Download effect of static compression on atp production of intervertebral disc

Effect of Static Compression on ATP Production of Intervertebral Disc
1
Wang, C.; 1,2 Yuan, T-Y, 2 Travascio, F.; 1Helbig, M. W.; 1Khoury, K.; 2 Gu, WY; 1 Huang, C.-Y.C.
1
Stem Cell and Mechanobiology Lab, 2Tissue Biomechanics Lab, Dept of Biomedical Engineering, University of Miami, Coral Gables, FL
[email protected]
MATERIALS AND METHODS
Lumbar spines of 4–6 month-old pigs were obtained within 2 - 8 h of
sacrifice. Functional spinal units (FSUs) were isolated from L1-L5
levels of the spine by making parallel transverse cuts through the
vertebrae at 0.5mm above and below the disc. The FSUs were placed in
custom made compression chambers and cultured in high glucose
Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen Corp.,
Carlsbad, CA) containing 10% fetal bovine serum (FBS, Invitrogen
Corp) and 1% antibiotic-antimycotic (Invitrogen Corp) in an incubator at
37°C overnight. The medium was continuously circulated at
0.37ml/min, which is about twice the rate of blood flow to the disc in
vivo. For the whole disc compression test, the disc height was measured
at 5 locations on the disc surface. For the compression group, the discs
were subjected to 15% compressive strain for 2 hours. The control group
was left undisturbed in the incubator for the duration of the experiment.
After the experiment, a transverse cut was made on each unit to expose
the annulus fibrosus (AF) and nucleus pulposus (NP). The tissue
samples (~10 mm3) were harvested from the locations shown in Figure
1. The samples obtained from AF and NP regions were used to
determine the lactate, ATP, and DNA contents. ATP and lactate were
released from tissue samples using perchloric acid treatment [2] and then
determined using the Luciferin-luciferase method (Sigma, St. Louis,
MO) and an enzymatic assay (Sigma), respectively. The DNA content
was measured based on the protocol described in a previous study [6].
The lactate and ATP contents were normalized by the DNA content. A
student t-test was performed to examine differences in ATP and lactate
contents between AF and NP regions and two experimental groups of
each region using Excel (Microsoft Inc, Redmond, WA).
AF
ATP content (nmol/ mg DNA)
10
*
(a)
Loading
Control
8
6
4
2
0
AF
NP
2
Lactacte content ( mmol/mg DNA)
INTRODUCTION
Degenerative disc disease is considered to be one of the most common
causes of low back pain. This poses a major socio-economic problem in
the US. Since the intervertebral disc (IVD) is the largest avascular tissue
in the human body, steep gradients in the concentration of nutrients (e.g.,
glucose and oxygen) may strongly influence the production of adenosine
triphosphate (ATP) [1]. Intracellular ATP serves as essential cellular
energy for the biosynthesis of extracellular matrix [2], which is
important for maintaining the integrity of tissue structure of IVD. A
previous theoretical study demonstrated that mechanical loading may
mediate cellular glucose consumption within IVD by altering the
transport of oxygen and lactate [3]. In addition, extracellular ATP
released from cells has been shown to mediate a wide variety of
biological responses by binding purinergic receptors [4]. Therefore, the
regulation of ATP production may play an important role in maintaining
normal disc function. The objective of this study was to investigate the
effect of static compression on ATP production in the IVD.
(b)
Loading
**
Control
1.5
1
0.5
0
AF
NP
Figure 2 Comparison of a) ATP and b) lactate contents between the
loading and control groups of AF and NP regions (n=9). *: p=0.043. **:
p=0.037.
DISCUSSION
To our knowledge, this is the first study to investigate the effect of static
compression on the ATP content in the IVD. This study demonstrated
that static compression affects cellular ATP metabolism in the IVD.
Lower ATP content and higher lactate content was found in the loading
AF group. A decrease in solute diffusivity (or tissue permeability)
caused by static compression [3] may increase extracellular lactate
accumulation and reduce local concentrations of oxygen and glucose.
This may result in a decrease in ATP production. The increase in lactate
accumulation in the AF region may decrease pH, creating a hazardous
condition for cells [1]. A significant increase in ATP content of the NP
loading group suggests that static compression may intrisically promote
ATP production in NP cells (Figure 2a). In concurrence with our
previous study [6], a significant difference was found in ATP content
between two anatomical regions, indicating that NP and AF cells may
produce ATP via different major pathways. Furthermore, extracellular
ATP can cause tissue calcification [7]. Significantly high ATP content in
the NP region may be a potential factor causing endplate calcification
and facilitating disc degeneration .
ACKNOWLEDGEMENTS
The authors would like to thank Mr. Andre Castillo for his help with
making the apparatus. This study was supported by the grants from NIH
NIAMS (AR056101 and AR050609).
NP
Figure 1 Harvest sites of AF and NP
RESULTS
Static compression significantly increased the ATP content of NP tissues
(p=0.043), but the ATP content of AF tissues tended to be lower under
static compression (Figure 2a). The lactate content of AF tissue was
significantly increased by static compression (p=0.037) (Figure 2b).
However, no significant difference was found in the lactate content of
NP tissues between the loading and control groups. Without static
loading, the lactate content of AF tissue was not significantly different
from that of NP tissue. With static compression, the lactate content of
AF tissue tended to be higher than that of NP tissue (p=0.09). The ATP
content of NP tissue was significantly higher than that of AF tissue for
both loading conditions (p<0.001).
REFERENCES
1. Bibby SR, Jones DA, Ripley RM, Urban JP, Spine 30:487-96, 2005.
2. Lee RB, Urban JP, Biochem J, 321: 95-102, 1997.
3. Huang CY, Gu WY, J Biomechanics, 41, 1184-96, 2008.
4. Burnstock G. Pharmacol Rev 58: 58-86, 2006.
5. Downs TR, Wilfinger WW. Anal Biochem. 131,538-547, 1983.
6. Czamanski J, Yuan T-Y, Gu WY, Huang C-YC. The 56th Annual
ORS Meeting, 2010.
7. Ryan LM, Kurup IV, Derfus BA, Kushnaryov VM. Arthritis Rheum
35: 1520-5, 1992.
Poster No. 723 • ORS 2011 Annual Meeting