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Spinal Traction Promotes Molecular Transportation in a Simulated
Degenerative Intervertebral Disc Model
Ya-Wen Kuo, PhD; Yu-Chun Hsu, MS; I-Ting Chuang, MS; Pen-Hsiu Grace Chao,
PhD; and Jaw-Lin Wang, PhD
Institute of Biomedical Engineering, College of Medicine and Engineering, National
Taiwan University, Taipei, Taiwan
Address correspondence and requests for reprints to
Jaw-Lin Wang, PhD.
Professor, Institute of Biomedical Engineering, College of Medicine and College of
Engineering, National Taiwan University, Adjunct Professor, Department of
Mechanical Engineering, College of Engineering, National Taiwan University
Address: 602 Jan-Shu Hall, 1 Section 4, Roosevelt Road, Taipei 10617, Taiwan, ROC
Phone: 886-2-33665269, Fax: 886-2-23687573, Email: [email protected]
*The first two authors contributed equally to this work.
The Manuscript submitted does not contain information about medical
device(s)/drug(s). National Science Council, Taiwan (NSC
101-2628-B-002-039-MY3) grant funds were received to support this work. No
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relevant financial activities outside the submitted work.
STRUCTURED ABSTRACT (word limit: 300)
Study Design. Biomechanical experiment using an in-situ porcine model.
Objective. To find the effect of traction treatment on anulus microstructure, molecular
convection and cell viability of degraded discs.
Summary of Background Data. Spinal traction is a conservative treatment for disc
disorders. The recognized biomechanical benefits include disc height recovery,
foramen enlargement, and intradiscal pressure reduction. However, the influence of
traction treatment on anulus microstructure, molecular transportation and cell viability
of degraded discs has not been fully investigated.
Methods.A total of 48 thoracic discs were dissected from 8 porcine spines (140 kg, 6
month old) within 4 hrs after sacrifice and then divided into 3 groups: intact, degraded
without traction, and degraded with traction. Each disc was incubated in a
whole-organ culture system and subjected to diurnal loadings for 7 days. Except for
the intact group, discs were degraded with 0.5 ml trypsin on Day 1 and a 5 hr fatigue
loadings on Day 2. From Day 4 to Day 6, half of the degraded discs received a 30 min
traction treatment per day (traction force: 20 kg, loading: unloading = 30 sec: 10 sec).
By the end of the incubation, the discs were inspected for disc height loss, anulus
microstructure, molecular (fluorescein sodium) intensity and cell viability.
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Results.Collagen fibers were crimped and delaminated, while the pores were
occluded in the anulus fibrosus of the degraded discs. Molecular transportation and
cell viability of the discs decreased after matrix degradation. With traction treatment,
straightened collagen fibers increased within the degraded anulus fibrosus, and the
anulus pores were less occluded. Both molecular transportation and cell viability
increased, but not to the intact level.
Conclusion.Traction treatment is effective in enhancing nutrition supply and
promoting disc cell proliferation of the degraded discs.
Key words:intervertebral disc; degeneration; fluid convection; nutrition; traction
therapy; whole disc culture system; trypsin; fluorescence profilometry; cell viability;
scanning electron microcope
Level of Evidence:N/A
MINI ABSTRACT (word limit: 50)
Traction treatment has been found to reduce pore occlusion of anulus fibrosus in
the radial and circumstantial dimension, enhancing molecular transportation through
the anulus fibrosus and cell viability within degraded discs. These findings show the
efficacy of traction treatment in promoting disc healing from matrix damages.
KEY POINTS
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Key point 1: Collagen fibers are crimped and delaminated, while the pores are
occluded in the anulus fibrosus of degraded discs.
Key point 2:Disc height, molecular convection and cell viability decrease in the
degraded disc.
Key point 3: With traction treatment, collagen fibers are straightened, and pores are
less occluded in the anulus fibrosus of the degraded disc. Meanwhile, pores and
cracks within the anulus fibrosus increase without disc height loss.
Key point 4: Molecular convection and cell viability of the degraded disc increase
with traction treatment, but not to the intact level.
INTRODUCTION (2700 words)
Spinal traction is a conservative treatment for pain and discomfort arising from
disc degeneration.1-4The nerve root is usually impinged after disc degeneration due to
spinal alignment changes and disc herniation. Traction treatment decompresses the
nerve root by increasing disc height, enlarging intervertebral foramen, and producing
negative intradiscal pressure that helps to retreat protruded disc materials.4-7However,
it is not known whether traction treatment inhibits the progression of disc
degeneration.
Degenerative discs are characterized by extracellular matrix degradation, anulus
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fibrosus (AF) destruction and cell apoptosis. Disc cells are responsive to anabolic and
catabolic activities within the disc. Extracellular matrix degradation, which results
from digestive enzyme activation or excessive fatigue loadings, accelerates disc cell
apoptosis. The anabolism and catabolism within the disc is unbalanced, progressing to
matrix degradation. Therefore, boosting cell viability to enhance matrix synthesis may
be one of the strategies for degeneration therapies.8
Cell viability is related to nutrition supply. Fluid transportation through AF is one
of the disc nutrient transportation pathways. The pressure difference between the
inside and outside of the disc determines the fluid flow direction. Fluid flows into the
disc with the decrease of loading.9-12The magnetic resonance (MR) techniques reveal
the signals of disc rehydration after bed rest.11-13Resuming disc volume from
compression decreases the inside pressure and draws fluid and nutrient flow. The
structural damages prevent the volume of degenerative discs from well recovering
once unloaded. The pressure gradient that drives the fluid inflow is thus decreased.
Furthermore, the dissolved nutrients must pass through the pores in the AF to reach
disc center.14The deforming and occlusion of anular pores due to fiber destruction
and collagen matrix debris would block the nutrition pathways within AF.
Traction therapy has been postulated to accelerate fluid flow in the disc by an in
vivo MRI observation,
15-17
forcing more nutrients to flow into the disc. The increase
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of nutrient supply helps to promote disc cell growth. The revival of disc cells
enhances extracellular matrix production, thus inhibiting the degeneration progression.
In addition, tensile force is crucial to disc cell biochemical responses. Tensile force
promotes type I collagen synthesis of anulus cells and type II collagen transcription in
nucleus pulposus (NP) cells.18Currently, little evidence supports whether traction
treatment increases the nutrient transportation, and hence activates cell proliferation in
degraded discs. Therefore, the purpose of this study is to find the effect of spinal
traction on the AF microstructure, molecular transportation, and cell viability using a
simulated degenerative disc model.
MATERIALS AND METHODS
Specimen Preparation.Eight spines were obtained from juvenile pigs (weight:
about 140 kg) within 4 hrs after sacrifice. Six lower thoracic discs were dissected
from each spine by cutting through the upper and lower vertebrae at the middle height.
The discs were irrigated with saline solution to remove clotted blood and bone debris
after cleaned off muscle and nerve tissue. The discs were then sterilized in
phosphate-buffered saline (PBS) (Biowest Co., France) supplemented with 0.5%
gentamicin (Biowest), 0.5% Amphotericin B (Gibco Invitrogen Co., Switzerland), and
betadine to prevent bacterial infection during incubation.
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Whole organ culture system. The whole disc culturing system contained a
bioreactor, a pneumatic loading system, and a media circulating system (Figure 1).
The bioreactor is made of a transparent polycarbonate chamber and a Teflon loading
piston with two porous stainless plates. Each disc was mounted to the bioreactor by
inserting 2 screws through the porous plates to the vertebrae center. The culture media
consisted of DMEM (4.5 g/L glucose, 110 mg/L sodium pyruvate, and L-glutamin),
supplemented with 3.7 g/L NaHCO3, 10% FBS, 0.02 M HEPES buffer, 1%
penicillin/streptomycin, 3 mL/L gentamicin, and 0.5% amphotericin B (Gibco
Invitrogen Co, Basel, Switzerland). The culture media was continuously circulated at
a rate of 200 l/min by a peristaltic pump through the silicone tubing, and exchanged
every 2 to 3 days. The pneumatic system gave daily diurnal loadings and cyclic
traction. A diurnal loading was given every day, including a 16 hrs dynamic loading
(0.2-0.8 MPa, 0.2 Hz) followed by an 8 hrs static rest (0.2 MPa).19The pressure acting
on the discs was calculated by dividing the axial force with individual discs
cross-sectional area predicted according to the ellipse area function (ab) which
required the measure of discs transverse width (2a) and anteroposterior length (2b) by
a caliper before the disc was amounted to the bioreactor. Once the disc cross-sectional
area is determined, the axial output force of the pneumatic loading system was
adjusted in order to achieve the pre-designated and desired pressure.
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Experimental protocols.The discs were randomly divided into 3 groups: intact,
degraded without traction and degraded with traction. Each disc, including the intact
and degraded discs was incubated in the whole-organ culture system, with sustained
diurnal loading and 5% CO2circulation at 37C for the duration of the experimental
period. A 7-day experimental protocol containing 2 phases was designed. The 1st
phase (Day1 to Day 3) aimed to create Grade II degenerative disc; the 2nd phase (Day
4 to Day 6) simulated 3 times of traction treatments within a week in the clinical
setting. Day 7 was seen as a rest day. On Day 1, all discs other than the ones in the
intact group were injected with a 0.5 ml trypsin solution (0.25%), which digested
proteins within the extracellular matrix. On Day 2, the trypsin-degraded discs were
subjected to 5 hrs of fatigue loading (peak to peak: 190 N to 590 N), which produced
micro injuries to the AF. The traction force was 20 kg. The duration of loading and
relaxation was 30 sec and 10 sec, respectively. Each traction treatment lasted for 30
min.
Noticeably, the diurnal loading was daily applied to all specimens during the
7-day incubation in the whole organ culture system for real-life situations, where
people either being healthy or sick are likely needing to carry on with their routine
daily activates. The duration of dynamic part of this diurnal loading was decreased to
accommodate the unique loading conduction for certain purpose. For example, on
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Day 3, the duration of dynamic loading was decreased from 16 hrs to 11 hrs for the
discs in degradation group to accommodate 5 hrs intense fatigue loading aiming to
create anulus fibrosus microfracture; on Day 4~6, the discs allocated to traction
intervention group were given 15.5 hrs dynamic loading to accommodate the 0.5 hr of
traction treatment.
By the end of Day 7, the discs from each group were retrieved out of the whole
organ culture system to inspect disc height (n=8), AF microstructure (n=4),
fluorescence profilometry (n=8) and cell viability (n=8) (Figure 2).
Disc height loss.The disc height was measured with a caliper. The initial disc
height was measured before culturing. The disc height loss was the difference of disc
height between Day 1 and Day 7. The x-ray image analysis is another popular disc
height measuring techniques and maybe regarded by some as more appropriate for
measuring disc heights.20-24For this reason, prior to the start of this experiment, a pilot
study was conducted to assess the reliability of caliper measurement against those
measured with x-ray image analysis technique. The Pearsons correlation coefficient
between the caliper measures and x-ray image measures was 0.911 (p
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image analysis techniques.
AF microstructure. The disc was first dissected into 2 halves along the sagittal
line. Then the middle portion of the anterior or posterior part of AF tissue was
dissected and trimmed into a cubic sized as 2x4x2 mm. The AF cuboids were fixed in
gluteraldehyde solution (2.5%) for 2 hrs and then frozen at -20C. The frozen AF
cuboids were sliced into 300 m thick samples. After immersion in osmium tetroxide
solution (OsO4) at 4C for 12 hrs, the samples were dehydrated through sequential
immersion in 30, 50, 70, 85, 90, 95, 100% ethanol solution and propanone solution.
Lastly, the samples were dried in a critical point dryer (Hitachi HCP-2) and
sputter-coated with gold. The samples were photographed by a scanning electron
microscope (SEM, FEI Inspect S) from the axial and radial view.
Fluorescence profilometry.The molecular transportation capacity of disc was
represented by fluorescence profilometry. The specimen was removed the upper/lower
vertebral bodies and then returned to the bioreactor. A 50 ml of fluorescein sodium
(FS) solution (100 M) was added in the medium collecting bottle and circulated in
the bioreactor for extra 1 hr. Thereafter, the specimen was taken out, casted and frozen
at -20C. The frozen specimen was cut along the sagittal plane and then exposed to
blue light (length: 490 nm). The green light (length: 514 nm) emitted by FS after
excitation was photographed. The disc area of the fluorescence image was extracted
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with an image process software (Figure 3A). The mid anterior-posterior length of disc
was normalized with the anterior end as 0 and the posterior end as 1 (Figure 3B). The
fluorescence intensity of each pixel with respect to their position was measured. The
outer anterior AF (AOAF), inner anterior AF (AIAF), NP, inner posterior AF (PIAF),
and outer posterior AF (POAF) were located at 0~0.2, 0.2~0.4, 0.4~0.8, 0.8~0.9, and
0.9~1 along the disc profile, respectively. The mean fluorescence intensity, i.e., the
image brightness, of these five regions were calculated (Figure 3C). We performed a
linear calibration test between the concentration of FS solution and its fluorescence
intensity.25The result showed that their linear relationship is significant (r2= 0.9645,
p
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Statistics Analysis. Two-way ANOVA was used to evaluate the effect of the
treatment (3 levels: intact, degradation without traction, and degradation with traction)
and disc regions (5 levels: AOAF, AIAF, NP, PIAF, and POAF) on fluorescence
intensity of FS. Two-way ANOVA was used to evaluate the effect of the treatment (3
levels: intact, degradation without traction, and degradation with traction) and disc
regions (5 levels: AOAF, AIAF, NP, PIAF, and POAF) on fluorescence intensity of FS.
Treatment and disc regions are both considered as between-subject factors. One-way
ANOVA was performed to test the effect of treatment on disc height loss and cell
viability. The LSD was used for post-hoc analysis for both ANOVA tests. A significant
difference was defined as p
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alternatively aligned, with one containing pores and another consisting of collagen
mass. The pores were clustered in strips of bands. The pores were oval in shape and
had a smooth contour (Figure 4A). In the radial section, longitudinal collagen fibers
were aligned. Only a few narrow fissures were observed (Figure 4D). For the
degraded disc without traction, delamination was observed in the axial section. The
pores were massively occluded by the collapse of surrounding collagen mass (Figure
4B). In the radial section, collagen fibril bundles were crimped and torn apart. Micro
cracks were seen between fibers (Figure 4E). For the degraded disc with traction, the
distinct lamination still existed. The pores were not occluded and the number of pores
increased. The pore shape was irregular and the contour was coarse (Figure 4C). In
the radial section, the crimped collagen fibers were straightened. Many irregular
cracks appeared among parallel collagen fibril bundles (Figure 4F).
Fluorescence profilometry. The fluorescence intensity was highest in the intact
disc, dramatically decreased in the degraded disc without traction, and partially
recovered in the degraded disc with traction. The result of 2-way ANOVA showed that
both of disc treatment (p
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was significantly different from one another (all p0.05), but significantly lower than that of POAF (all p
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microstructure and causes fluid leakage. The maximal force of fatigue loading (590 N)
resulted in a pressure of 1.1 MPa on the porcine discs with a cross-sectional area of
550(30) mm2in this study. This pressure is less than the reported ultimate
compressive loading of 6-month old porcine disc (17.5 MPa).35A 2 hrs fatigue
loading (peak-to-peak 190 N-to-590 N, 5Hz) squeezes the disc fluid and injures disc
integrity.36With the same loading magnitude and loading cycles, the fatigue loading
of this study would create unrecoverable injuries in the discs, especially in the AF
region. In clinical settings, traction treatment is often prescribed for conditions
associated with degenerated disc. One published study37provided a disc degradation
protocol which was able to simulate natural Grade II disc degeneration with evidence
of comparable rheological alterations, histologic damages and biochemical
compositions reduction. By following the same protocol, the in-vitro disc
degeneration model of the current study is conceivable for evaluating traction
treatment efficiency.
The fluorescence profilometry across the disc was used to manifest the molecular
transportation within the AF. The decrease of molecular transportation in the degraded
disc without traction resulted from the AF destruction and pore occlusion. The AF
destruction reduced disc height and prevented the resuming of disc volume during the
unloading period of diurnal loading. This decreased the reduction level of intradiscal
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pressure. The occlusion of pores resulted from the cleaved collagen and extracellular
matrix substance due to enzyme and fatigue loading. The transportation of FS
decreased with the increasing distance from the AF. However, in the intact disc, FS
accumulated in the NP due to the abundant undigested proteoglycan. Proteoglycan
holds water with the negative charges, so the dissolved FS was retained
simultaneously. Lower molecular transportation implies the reduction of nutrient
supply thus accelerating the apoptosis of disc cells.
The correlation between the external traction force and internal intradiscal
pressure in an in vivo clinical setting has less been validated. In this study, we
designed the traction force by the following rational. Wilke et al.38reported that the
intradiscal pressure is 0.1 MPa for a 70 kg person in prone position. Assuming the
disc area to be 1000 mm2,39the internal force due to soft tissue could be estimated at
100 N (i.e. 10 kg) in compression. The clinical setting of traction force for the lumbar
is 25% to 50% of body weight.40The external traction force for an 80 kg adult39
would be ranged from 20 kg to 40 kg in tension, hence the correspondent in vivo
internal traction force at disc may range from 10 kg to 30 kg. Therefore, we use 20 kg
as the traction force for the disc. However, given the smaller cross-section area of
tested specimens (area=550 mm2), the traction stress of this study may still be located
at the upper level of clinical setting.
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Few limitations about this study should be addressed. Clinically, traction
treatment is practiced 2-3 times per week and 4-6 weeks in a row. We only provided a
week of traction protocol. Though the results seem promising, the long term outcome
remains unknown. Secondly, the effect of traction treatment on disc cell through
increasing nutrient convection is revealed by higher cell viability compared to the
degraded disc without traction treatment. However, this finding could not tell whether
the living cells function to increase cell proliferation, regenerate disc matrix and
suppress abnormal matrix degradation, but only imply the reduction of cell apoptosis
or increase of cell growth after the first 3 sessions of traction treatment. Thirdly, part
of nutrient transportation within the disc is through endplate. In this study, we did not
examine the microstructure, e.g., the deformation or occlusion, of endplate due to
degradation or traction. Since the circulation of fluorescence was not limited to the
axial or radial direction of the disc, the outcomes of FS intensity and cell viability
should reflect the capability of nutrient transportation of endplate and AF in total. The
mechanical response of endplate due to the degradation and traction should be similar
to that of AF. Lastly, the porcine disc is a commonly used model for the human spine
biomechanics due to the similarities in AF structure, cell number and biochemical
component.41The porcine discs used here were removed of the posterior element and
soft tissue, which reduces their differences from the human discs in musculature,
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loading direction and horizontal positioning. However, considering the subtle
differences in notochordal cells,42,43caution should be taken when applying the results
of this study to human disc.
In conclusion, the matrix degradation leads to disc destruction and obstructs the
pores in the AF, which in turn interferes with nutrient transportation thus decreases
cell viability. With spinal traction, disc height is maintained, and the debris in the
pores of the AF are expelled. The nutrient transportation and cell viability are thus
enhanced, hence relieving the degeneration process.
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CAPTIONS OF FIGURES
Figure 1. A schematic diagram of a whole-organ culture system with culture medium
circulated within. The arrows indicated the direction of culture medium circulation.
The pneumatic cylinder provides external loadings to specimens.
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Figure 2.Experimental protocols.
Figure 3. The process of measuring disc fluorescence profilometry. (A) The disc
contour is segmented from the fluorescence image based on the regular image. (B)
The disc profile was normalized as anterior end to be 0 and posterior end to be 1. (C)
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A fluorescence profilometry was categorized into five regions, i.e., the anterior outer
AF (AOAF), anterior inner AF (AIAF), NP, posterior inner AF (PIAF), and posterior
outer AF (POAF) along the 0~0.2, 0.2~0.4, 0.4~0.8, 0.8~0.9, and 0.9~1 disc profile,
respectively.
Figure 4. SEM images of AF in axial section (upper row) and radial section (lower
row). The column displays the AF of intact disc (A, D), the degraded discs without
traction (B, E), and the degraded disc with traction (C, F). The arrows indicate the
pores occluded by collagen mass. The hollow ovals show the increased number of
pores/cracks in AF.
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Figure 5.The fluorescence intensity (brightness) among the five disc regions (AOAF,
AIAF, NP, PIAF, POAF) of intact discs, degraded discs without traction, and degraded
disc with traction.
Figure 6.Cell viability of NP and AF of the intact disc, degraded disc without traction,
and degraded disc with traction.