During treatment, acupuncture therapists aim to achieve "needle grasp" as a sensory marker of an appropriate degree of needle manipulation. Recent studies suggest that needle grasp occurs when collagen fibers in the subcutaneous connective tissue attach to and wind around the needle, thus imposing a local stress and strain field on the surrounding tissue [6
]. In this paper, we imaged a simple, in vitro, acellular collagen gel system using polarized light microscopy during acupuncture needle rotation and measured the degree of winding in terms of fiber alignment to identify relationships between collagen concentration, crosslinking, and winding, as well as the failure of the gels.
We found that both collagen concentration and crosslinking influenced the response to controlled acupuncture needle rotation. Alignment increased with increasing collagen concentration, but decreased in gels that were crosslinked with formalin. Crosslinked gels also failed at a significantly lower number of needle rotations than untreated gels. Failure consistently occurred ~0.25 mm-1.0 mm away from the needle, and corresponded to the point where circumferential alignment of collagen fibers wound around the needle transitioned to radial alignment from fibers in the periphery of the gel being pulled into the needled area. The acute change in fiber geometry likely introduced a stress concentration that ultimately caused a tear in the collagen gel.
Altering the collagen concentration increases fiber density, which has potential indirect and direct consequences on the polarized light microscopy. First, the increase in fiber density could affect the mechanical properties simply by increasing the number of structural elements to carry load and/or by increasing fiber-fiber interactions, which would affect the tissue response to needle rotation. The increase in fiber density would also influence the degree of alignment, as measured with polarized light microscopy. An increase in fiber density implies that a greater number of fibers would be aligned for the same number of revolutions, and could therefore more efficiently rotate the polarization state of the incident light.
Crosslinking the gel decreases the flexibility of individual and aggregate fibers, which had a marked effect on the mechanical properties of the gels, increasing the storage modulus in shear by about an order of magnitude. The increased rigidity of crosslinked fibers increased the resistance to winding and deformation, and also increased the stress generated with winding, thereby leading to less alignment and earlier failure compared to untreated gels of the same concentration.
In addition to altering the mechanical properties of the gels, changing collagen concentration and crosslinking the gels could have influenced interactions and adhesion with the needle and/or the polystyrene dish. However, no gels failed at either the needle interface or the dish interface, and we believe that differences in adhesion among conditions played a minimal role in the bulk of the observed temporal response, except, perhaps, for the initial lag period in the alignment curves that represents initiation of alignment.
The differences between the response of untreated and crosslinked collagen gels to in vitro acupuncture, and particularly the earlier failure of crosslinked gels, suggests that mechanostructural differences in the soft tissues that contact an acupuncture needle during therapy may be responsible for the selective coupling and winding of collagen fibers in specific soft tissue layers during needle rotation. In vivo and explant studies have demonstrated that, although an acupuncture needle is inserted through the epidermis and dermis and into subcutaneous fat and muscle, only the subcutaneous loose connective tissue appears to specifically wind around the needle [3
]. The resulting recruitment of loose connective tissue fibers towards the needle can thicken that layer and subsequently compress the overlying and underlying tissue layers, but the characteristic whorl pattern is only seen in the loose connective tissue.
The tissue properties that govern this selective adherence and winding are not yet known. However, a recent study by Iatridis et al. documented the mechanical properties in uniaxial tension of loose connective tissue from mouse explants, and noted important distinctions between loose connective tissue and other load bearing soft tissues, including skin [16
]. Most soft tissues demonstrate significant non-linear stiffening above a certain strain – typically between 1% and 20%. For example, skin has a low strain modulus on the same order as loose connective tissue (2.75 kPa) up to about 10% strain [17
], after which the modulus increases to ~240 kPa [17
]. In contrast, loose connective tissue demonstrated a highly linear elastic response up to 50% strain [16
]. Thus, as tissue begins to wind around the needle and deform, significantly greater stress will be generated in skin vs. loose connective tissue, and, similar to the response of our crosslinked gels, we would expect the failure stress to be reached at a lower number of revolutions. The network of collagen fibers in the dermis may be too stiff to effectively respond to acupuncture needle rotation.
In clinical acupuncture, the thickness of the connective tissue layer varies, often with the thickness of subcutaneous fat, and it is especially thick at intermuscular cleavage planes. These planes correlate anatomically to acupuncture points and meridians[14
], and Langevin has shown that the resistant force to needle rotation at acupuncture points is greater than at control points, where the connective tissue layer is thinner. It was suggested that needling in locations where connective tissue is more pronounced enhances the mechanical response of fibroblasts residing within this tissue [1
]. In investigating how the depth of needle insertion and gel thickness may affect the response of a homogeneous tissue, we found that both the relative depth as well as the absolute depth of insertion into the collagen gel were important factors in the failure and alignment responses. Thinner gels were able to withstand more needle rotations than thick ones. Interestingly, the failure point was reached earlier as the depth of insertion was increased in 4 mm-thick gels, but no real trend was observed in 6 mm-thick gels. For both 4 mm-thick and 6 mm-thick gels, more alignment was recorded when the depth of insertion was greater. The last observation is consistent with an increase in the number of fibers subjected to rotation via contact with the needle. We also observed that more alignment was generated for the same depth of insertion in thin gels versus thick ones. The greatest amount of alignment was observed with the greatest absolute coverage of the needle by the collagen gel – 4.5 mm insertion into a 6 mm-thick gel. We believe that the increase in thickness of collagen below the needle increases the physical resistance to drawing individual fibers up and in towards the needle, thereby creating more stress to stimulate resident cells. We also note that the tip of the acupuncture needle is tapered. The length of the tapered tip represents a greater proportion of the inserted needle at shallower insertion depths than deeper insertions. The biomechanical response of the gel, particularly the initial adhesion of the collagen fibers to the needle, may be influenced by needle diameter [14
], which would be embedded in our needle depth results.
The differences with collagen concentration and crosslinking, as well as the empirical differences in alignment from separate batches of collagen (compare 2.0 mg/ml plot in Figure to 3 mm insertion into 4 mm-thick gels in Figure ), suggest that subtle changes in tissue composition and structure may affect the biomechanical response to needle rotation in vivo, and potentially the efficacy of acupuncture therapy. It is well known that the collagen content of human skin throughout the body is non-uniform [9
], and the matrix components of skin can be crosslinked, degraded, and or damaged by any number of environmental factors, including exposure to ultraviolet light, disease states, such as glycation associated with type 1 diabetes mellitus [19
], and normal physiological processes, such as wound healing. There have been relatively few studies of loose connective tissue of any kind, though it is likely that the biophysical properties of this tissue also vary among individuals and with location in one individual, and may dictate, in part the efficacy of acupuncture in a particular patient or at a particular location.
It is important to keep in mind that the in vitro system developed in this work is only a first step and differs significantly from the loose connective tissue involved in acupuncture, a cellular tissue comprising primarily fibroblasts embedded in an extracellular matrix of collagen and elastin fibers and proteoglycans. We chose to begin with an acellular collagen gel, representing the most significant structural component of the extracellular matrix, to establish a baseline for further study before proceeding to the more complex cellular system, in which a number of variables can change dynamically due to fibroblast-mediated compaction, matrix synthesis, and degradation. We also chose a rotational velocity (0.3 rev/sec) significantly slower than typically applied clinically to facilitate image acquisition and reduce viscoelastic effects.