assess bone formation in the context of bone regeneration using multimodal analyses. It aims to provide qualitative and quantitative information on new bone formation, enhancing the rigor and validity of basic and pre-clinical investigations.

人体软组织的生物力学特性采用压痕和拉伸试验

摘要

探索更多视频

此视频中的章节

表征多层鱼鳞（中