School of Engineering and Materials Science, Queen Mary University of London, Mile End Rd, London E1 4NS, United Kingdom; Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, The Netherlands.
School of Engineering and Materials Science, Queen Mary University of London, Mile End Rd, London E1 4NS, United Kingdom; Comparative Biomedical Sciences, Royal Veterinary College, Royal College Street, London NW1 0TU, United Kingdom.
Acta Biomater. 2018 Apr 1;70:281-292. doi: 10.1016/j.actbio.2018.01.034. Epub 2018 Feb 2.
Structure-function relationships in tendons are directly influenced by the arrangement of collagen fibres. However, the details of such arrangements in functionally distinct tendons remain obscure. This study demonstrates the use of quantitative polarised light microscopy (qPLM) to identify structural differences in two major tendon compartments at the mesoscale: fascicles and interfascicular matrix (IFM). It contrasts functionally distinct positional and energy storing tendons, and considers changes with age. Of particular note, the technique facilitates the analysis of crimp parameters, in which cutting direction artefact can be accounted for and eliminated, enabling the first detailed analysis of crimp parameters across functionally distinct tendons. IFM shows lower birefringence (0.0013 ± 0.0001 [-]), as compared to fascicles (0.0044 ± 0.0005 [-]), indicating that the volume fraction of fibres must be substantially lower in the IFM. Interestingly, no evidence of distinct fibre directional dispersions between equine energy storing superficial digital flexor tendons (SDFTs) and positional common digital extensor tendons (CDETs) were noted, suggesting either more subtle structural differences between tendon types or changes focused in the non-collagenous components. By contrast, collagen crimp characteristics are strongly tendon type specific, indicating crimp specialisation is crucial in the respective mechanical function. SDFTs showed much finer crimp (21.1 ± 5.5 µm) than positional CDETs (135.4 ± 20.1 µm). Further, tendon crimp was finer in injured tendon, as compared to its healthy equivalents. Crimp angle differed strongly between tendon types as well, with average of 6.5 ± 1.4° in SDFTs and 13.1 ± 2.0° in CDETs, highlighting a substantially tighter crimp in the SDFT, likely contributing to its effective recoil capacity.
This is the first study to quantify birefringence in fascicles and interfascicular matrix of functionally distinct energy storing and positional tendons. It adopts a novel method - quantitative polarised light microscopy (qPLM) to measure collagen crimp angle, avoiding artefacts related to the direction of histological sectioning, and provides the first direct comparison of crimp characteristics of functionally distinct tendons of various ages. A comparison of matched picrosirius red stained and unstained tendons sections identified non-homogenous staining effects, and leads us to recommend that only unstained sections are analysed in the quantitative manner. qPLM is successfully used to assess birefringence in soft tissue sections, offering a promising tool for investigating the structural arrangements of fibres in (soft) tissues and other composite materials.
肌腱中的结构-功能关系直接受到胶原纤维排列的影响。然而，功能不同的肌腱中这种排列的细节仍然不清楚。本研究利用定量偏光显微镜（qPLM）在介观尺度上识别两种主要肌腱区室的结构差异：束和束间基质（IFM）。它对比了功能不同的位置和储能肌腱，并考虑了年龄的变化。值得注意的是，该技术便于分析卷曲参数，其中可以考虑和消除切割方向的人为因素，从而能够首次对功能不同的肌腱进行卷曲参数的详细分析。IFM 的双折射度（0.0013±0.0001[-]）低于束（0.0044±0.0005[-]），表明 IFM 中的纤维体积分数必须低得多。有趣的是，在马的储能浅表指深屈肌腱（SDFTs）和位置指浅伸肌腱（CDETs）之间没有发现明显的纤维定向分散的证据，这表明肌腱类型之间可能存在更微妙的结构差异，或者变化集中在非胶原蛋白成分中。相比之下，胶原卷曲特征在很大程度上是肌腱类型特异性的，这表明卷曲专门化在各自的机械功能中是至关重要的。SDFTs 的卷曲度比位置性 CDETs （135.4±20.1μm）要精细得多（21.1±5.5μm）。此外，与健康的对应物相比，受伤肌腱的卷曲度更细。肌腱卷曲度在肌腱类型之间也有很大差异，SDFTs 的平均卷曲角度为 6.5±1.4°，CDETs 的平均卷曲角度为 13.1±2.0°，这突出表明 SDFT 的卷曲度要紧密得多，这可能有助于其有效的回弹能力。
这是第一项定量研究功能不同的储能和位置肌腱的束和束间基质的双折射的研究。它采用了一种新的方法——定量偏光显微镜（qPLM）来测量胶原卷曲角，避免了与组织切片方向相关的人为因素，并首次直接比较了不同年龄的功能不同的肌腱的卷曲特征。对匹配的苦味酸红染色和未染色肌腱切片的比较确定了非均匀染色效果，并促使我们建议仅对未染色的切片进行定量分析。qPLM 成功地用于评估软组织切片的双折射，为研究（软组织）和其他复合材料中的纤维结构排列提供了一种很有前途的工具。
