efectos moleculares de la traccion en el disco

8/12/2019 Efectos Moleculares de La Traccion en El Disco

1/28

AC

CEPTE

D

Copyright Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.

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


2/28

AC

CEPTE

D


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.


3/28

AC

CEPTE

D


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


4/28

AC

CEPTE

D


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


5/28

AC

CEPTE

D


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


6/28

AC

CEPTE

D


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.


7/28

AC

CEPTE

D


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.


8/28

AC

CEPTE

D


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


9/28

AC

CEPTE

D


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


10/28

AC

CEPTE

D


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


11/28

AC

CEPTE

D


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


12/28

AC

CEPTE

D


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


13/28

AC

CEPTE

D


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


14/28

AC

CEPTE

D


was significantly different from one another (all p0.05), but significantly lower than that of POAF (all p


15/28

AC

CEPTE

D


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


16/28

AC

CEPTE

D


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.


17/28

AC

CEPTE

D


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,


18/28

AC

CEPTE

D


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.

Reference

1. Moustafa IM, Diab AA. Extension traction treatment for patients with discogenic

lumbosacral radiculopathy: a randomized controlled trial. Clinical rehabilitation

2013;27:51-62.

2. Beurskens AJ, de Vet HC, Koke AJ, et al. Efficacy of traction for non-specific

low back pain: a randomised clinical trial.Lancet1995;346:1596-600.

3. Diab AA, Moustafa IM. The efficacy of lumbar extension traction for sagittal

alignment in mechanical low back pain: A randomized trial.Journal of back and

musculoskeletal rehabilitation2013;26:213-20.

4. Chung CT, Tsai SW, Chen CJ, et al. Comparison of the intervertebral disc spaces


19/28

AC

CEPTE

D


between axial and anterior lean cervical traction.European spine journal : official

publication of the European Spine Society, the European Spinal Deformity Society,

and the European Section of the Cervical Spine Research Society2009;18:1669-76.

5. Apfel CC, Cakmakkaya OS, Martin W, et al. Restoration of disk height through

non-surgical spinal decompression is associated with decreased discogenic low back

pain: a retrospective cohort study.BMC Musculoskelet Disord2010;11:155.

6. Gose EE, Naguszewski WK, Naguszewski RK. Vertebral axial decompression

therapy for pain associated with herniated or degenerated discs or facet syndrome: an

outcome study.Neurol Res1998;20:186-90.

7. Ramos G, Martin W. Effects of vertebral axial decompression on intradiscal

pressure.J Neurosurg1994;81:350-3.

8. Bae WC, Masuda K. Emerging technologies for molecular therapy for

intervertebral disk degeneration. The Orthopedic clinics of North America

2011;42:585-601, ix.

9. Kuo YW, Wang JL. Rheology of intervertebral disc: an ex vivo study on the

effect of loading history, loading magnitude, fatigue loading, and disc degeneration.

Spine (Phila Pa 1976)2010;35:E743-52.

10. Lin LC, Hedman TP, Wang SJ, et al. The analysis of axisymmetric

viscoelasticity, time-dependent recovery, and hydration in rat tail intervertebral discs


20/28

AC

CEPTE

D


by radial compression test.Journal of applied biomechanics2009;25:133-9.

11. Matsumura Y, Kasai Y, Obata H, et al. Changes in water content of intervertebral

discs and paravertebral muscles before and after bed rest.Journal of orthopaedic

science : official journal of the Japanese Orthopaedic Association2009;14:45-50.

12. Malko JA, Hutton WC, Fajman WA. An in vivo magnetic resonance imaging

study of changes in the volume (and fluid content) of the lumbar intervertebral discs

during a simulated diurnal load cycle. Spine (Phila Pa 1976)1999;24:1015-22.

13. Malko JA, Hutton WC, Fajman WA. An in vivo MRI study of the changes in

volume (and fluid content) of the lumbar intervertebral disc after overnight bed rest

and during an 8-hour walking protocol.J Spinal Disord Tech2002;15:157-63.

14. Travascio F, Jackson AR, Brown MD, et al. Relationship between solute

transport properties and tissue morphology in human annulus fibrosus.Journal of

orthopaedic research : official publication of the Orthopaedic Research Society

2009;27:1625-30.

15. Guehring T, Unglaub F, Lorenz H, et al. Intradiscal pressure measurements in

normal discs, compressed discs and compressed discs treated with axial posterior disc

distraction: an experimental study on the rabbit lumbar spine model.Eur Spine J

2006;15:597-604.

16. Guehring T, Omlor GW, Lorenz H, et al. Disc distraction shows evidence of


21/28

AC

CEPTE

D


regenerative potential in degenerated intervertebral discs as evaluated by protein

expression, magnetic resonance imaging, and messenger ribonucleic acid expression

analysis. Spine (Phila Pa 1976)2006;31:1658-65.

17. Kramer J. Pressure dependent fluid shifts in the intervertebral disc. The

Orthopedic clinics of North America1977;8:211-6.

18. Li S, Jia X, Duance VC, et al. The effects of cyclic tensile strain on the

organisation and expression of cytoskeletal elements in bovine intervertebral disc

cells: an in vitro study.European cells & materials2011;21:508-22.

19. Gantenbein B, Grunhagen T, Lee CR, et al. An in vitro organ culturing system

for intervertebral disc explants with vertebral endplates: a feasibility study with ovine

caudal discs. Spine (Phila Pa 1976)2006;31:2665-73.

20. Chen WH, Liu HY, Lo WC, et al. Intervertebral disc regeneration in an ex vivo

culture system using mesenchymal stem cells and platelet-rich plasma.Biomaterials

2009;30:5523-33.

21. Kroeber MW, Unglaub F, Wang H, et al. New in vivo animal model to create

intervertebral disc degeneration and to investigate the effects of therapeutic strategies

to stimulate disc regeneration. Spine (Phila Pa 1976)2002;27:2684-90.

22. Frobin W, Brinckmann P, Biggemann M, et al. Precision measurement of disc

height, vertebral height and sagittal plane displacement from lateral radiographic


22/28

AC

CEPTE

D


views of the lumbar spine. Clin Biomech (Bristol, Avon)1997;12 Suppl 1:S1-S63.

23. Roberts S, Menage J, Sivan S, et al. Bovine explant model of degeneration of the

intervertebral disc.BMC Musculoskelet Disord2008;9:24.

24. Kunkel ME, Herkommer A, Reinehr M, et al. Morphometric analysis of the

relationships between intervertebral disc and vertebral body heights: an anatomical

and radiographic study of the human thoracic spine.Journal of anatomy

2011;219:375-87.

25. Liu T, Wang R, Hsiao J, et al. Development of a cost effective fluorescent

photographic system for small animal and disc study. Orthopaedic Research Society

(ORS). Long Beach: Orthopaedic Research Society (ORS), 2011:689.

26. DiFabio JL, Pearce RH, Caterson B, et al. The heterogeneity of the

non-aggregating proteoglycans of the human intervertebral disc. The Biochemical

journal1987;244:27-33.

27. Jahnke MR, McDevitt CA. Proteoglycans of the human intervertebral disc.

Electrophoretic heterogeneity of the aggregating proteoglycans of the nucleus

pulposus. The Biochemical journal1988;251:347-56.

28. Lipson SJ, Muir H. 1980 Volvo award in basic science. Proteoglycans in

experimental intervertebral disc degeneration. Spine (Phila Pa 1976)1981;6:194-210.

29. Berg A, Singer T, Moser E. High-resolution diffusivity imaging at 3.0 T for the


23/28

AC

CEPTE

D


detection of degenerative changes: a trypsin-based arthritis model. Investigative

radiology2003;38:460-6.

30. Urban JP, Roberts S. Degeneration of the intervertebral disc.Arthritis research &

therapy2003;5:120-30.

31. Lyons G, Eisenstein SM, Sweet MB. Biochemical changes in intervertebral disc

degeneration.Biochim Biophys Acta1981;673:443-53.

32. Antoniou J, Mwale F, Demers CN, et al. Quantitative magnetic resonance

imaging of enzymatically induced degradation of the nucleus pulposus of

intervertebral discs. Spine (Phila Pa 1976)2006;31:1547-54.

33. Mwale F, Demers CN, Michalek AJ, et al. Evaluation of quantitative magnetic

resonance imaging, biochemical and mechanical properties of trypsin-treated

intervertebral discs under physiological compression loading.Journal of magnetic

resonance imaging : JMRI2008;27:563-73.

34. Jim B, Steffen T, Moir J, et al. Development of an intact intervertebral disc organ

culture system in which degeneration can be induced as a prelude to studying repair

potential.European Spine Journal2011:1-11.

35. Lundin O, Ekstrom L, Hellstrom M, et al. Exposure of the porcine spine to

mechanical compression: differences in injury pattern between adolescents and adults.

European spine journal : official publication of the European Spine Society, the


24/28

AC

CEPTE

D


European Spinal Deformity Society, and the European Section of the Cervical Spine

Research Society2000;9:466-71.

36. Wang JL, Wu TK, Lin TC, et al. Rest cannot always recover the dynamic

properties of fatigue-loaded intervertebral disc. Spine (Phila Pa 1976)

2008;33:1863-9.

37. Hsu YC, Kuo YW, Chang YC, et al. Rheological and Dynamic Integrity of

Simulated Degenerated Disc and Consequences After Cross-linker Augmentation.

Spine (Phila Pa 1976)2013;38:E1446-53.

38. Wilke HJ, Neef P, Caimi M, et al. New in vivo measurements of pressures in the

intervertebral disc in daily life. Spine (Phila Pa 1976)1999;24:755-62.

39. Gilsanz V, Boechat MI, Gilsanz R, et al. Gender differences in vertebral sizes in

adults: biomechanical implications.Radiology1994;190:678-82.

40. Borman P, Keskin D, Bodur H. The efficacy of lumbar traction in the

management of patients with low back pain.Rheumatology international

2003;23:82-6.

41. Cho H, Park SH, Lee S, et al. Snapshot of degenerative aging of porcine

intervertebral disc: a model to unravel the molecular mechanisms. Experimental &

molecular medicine2011;43:334-40.

42. Alini M, Eisenstein SM, Ito K, et al. Are animal models useful for studying


25/28

AC

CEPTE

D


human disc disorders/degeneration?Eur Spine J2008;17:2-19.

43. Hunter CJ, Matyas JR, Duncan NA. The notochordal cell in the nucleus

pulposus: a review in the context of tissue engineering. Tissue Eng2003;9:667-77.

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.


26/28

AC

CEPTE

D


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)


27/28

AC

CEPTE

D


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.


28/28

AC

CEPTE

D

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.

efectos moleculares de la traccion en el disco

Documents