Historically, specimens were sectioned and traced, and the areas are measured using a planimeter [
27]. This method is inherently destructive and prevents further testing of the sample. Chatzistergos et al. [
124] measured tendon CSA by taking sections of the tissue post-rupture. This assumes no plastic deformation has occurred during testing, as this may influence the final shape of the tissue. More recently, Iriuchishima et al. [
125] evaluated CSA of ACL versus the grafts used to replace them. The authors revisited Cronkite’s technique and sectioned ACL at the bone attachments and through the midsection. With the use of digital photography and image processing they were able to measure CSA with a higher degree of accuracy and reproducibility. The authors did not discuss a method for measuring CSA of the grafts in a non-destructive fashion, limiting the clinical value of the study. It was, however, noted that a 3D measurement system would provide a higher degree of anatomical accuracy due to the natural path of the ligament.
‘By estimation’ techniques have ranged from estimating the shape of the specimens and measuring the height and width of the sample to ‘fit’ the shape, to the gravimetric method of calculating the area based on the length and volume and even ocular micrometry [
126]. These techniques often assume uniformity within the tissue and, while simple, can introduce errors such as those discussed by Seitz et al. [
58].
Area micrometry allows for irregularity in the shape of soft tissue by using adjustable blocks to compress the tissue into a channel with known size, from which the volume can be calculated [
126‐
128]; however, these measurements generally underestimate CSA [
59].
Race and Amis [
129] approached the measurement of CSA differently, taking a silicone rubber cast and creating poly-methyl methacrylate replicas of the tissue for analysis. This technique was developed further by Goodship and Birch [
130] and Schmidt and Ledoux [
131] using new materials and improved techniques. Images were taken of the replicas and then analysed in silico to measure the area. These newer techniques were shown to improve measurement accuracy compared to existing methods. The casting method offers the advantage of being able to revisit measurements as the cast can be preserved even after destructive testing of the tendon.
Non-contact methods of measuring tendon dimensions offer significant advantages in terms of speed and usability. Shadow amplitude was developed in the 1960s to measure whole tissues. Of the aforementioned techniques, it was identified as the only non-destructive method able to be adapted to measure local CSAs [
126]. It was also noted that there was an inherent need for refinement in the measuring of CSAs, due to the poor repeatability of the technique and inability to identify concavities.
Technological changes have led to improved non-contact devices, such as laser micrometres developed by Lee and Woo [
4,
132], video dimension analysers [
133], and charge-coupled device (CCD) laser sensors [
134], as well as advances in medical imaging, including CT [
69,
135,
136], MRI [
137,
138], and US [
114]. When evaluating their new technique, Race and Amis [
129] pointed out that laser micrometry is potentially the most precise method of measuring the tendon; however, it is affected by specimen geometry and concavities, which also make it potentially the least accurate when dealing with complex shapes, leading researchers to develop new ways to measure CSA as technology improved.
Langelier et al. [
60] developed a new computer-controlled laser micrometre based on the work of Lee and Woo [
132]. The system utilised a 10-μm laser and was found to be accurate and highly repeatable, but unable to identify concavities in the tissue. Liu et al. [
139] proposed the use of a coordinate measurement machine, utilising laser micrometry of 1 μm to scan the tendon. This method was shown to be more accurate than that developed by Langelier et al. [
60] when scanning a standard block (0.4%, with 1.6% repeatability); however, the tendon measurements were only compared to the less accurate shape-fitting technique.
Translucency of tendon is a known issue in microscopic investigation of tissue and may inhibit laser-based measurement due to refraction of light at the surface. Langelier et al. [
60] discussed this issue and attributed it to the density of the sample being insufficient to interrupt the laser beam; however, this does not exclude the issue of refraction playing a part. Neither Langelier et al. [
60] nor Liu et al. [
139] evaluated their techniques against reliable existing methods; thus, the accuracy and repeatability of their experiments may be lower than reported when applied to hydrated soft tissue.
Moon et al. [
134] trialled CCD laser sensors to address some of these issues, finding that the new system was able to measure concavities in an accurate and repeatable fashion. It was, however, susceptible to underestimation of CSA due to laser penetration of the semi-transparent surface of the tissue. Therefore, the tendon was stained with Indian ink to provide a reflective coating. The system was also limited to objects with CSA larger than 20 mm
2. It was noted that the improvements over other measurement techniques were negligible for rabbit ligaments and tendon [
134].
Salisbury et al. [
140] sought to develop a new method for characterising CSA, using a laser-slice method. The technique was effective at measuring the concavities in CSA profile and did not require any surface modification. The tendon was required to be rotated almost perfectly vertical in order to accurately measure CSA, which limited the potential use on tissues which have a 3D anatomical path. The accuracy was comparable to other methods but was deemed cheaper and more reliable when dealing with cavities than other methods.
The importance of understanding the local variations in shape and area in soft tissues has previously been identified in relation to the development of CSA measurement systems [
60,
141]. These papers discussed calculating local stresses and strains based on the local shape data. This information improves understanding of how the tissue changes with load and may be used to create more accurate FEA models. To date, almost all methods have required researchers to measure CSA outside of a materials testing system (MTS). This can often mean the condition of the sample can change between the measurement of CSA and the final testing procedure [
59]. It is not necessarily practical to measure the tendon immediately prior to testing, such as when using cryo-grips [
59]. Therefore, in an ex vivo setting, a measurement system capable of integration with an MTS is desirable. The importance of measuring the instantaneous CSA so that true stress can be calculated has previously been discussed [
59].
Pokhai et al. [
59] developed a laser reflectance system for an MTS; however, it is sensitive to opacity, reflectivity, and orientation, as well as small specimen size and small concavities. Vergari et al. [
64] developed a linear scanner to measure CSA of the tendon. While this method is much faster (under 2 s per measurement) than existing techniques, and also highly accurate (less than 2% error), it is limited in the shapes that are measurable, due to the linearity of the measurements. As with previous techniques, it is only able to measure one region at a time, meaning that whole tendon shape data are not available during mechanical testing. Heuer et al. [
142] developed a 2D laser scanner to measure the deformation of an intervertebral disc in three dimensions. This scanner cannot distinguish tissue morphological complexities such as concavities and has a relatively limited viewing window.
Recent developments in 3D laser and structured light scanning (SLS), as well as advanced digital image correlation (DIC), have made these techniques affordable and suitable options for research.
Hashemi et al. [
143] utilised a commercially available 3D photographic scanner to scan the ACL. The scanning process was approximately 30 min, from which a 3D model was generated. The accuracy was similar to that seen with early laser micrometres; however, it also lacked the ability to detect concavities. The advantage of this technique over the laser-based systems is that CSA can be calculated at any point along the length of the tissue.
Three-dimensional structured white light (SWL) scanners have been used by Nebel [
144] to create photorealistic 3D models of human bodies with an accuracy of 1 mm. These models were then converted to FEA-compatible models. More recently, Ahn et al. [
145] used a 3D SWL scanner to evaluate the changes in the dentoalveolar protrusion in patients before and after orthodontic work. This involved scanning the face from three angles simultaneously and reconstructing the model to ensure that any change between angles was not a product of patient movement or positioning.
Hayes et al. [
146] reported a technique, using SLS, to measure CSA of biological tissue. The technique was shown to be fast, simple, and accurate, with minimal sample preparation. The technique demonstrated a high degree of repeatability and was able to capture the entire geometry, thereby enabling true stresses to be calculated along the sample.
DIC has been used to investigate the deformation of biological materials under load [
147‐
153]. The principle of DIC is to detect gradient differences in a greyscale image to find patterns which can subsequently be tracked between images. This often requires application of an irregular pattern of similar-sized dots to the material. This has led to DIC sometimes being referred to as ‘speckle imaging’. There are currently methods of calculation able to determine sub-pixel resolution of the strain fields [
154]. Commercial packages are available that utilise stereophotogrammetry to map the strain field in 3D. The limitation of this system is usually in applying the speckle pattern, which must be fine and irregular, but with good contrast to enable visualisation of the gradient. The technique allows for the use of high resolution or high-speed cameras to maximise the quality of data captured. However, a big advantage of DIC over other modalities is the ability to record local strain in addition to calculating the shape data. Evans et al. [
155] have previously discussed the advantages of using DIC in mechanical testing, as it provides more information, such as differences between regions of the test sample, which would otherwise be unmeasurable.