To test the proposal presented in section 5.1 stating that skeletal muscle perimysium is hierarchical with two distinct orders of collagen fibre organisation which respond differently to applied deformation, collagen fibre orientation in transverse sections of chicken pectoralis, porcine biceps femoris, and porcine longissimus dorsi muscles under different deformation conditions were analysed and are illustrated in Figure 5.6i-iii respectively. For each condition, a series of images from different specimens are presented.
In undeformed chicken samples, as shown in Figure 5.6i(a), the perimysium in the transverse plane (w1) is wavy since there is no load on the tissue.
Where the chicken tissue was compressed along the direction of the muscle fibres (Comp-F), collagen fibre organisation in perimysium, viewed in cross section (w1) is straight, (see Figure 5.6i (b)). Comp-F causes shortening of the tissue in the direction of the muscle fibres and accordingly expansion in cross sectional plane resulting in a larger block cross section. Therefore, perimysium stretches due to the increase in cross section area of the tissue showing straighter collagen fibrils (w1) compared to the control case.
Where the tissue was compressed perpendicular to the muscle fibres (Comp-XF), the tissue expanded in the muscle fibre direction (indicated as L in Figure 5.1) and other perpendicular direction (T′, Figure 5.1). Where perimysium in the transverse plane is parallel to compressive deformation (T), the collagen fibrils were wavy with dramatic change in the aspect ratio of muscle fibres (see Figure 5.6i(c), top image). Conversely, where perimysium was aligned perpendicular to the deformation direction the collagen fibrils straightened (see Figure 5.6i(c), two bottom images).
In tension in the muscle fibre direction (Ten-F), the tissue cross section becomes smaller resulting in wavy collagen fibrils (w1) in perimysium.
In tension applied perpendicular to the muscle fibres (Ten-XF), the collagen fibrils in the transverse plane aligned with the load direction became straight (see Figure 5.6i(e), three top images), because the tissue elongated in the deformation direction, but the perimysium perpendicular to the tensile deformation was relieved and showed waviness (see Figure 5.6i(e), bottom image).
Cross-species comparison shows that the same collagen reorientation (w1) was seen in both porcine tissues, i.e. wavy collagen in control but less wavy than chicken tissue; straight collagen fibrils in Com-F (see Figure 5.6ii & iii, Comp-F); less wavy collagen in Comp-XF where the perimysium is perpendicular to the direction of deformation (see Figure 5.6iii(c), the top image), and wavy
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perimysium where perimysium is aligned with the direction of deformation(see Figure 5.6iii(c), bottom image); wavy structure in Ten-F (see Figure 5.6ii & iii, Ten-F); and straight collagen fibrils in Ten-XF where the perimysium aligned with the direction of deformation (see Figure 5.6ii(d) and Figure 5.6iii(e), three top images), and less wavy collagen fibrils where perimysium is perpendicular to the direction of deformation. The collagen waviness observed in Ten-F case for porcine BF muscle is greater than in the case of LD muscle.
i. Chicken
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ii. Porcine
BF
iii. Porcine
LD
Figure 5. 6-Visualisation of w1 in collagen fibrils of perimysium in transverse sections for undeformed, and externally applied compression and tensile deformations in chicken pectoralis (i), porcine biceps femoris (ii), and porcine
longissimus dorsi (iii) muscles.
Several example micrographs for control (a), Comp-F (compression in the muscle fibre direction, b), Comp-XF (compression in the muscle cross-fibre direction, c), Ten-F (tension in the muscle fibre direction, d), and Ten-XF (tension in the muscle cross-fibre direction, e) conditions, visualised by confocal microscopy in the transverse plane. Red arrows
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represents the direction of applied deformations in Comp-XF and Ten-XF cases. Manually drawn curves/lines in yellow indicate the condition of collagen fibril waviness viewed in the transverse plane. Scale bars indicate 20 µm.
Changes in w2 were viewed and analysed in longitudinal sections of chicken muscle tissue under different deformation conditions, presented in Figure 5.7. Perimysium is observed to be wavy in control (a) as there is no load applied on the tissue. It is also observed that the collagen fibrils in Comp- F (b) are also wavy as the tissue shortens in the direction of muscle fibres in this deformed state. The collagen fibrils in Comp-XF (c) stretched due to the induced deformation resulting in less wavy collagen fibrils in the longitudinal direction (w2), see yellow lines. In Ten-F (d) the collagen fibrils were straight (yellow lines) because there is a direct deformation along the perimysium in the muscle fibre direction. Since in Ten-XF (e) the tissue shortening is induced in the longitudinal direction, collagen waviness was observed in this direction (w2).
Figure 5. 7-Visualisation of w2 in collagen fibrils of perimysium in longitudinal sections for undeformed, and externally applied compression and tensile deformations in chicken pectoralis muscle.
Several example micrographs for control (a), Comp-F (compression in the muscle fibre direction, b), Comp-XF (compression in the muscle cross-fibre direction, c), Ten-F (tension in the muscle fibre direction, d), and Ten-XF (tension
in the muscle cross-fibre direction, e) conditions, visualisedby confocal microscope in the longitudinal direction. Manually drawn curves/lines in yellow indicate the condition of collagen fibril waviness viewed in the lonitudinal
direction. Scale bars indicate 20 µm.
The hierarchical organisation of muscle perimysium was proposed to change under deformation and contributes to the tension/compression asymmetry observed in stress-strain behaviour of skeletal muscle. This was tested through examining the confocal images of chicken and porcine tissues in different mechanical conditions. Figure 5.8 illustrates the three-dimensional
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reorganisation/reorientation of perimysium collagen fibrils when the tissue is exposed to externally applied deformations showing that collagen fibrils in perimysium responds to the deformation differently in different planes; these findings supported the proposal. Therefore, the straightness in collagens represents load resistance in the structure because when no load is borne in a particular plane, the collagen is wavy in that plane.
i. Chicken
pectoralis
ii. Porcine BF
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iii. Porcine LD
Figure 5. 8-The three-dimensional reorganisation/reorientation of perimysium collagen fibrils when the tissue is exposed to externally applied deformations in chicken pectoralis (i), porcine biceps femoris (ii), and porcine longissimus
dorsi (iii) muscles.
Sample micrographs in Comp-F (compression in the muscle fibre direction), Comp-XF (compression in the muscle cross-fibre direction), Ten-F (tension in the muscle fibre direction), and Ten-XF (tension in the muscle cross-fibre direction) conditions were taken by confocal microscopy. w1 and w2 (dark green) represent the condition of the perimysium viewed in the cross sectional plane and longitudinal direction respectively. Black arrows in cartoons and red
arrows in the images show the direction of applied deformation. Observed w1 (in blue for partly wavy pattern, and purple for partly straight pattern), and w2 (in orange) are shown here in the deformed conditions. Scale bars indicate 20
µm.
This approach investigates how the mechanical response relates to microstructural organisation (summarised in Table 5.2). It should be noted that the data from longitudinal sections of porcine tissue are missing due to technical issues associated with generating longitudinal sections (see Section 5.4.4, point 2).
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Table 5. 2-Summary of perimysium behaviour (straightness/waviness) at transverse plane and longitudinal direction.
w1 (at cross sectional plane)
w2 (in longitudinal
direction) Deformation
condition Chicken Porcine BF Porcine LD Chicken
Control wavy wavy wavy wavy
Comp-F straight straight straight wavy
Comp-XF partly straight/partly wavy -- partly straight/partly wavy less wavy
Ten-F wavy wavy wavy straight
Ten-XF partly straight/ partly wavy partly straight partly straight/ partly wavy wavy
5.4.
Discussion
Given that investigating the structural changes in skeletal muscle to interpret mechanical response of passive skeletal muscle to deformation requires microscopic analysis, collagen fibril orientation in perimysium under different loading conditions was viewed in transverse and longitudinal section planes in two different species to observe the structural properties of perimysium.
Overall the organisation and pattern of perimysium and endomysium was similar between chicken and porcine samples with some notable differences. In general, perimysium in the porcine muscle examined was larger (Figure 5.5) and porcine tissues also revealed a combination of more intensely stained and less intensely stained endomysium within the same fascicle in porcine LD and BF muscles, whereas in chicken pectoralis muscle the fibres surrounded with evenly stained endomysium were observed. Two types of muscle fibre can be found in skeletal muscle; slow-twitch2 and fast-twitch
fibres. Slow-twitch fibres use primarily oxidative respiration (uses oxygen to make energy) while fast- twitch fibres use primarily anaerobic respiration (Plowman and Smith, 2013). It was previously shown that the slow tonic muscles such as pectoralis and soleus have higher amounts of collagen as they correspond to postural control rather than fast activities (Velten and Welch Jr, 2014, KOVANEN et al., 1980, Rodrigues et al., 1996). Kovanen et al. (1984) attempted to show whether the difference observed between muscle types in terms of collagen content is also observable at the level of individual muscle fibres (fast-twitch and slow-twitch). Through histochemical staining they showed that perimysium and endomysium stained more strongly in the slow twitch area of rat gastrocnemius muscle than in the fast twitch area. Therefore the more intensely stained endomysium observed in porcine tissue in the current study may represent slow-twitch muscle fibres. Indeed LD and BF are
2The slow-twitch fibres are usually smaller in diameter, contain more mitochondria, use aerobic respiration for ATP supply, fire slowly but
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mixed-type muscles, whereas chicken pectoralis muscle is reported to contain mostly slow-twitch fibres (Velten and Welch Jr, 2014).