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An in vitro assay of collagen fiber alignment by acupuncture needle rotation.

Julias M, Edgar LT, Buettner HM, Shreiber DI - Biomed Eng Online (2008)

Bottom Line: 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.For the same depth of insertion, alignment was greater in thinner gels.

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Affiliation: Department of Biomedical Engineering, Rutgers, The State University of New Jersey, Piscataway, NJ, USA. tikus@eden.rutgers.edu

ABSTRACT

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.

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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.
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Figure 2: 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.

Mentions: In all cases, PLM generated images with a '4-leaf clover' morphology of birefringence that emerged and increased in area with increasing needle rotation, extending beyond the field of the captured image, until gel failure, at which point the intensity decreased (Figure 2). The PLM images were imported into MATLAB to quantify the birefringence. First, the failure point for each individual experiment was identified by plotting the number of pixels with intensity greater than a given threshold value, determined as described below, against the number of needle revolutions and identifying the global maximum of the curve (Figure 2F). This point was confirmed by visual inspection of the image sequence. Failure points were compared statistically using a two-way ANOVA, with collagen concentration and crosslinking as fixed effects (SPSS 12.0, Chicago, IL). Significance levels were set at P < 0.05. The earliest failure point (rounded down to the nearest integer) among all experiments was 4 revolutions for untreated samples and 2 revolutions for crosslinked samples.


An in vitro assay of collagen fiber alignment by acupuncture needle rotation.

Julias M, Edgar LT, Buettner HM, Shreiber DI - Biomed Eng Online (2008)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2483714&req=5

Figure 2: 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.
Mentions: In all cases, PLM generated images with a '4-leaf clover' morphology of birefringence that emerged and increased in area with increasing needle rotation, extending beyond the field of the captured image, until gel failure, at which point the intensity decreased (Figure 2). The PLM images were imported into MATLAB to quantify the birefringence. First, the failure point for each individual experiment was identified by plotting the number of pixels with intensity greater than a given threshold value, determined as described below, against the number of needle revolutions and identifying the global maximum of the curve (Figure 2F). This point was confirmed by visual inspection of the image sequence. Failure points were compared statistically using a two-way ANOVA, with collagen concentration and crosslinking as fixed effects (SPSS 12.0, Chicago, IL). Significance levels were set at P < 0.05. The earliest failure point (rounded down to the nearest integer) among all experiments was 4 revolutions for untreated samples and 2 revolutions for crosslinked samples.

Bottom Line: 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.For the same depth of insertion, alignment was greater in thinner gels.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biomedical Engineering, Rutgers, The State University of New Jersey, Piscataway, NJ, USA. tikus@eden.rutgers.edu

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus