1. • Acellular collagen gels were prepared at a final concentration of 2.5
mg/ml using previously published methods2
• Gels were prepared in Petri dishes lined with plasma-treated, porous
polyethylene rings to prevent the gel from slipping during needling
• Gels were secured and centered on the bottom plate of a Kinexus Ultra
Rheometer (Malvern Instruments)
• Stainless steel, 250 μm-diameter acupuncture needles were coupled to
the rheometer with a custom chuck and inserted into the gel to a
consistent depth
• The needle was rotated at 10 revolutions per minute for 3 rotations,
during which the reaction torque on the needle was recorded
• During the torque-relaxation tests, one gel of each type was needled until
failure in order to serve as a calibration measure, and the maximum
attained torque was recorded. The other gels were needled until 75% of
the previously attained maximum torque was reached and were then
allowed to relax until the torque leveled off
• The influence of gel diameter and shape on torque were examined
according to Table 1
In Vitro Assay of Time-Dependent Torque During Collagen Fiber
Acupuncture Needle Rotation
Alicia Lee, Joshua Hogate, David Shreiber
Department of Biomedical Engineering, Rutgers, The State University of New Jersey, Piscataway, NJ
INTRODUCTION
REFERENCES
1. Langevin HM, Churchill DL, Wu J, et al. Evidence of connective tissue involvement in
acupuncture. FASEB J. 2002; 16:872-874. [PubMed: 12467083]
2. Julias M, Edgar LT, Buettner HM, Shreiber DI. An in vitro assay of collagen fiber
alignment by acupuncture needle rotation.BioMedical Engineering OnLine 2008, 7:19.doi:
10.1186/1475-925X-7-19
3. Julias, M., Buettner, H. M. and Shreiber, D. I. (2011), Varying Assay Geometry to Emulate
Connective Tissue Planes in an In Vitro Model of Acupuncture Needling. Anat Rec,
294: 243–252. doi: 10.1002/ar.21308
4. Hogate J., Shreiber DI. In Vitro Assay of Reaction Torque and Fiber Alignment During
Collagen Fiber Acupuncture Needle Rotation. Poster session presented at: Aresty
Undergraduate Research Symposium; 2014 Apr 24; New Brunswick, NJ.
DISCUSSION AND IMPLICATIONS
Although acupuncture is a recognized form of treatment for many disorders, its
mechanism of action remains unknown. Evidence suggests that
mechanotransduction via mechanically stressed connective tissue may play a
role. Dr. Helene Langevin of the University of Vermont has shown that the
collagen fibers within loose connective tissue couple with the needle during
routine acupuncture needle rotation (Figure 1),
Elliptical gels and gels with increased diameter take longer to reach failure and are
able to attain a higher maximum torque than the control gels. More samples will be
collected to confirm these results, which are consistent with previous
experiments4. More samples will also be collected to further explore the torque-
relaxation behavior of various types of gels.
Future studies will entail:
1) Determining the influence of varying needle rotation speeds and crosslinking
gels with 4% paraformaldehyde on torque, alignment, and gel relaxation
behavior
2) Characterizing torque in cellular gels
3) Delivering and holding specific mechanical signals to resident cells to mimic
clinical acupuncture and study mechanotransduction
Figure 1: Ultrasound acoustic
imaging of rat subcutaneous
tissue deformation due to
acupuncture needle rotation
in situ1. (A) Prior to needle
rotation; (B) After needle
rotation.
Figure 4: A rotational
rheometer was
adapted to perform in
vitro acupuncture
with high positional
and torque sensitivity.
METHODS: Collagen Hydrogel Preparation
Table 1: Experimental Conditions for torque measurement
during in vitro acupuncture
Diameter Shape
Normal (Control) Condition 1/2" Circular
Increased Diameter 7/8” Circular
Elliptical 1/2”/7/8” Elliptical
Figure 3: Similar to in situ
observation, fibroblasts entrapped
within collagen gels align with the
gel after acupuncture needling3. (A)
Before needling; (B) After needling
in a circular gel; (C) After needling
in an elliptical gel; (D) Quantitative
comparison of average alignment
which results in an increasing measured force on
the needle and the “needle grasp” sensation that
is experienced and utilized by acupuncture
therapists1. Using type I collagen gels to mimic
loose connective tissue, we have developed in
vitro models to quantitatively study the collagen
fiber alignment that results from this coupling
(Figures 2 & 3) 2,3 and to measure the needle
grasp, quantified as reaction torque, using a
rotational rheometer (Figure 4). As demonstrated
in previous experiments, reaction torque
accurately reflects the progression of fiber
alignment and is directly related to alignment4.
Consequently, we can utilize reaction torque as a
control signal to accurately control the degree to
which we subject cells to acupuncture in future
studies, thereby allowing us to systematically
analyze the effects of acupuncture. The objective
of this research was to determine the effect of gel
diameter and shape on the reaction torque in
order to model the mechanical behavior of
different types of body tissue during acupuncture
needling. We also examined the relaxation
behavior of needled gels in order to determine the
mechanical behavior of tissue during clinical
acupuncture.
RESULTS
Figure 2: Polarized light micrographs of fiber alignment following in
vitro acupuncture of collagen gels after (A) one revolution and (B)
four revolutions. (C) The area of alignment increases non-linearly with
needle rotation until the gel fails.2
A B
C
Reaction
plate
3D-printed fixture
for centering and
holding dish
Matek, glass-bottom
Petri dish with
porous polyethylene
insert to hold gel
Stainless steel
acupuncture needle
Drill chuck
“Universal”
fixture
To servomotor of
Malvern Instruments
Kinexus Ultra
Rotational Rheometer
Figure 5: Reaction torque over time.
Note that the elliptical and wide gels
take longer to couple and reach failure
than the control gels
Figure 6: Reaction torque during
torque-relaxation tests. The control gels
attain maximum torque more quickly
than the elliptical and wide gels
Table 2: Results from Needle Stimulation of Gels until Failure
Avg. Time to Attain
Maximum Torque
Average Maximum
Torque Attained
Normal (Control) Condition 16.1 seconds 4.1e-5 N*m
Increased Diameter 24.9 seconds 7.1e-5 N*m
Elliptical 29.4 seconds 7.1e-5 N*m
Table 3: Results from Torque-Relaxation Tests
Avg. Time to Attain Maximum
Torque
Normal (Control) Condition 7.7 seconds
Increased Diameter 15.4 seconds
Elliptical 10.6 seconds