• 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