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Background: During traditional acupuncture therapy, soft tissues attach to and wind around the acupuncture needle. To study this phenomenon in a controlled and quantitative setting, we performed acupuncture needling in vitro.

Methods: Acupuncture was simulated in vitro in three-dimensional, type I collagen gels prepared at 1.5 mg/ml, 2.0 mg/ml, and 2.5 mg/ml collagen, and either crosslinked with formalin or left untreated. Acupuncture needles were inserted into the gels and rotated via a computer-controlled motor at 0.3 rev/sec for up to 10 revolutions while capturing the evolution of birefringence under cross-polarization.

Results: Simulated acupuncture produced circumferential alignment of collagen fibers close to the needle that evolved into radial alignment as the distance from the needle increased, which generally matched observations from published tissue explant studies. All gels failed prior to 10 revolutions, and the location of failure was near the transition between circumferential and radial alignment. Crosslinked collagen failed at a significantly lower number of revolutions than untreated collagen, whereas collagen concentration had no effect on gel failure. The strength of the alignment field increased with increasing collagen concentration and decreased with crosslinking. Separate studies were performed in which the gel thickness and depth of needle insertion were varied. As gel thickness increased, gels failed at fewer needle revolutions. For the same depth of insertion, alignment was greater in thinner gels. Alignment increased as the depth of insertion increased.

Conclusion: These results indicate that the mechanostructural properties of soft connective tissues may affect their response to acupuncture therapy. The in vitro model provides a platform to study mechanotransduction during acupuncture in a highly controlled and quantitative setting.

With this in mind, we have developed an in vitro approach to examine the first part of the proposed therapeutic mechanism, i.e., the link between tissue properties and collagen fiber winding, which can be used to guide further in vivo investigation. Specifically, we used type I collagen gels to study the effects of matrix properties on collagen fiber response to acupuncture needle rotation. We subjected gels with different collagen concentrations and degrees of crosslinking to computer-controlled needle rotation and used polarized light imaging to monitor the change in collagen fiber alignment during needle rotation. In separate gels, we varied the thickness and depth of needle insertion. Our results demonstrate that the winding response of fibrillar collagen to needle rotation resembles that of loose connective tissue and varies with network density and stiffness and the depth of needle insertion. The quantitative approach developed in this work provides a useful new tool to aid in elucidating tissue-level mechanisms of acupuncture.

A computer-controlled motor (MicroMo Electronics, Inc.) was used to needle the collagen gels. A 250-μm stainless steel acupuncture needle (Seirin, Tokyo, Japan) was attached to the motor and inserted perpendicular to the surface of the gel to a depth of 3 mm using a calibrated micromanipulator. The needle was rotated at 0.3 rev/s for either 2 or 4 revolutions in samples used for confocal imaging, or for 10 revolutions in samples used for continuous recording of the evolution of fiber alignment with polarized light microscopy.

Confocal microscopy was used to examine the fibril alignment pre- and post- needle rotation. The MatTek dish was covered with a 3 mm-thick sheet of poly(dimethyl siloxane) (PDMS), through which the acupuncture needle was inserted prior to entering the gel. The needle was rotated either 2 or 4 revolutions with the motor, after which the needle was de-coupled from the motor. Insertion through the PDMS prevented the needle from recoiling when detached from the motor and allowed the transfer of the dish to the confocal microscope. The gel was imaged with a 63 objective using a laser wavelength of 488 nm to visualize the fluorescent collagen fibers (excitation 497 nm, emission 520 nm). The confocal images were compared to bright field and polarized light images taken during the needling procedure.

Schematic of polarized light microscopy system. A dissection stereomicroscope with a USB camera was mounted upside-down to a bench top. A fiber-optic ring light was attached to the motor housing providing a light source to the sample without hindrance from the motor. The polarizer was placed on top of the sample dish, and the analyzer was placed on the microscope as shown with the axis of polarization orthogonal to the axis for the polarizer. A small hole in the polarizer allowed free insertion and rotation of the acupuncture needle in the sample.

Winding and failure of collagen gels during in vitro acupuncture. (A) PLM image of the gel immediately before the onset of tearing. The characteristic '4-leaf clover' pattern of birefringence increases in size up to the point of failure as the gel becomes increasingly aligned due to winding around the needle. (B-E) Development of gel failure at 0.5 sec (0.15 rev) intervals. At the onset of tearing (B), a weakening of the birefringence can be observed near the needle where the dense, circumferentially wound center transitions to radially aligned fibers (arrow). As failure ensues, a hole is observed in the gel (C-E), and the residual stress in the remainder of the gel is enough to bend the needle, as indicated by the shift in needle position, Δ, directed away from the tear. The increasing size of the tear results in a decreasing area of birefringence. (F) Images A-E marked on a plot of the area above a threshold intensity vs. needle revolutions. The peak represents the image taken at maximum alignment immediately prior to the onset of failure. Bar: 1 mm.

Revolutions to failure (average +/- standard error) during in vitro acupuncture. The number of revolutions before gel tearing was identified from alignment area curves and verified visually from the image sets. Crosslinking the collagen significantly decreased the ability of the collagen gels to withstand needle rotation without tearing (*, 2-way ANOVA, P < 0.001), whereas changing the collagen concentration had no effect (P = 0.274)

The depth of insertion study was performed with a new shipment of collagen, and preliminary experiments with collagen from the crosslinking and collagen concentration studies indicated that the new batch presented significantly less alignment than the old batch, but that trends in the response were the same. As such, experiments in the depth of insertion studies were performed exclusively with the new batch of collagen and analyzed separately from the crosslinking and collagen concentration studies. Changing the depth of needle insertion significantly affected the failure of the gels (Figure 9). Thicker gels failed at fewer revolutions than thinner gels. For 4 mm-thick gels, increasing the depth/percentage of needle insertion decreased the revolutions before failure, but this was not observed consistently with the 6 mm-thick gels. The alignment of gels was also affected by insertion depth and percentage (Figure 10). In vitro acupuncture with needles inserted the same depth in gels of different thickness generated more alignment in thinner gels, indicating that the fraction of the gel that is subjected to needle rotation is an important parameter in dictating the response. Maintaining the same percentage of insertion at gels of different thickness produced greater alignment in thicker gels than thinner gels. For example, inserting a needle 3 mm into a 4 mm-thick gel and rotating the needle produced more alignment than the same procedure in a 6 mm-thick gel (inverted triangles, Figure 10A and 10B). However, inserting a needle 75% into a 6 mm-thick gel (squares, Figure 10B) produces more alignment than 75% into a 4 mm gel (inverted triangles, Figure 10A).

Effects of gel height and depth of needle insertion on revolutions to failure during in vitro acupuncture. Thin gels were able to withstand significantly more needling than thick ones (P < 0.001). For 4 mm-thick gels, revolutions to failure decreased as the depth of needle insertion increased, but this was not observed for 6 mm-thick gels.

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. 59ce067264

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