A relatively unrecognized aspect of subcutaneous tissue is its biomechanical properties. As the intermediate layer between the muscle and skin, the subcutaneous tissue frequently experiences shear and lateral stresses whenever the body is in motion (Hermanns-Lê et al., 2004). Even during simple movements such as respiration and hand motion, muscles and bones move laterally with respect to the skin(Guimberteau et al., 2005). As a consequence, the subcutaneous tissue must be sufficiently malleable to permit unconstrained movement while being rigorous enough to avoid complete untethering of the two layers. Moreover, it must provide adequate tensile resistance to preserve the structural integrity of the nerves and vessels that traverse it. These biomechanical characteristics are arguably integral for the proper functioning of subcutaneous tissue yet remain difficult to evaluate particularly within the in vivo setting (Gibson et al., 2006).

According to past studies, biological tissues are structurally dynamic and adaptive – responding and accommodating to mechanical forces. For instance, cells such as fibroblasts and vascular smooth muscles orient in the direction of principal strain (Bischofs and Schwarz, 2003, Kanda and Matsuda, 1994). Collagenous networks similarly align along the axis of tensile strain and form macroscopic asymmetries (Girton et al., 2002, Vader et al., 2009). This structural response to mechanical stimuli ensures that the tissue is equipped for future, comparable forces and exemplifies the notion that ‘structure’ follows ‘function’. For this reason, structural properties, such as spatial anisotropy, may be an effective surrogate for the summative, tensile forces experienced by biological tissue and thereby serve as a useful means to investigate the biomechanical behavior of subcutaneous tissue (Markenscoff and Yannas, 1979). Spatial anisotropy is a method to quantify the amount of preferential orientation or alignment present in a substance or image.

As shown in Table 1A and 1B, the mean spatial anisotropy (the average anisotropy across the video sweep) ranged from 0.05 to 0.24, while the maximum spatial anisotropy (the maximum anisotropy within the sweep) ranged from 0.06 to 0.28. No image was associated with a negative global anisotropy – i.e., a vertical or shallow-to-deep oriented anisotropy. In general, greater spatial scales were associated with greater mean and maximum anisotropy; the calf region was associated with greater anisotropies compared to the thigh and arm; and the longitudinally oriented images were associated with greater anisotropies compared to transverse images. This latter observation was confirmed by the Wilcoxon Ranked Sum test which showed statistically significant differences between the longitudinal and transverse images particularly at larger spatial scales. These differences between the two probe orientations were accentuated further when the maximal anisotropy values were considered.

Multivariable statistical analyses for anisotropy at spatial scale of 1.0 mm showed that subcutaneous thickness, body site, and probe orientation were all significantly associated with spatial anisotropy. Greater subcutaneous thickness is associated with reduced mean and maximum spatial anisotropy. After accounting for subcutaneous thickness and probe orientation, the calf was associated with greater anisotropy whereas the arm was associated with lower anisotropy by both measures. Longitudinal probe orientation is associated with greater anisotropy, particularly for maximum spatial anisotropy.

Few animal studies, however, have provided some relevant insights into subcutaneous tissue. Based on ex-vivo rat subcutaneous tissue samples under uniaxial tension, the instantaneous elastic response of subcutaneous tissue was found to be highly linear up to 50% strain (Iatridis et al., 2003). This is unlike other specialized connective tissues, such as cartilage or tendons, where the elastic response is decidedly non-linear (e.g. ever intensifying tension with increasing strain) due to the increasing recruitment of fibers as tensile forces rise. The linear elastic response of subcutaneous tissue suggests that nearly most of the structural components within the tissue are already recruited - sensing the tensile forces within the tissue– even at low strain levels and thus theoretically exhausted of any extra reserve to resist additional strain. In addition, the elastic modulus of subcutaneous tissue was approximately 2.75 kPa, several orders of magnitude lower than many other solid tissues in the body - indicating that as biological tissues go, subcutaneous tissue is not especially stiff (Iatridis et al., 2003). Moreover, the relatively short viscoelastic relaxation time seen in the study revealed that subcutaneous tissue quickly accommodated to each additional strain by reducing its resistive tension.

Based on these findings, one would understandably conclude that subcutaneous tissue is simply ill-equipped to resist any tensile/shear forces and thus mechanically incapable of protecting the internal nerves/vessels during physical activity or trauma. However, the rat subcutaneous tissue used in the study is notably different from the human subcutaneous layers analyzed in this study in one important respect: the subcutaneous muscle of the rat ventral/lateral wall was removed prior to the biomechanical tests. Although subcutaneous muscles do not typically exist in humans (except for areas in the head, neck, and hand – e.g. platysma), in our samples, the echogenic bands within the subcutaneous layer may act to resist tensile forces much in the way that the subcutaneous muscle and its adjacent connective tissue would have probably done in the rat subcutaneous sample. Indeed, the echogenic bands were the structures highly correlated with increased spatial anisotropy across multiple scales whereas areas of high adipose (and low band) content showed no consistent change in anisotropy. Furthermore, the spatial anisotropy at band locations frequently increased with greater spatial scales suggesting that the echogenic bands operate at a macroscopic level - likely beyond the 1 mm range evaluated in this study - to resist tensile force along the span of an extremity.

The functional significance of these echogenic bands is presently unclear. Histologically, these bands are composed of collagenous fibers surrounded by mucinous proteoglycans and glycosaminoglycans. Considering collagen’s role in tensile resistance and the evident association between echogenic bands and spatial anisotropy, these bands likely provide the tensile protection to the internal structures that is inadequately provided by the adipose tissue alone (Knight et al., 1990). The mucinous, gel-like properties of the neighboring glycosaminoglycan may also facilitate sliding between collagen fibers and thereby provide the shear plane needed for the skin to slide over muscle during movement. Interestingly, the subcutaneous collagen of rodents has been observed to assume this shearing role with elastin serving the important role of maintaining elasticity (Kawamata et al., 2003).

If indeed spatial anisotropy is correlated with the direction and intensity of mechanical stress as we posit, this study provides some revealing insights into the mechanical behaviors of the extremity and its subcutaneous tissue. The statistically significant increase in longitudinal anisotropy as compared to transverse anisotropy indicates that the subcutaneous layer is not isolated from the mechanical stress induced by muscle activity. At all three investigated sites, the subcutaneous tissue overlied muscles that effectively ran parallel to the longitudinal axis of the extremity and thus plausibly experienced greater tensile force in the longitudinal direction during muscle contraction. Based on the ultrasound images of the subcutaneous layer, the echogenic bands form sheets spanning the length and circumference of the extremities. The consistent identification of greater spatial anisotropy in one direction was not naturally anticipated and speaks to both the nature in which these collagenous bands are organized (much like a tablecloth crimped when two points are tugged) and the sensitivity of our anisotropic measure.

In both our univariable and multivariable analyses, the subcutaneous layers of the calf was significantly associated with greater anisotropy compared to those at the thigh and arm, whereas the subcutaneous tissue of the thigh had greater anisotropy compared to the arm’s after accounting for subcutaneous thickness and probe orientation. Based on these results, gastrocnemius muscles may generate greater subcutaneous stresses compared to the Sartorius and Vastus Medialis, while these latter muscles may generate greater biomechanical stresses compared to the brachioradialis and biceps brachii. Although these anisotropy measures do not directly assess actual, immediate muscular stress, they may conceivably represent the composite forces generated by the muscle over the span of the day or of even greater periods of time. Considering the importance of leg muscles in ambulation (a daily activity), the study’s results are logical and consistent with this hypothesis. Nevertheless, studies that directly measure muscle stress are needed to confirm our supposition.

The inverse relationship between spatial anisotropy and subcutaneous thickness also indicates that mechanical stress is distributed across the subcutaneous tissue. Thicker subcutaneous tissues are more likely to disperse the mechanical forces and thus are associated with reduced spatial anisotropy. Thinner subcutaneous tissues, on the hand, are subjected to similar forces but over a smaller area. As a consequence, thinner subcutaneous layers are generally associated with greater echogenic band densities and thus spatial anisotropy. These results may carry implications for plastic surgeons interested in optimizing the amount of collagenous bands in the subcutaneous tissue to reduce development of “cellulite” (Gasperoni and Salgarello, 1995) or for massage therapists seeking to understand how manual interventions can affect subcutaneous tissue composition in various individuals. Indeed, deep mechanical massage has been shown to increase the amount of subcutaneous collagen bands in a porcine model (Adcock et al., 2001). This corroborates the assertion that collagenous bands are derivatives of mechanical stress but must also be understood within the context that subcutaneous thickness may significantly modify the distribution of mechanical stresses and therefore greatly moderate the response in band production and formation.

Based on our data, the ultrasound sweeps with longitudinal-oriented images were associated with greater variance in spatial anisotropy compared to the corresponding sweeps with transverse-oriented images. Not only did maximal anisotropy measures generate greater statistical differences between the two directions when compared to the statistical calculations using mean anisotropy, but the longitudinally-oriented sweeps generally revealed a visible peak in the spatial anisotropy vs. sweep distance map. The concept of longitudinal channels with greater mechanical stresses is not new and has been previously proposed by manual therapists. Variations on terms referring to this concept include “Anatomy Trains” (Myers, 2009) or “Myofascial Sequences” (Stecco, 2004) and are based on the notion that connective tissue structures serve as an “ectoskeleton” helping to mediate force transmissions across muscles and joints (Huijing, 1999a, Huijing, 1999b, Huijing, 2009, Benjamin, 2009). The fact these these peaks were significantly correlated with intermuscular spaces at the arm and calf intimate that these fascial planes help integrate mechanical stresses between muscles and transmit forces into more superficial layers such as the subcutaneous tissue. The lack of statistical significance at the thigh may be attributed to the somewhat oblique path of the Sartorius muscle and our determination of spatial anisotropy solely along the longitudinal axis of the extremity. This is in contrast with the calf where the intermuscular fascia between the gastrocnemius muscles aligned well with the longitudinal axis of the leg. Conceivably, calculating spatial anisotropy parallel to the Sartorius muscle would generate a more distinct correlation with the intermuscular plane.

This study relied on images directly acquired from the ultrasound device to calculate spatial anisotropy in the subcutaneous layer. As a result, only the spatial anisotropies in the transverse or longitudinal direction were calculated. For future studies, computational manipulation of video sweeps may be performed to reconfigure the images and generate anisotropies along different axes. This study had additional limitations. Ultrasonography inherently produces anisotropic images because it relies on impedance of acoustic waves traveling from skin to deeper layers. Vertical structures (spanning from superficial to deep) poorly reflect acoustic waves traveling in a vertical trajectory and thus are not well characterized on ultrasound images. Spatial anisotropy will nearly uniformly yield lateral anisotropies in ultrasonography and thus must be interpreted with this limitation in mind. Moreover, the lateral resolution of our ultrasound was approximately 0.5 to 1.0 mm – within the spatial range where the autocorrelations were performed. Although this spatial range was preliminarily tested and determined to have construct validity, our analyses clearly depends on the effectiveness of image processing program in interpolating data (adding pixels in the lateral direction). Furthermore, although no significant differences between two examiners were identified in this study, intra- and inter-examiner differences in ultrasound imaging acquisition must be considered since variations in ultrasound probe pressure can theoretically affect spatial anisotropy measures. In addition, this study is limited by the small sample size, limited number of body sites, and its focus on the relaxed state. Future studies should consider not only increasing the number of subjects and body sites but also diversifying the number of physical positions and states assessed (e.g. tensed muscle during flexion) to better grasp the generalizability of our findings. Finally, as stated previously, spatial anisotropy is merely a marker of structural tension and not a direct measure of biomechanical force.