RAT SPINAL MOTION SEGMENT IN ORGAN CULTURE: A CELL VIABILITY STUDY

+*Lim, T H; *Ramakrishnan, P S; *Kurriger G.; *Martin, J A; *Stevens, J W; *Mendoza, S A

*University of Iowa, Iowa City, Iowa
talim@engineering.uiowa.edu
INTRODUCTION:
The intervertebral disc (IVD) is a cartilaginous tissue with a fluidic 50 m
nucleus. This tissue provides the link between adjacent vertebral bodies
for articulation and also acts as a load bearing structure. IVD
degeneration is considered to be the major factor for low back pain.
Although the degenerative mechanism is poorly understood, mechanical
stress is thought to play a major role.
In vivo studies of mechanical stress effects on IVD have been a. b.
informative but there is a need to develop in vitro models to better
understand the effects of mechanical stress at the cellular level.
Maintaining the intact IVD within a motion segment (vertebral body-
IVD-vertebral body) offers the best chance to achieve near physiologic
conditions in vitro. However, a complete evaluation of this whole organ
culture system will be imperative.
Although recent studies have demonstrated the feasibility of keeping
cells viable in organ culture on rat and rabbit disc tissues for a period of c. d.
21 days[1-3], to our knowledge, there have been no similar studies on
cell viability in cultured motion segments. Therefore, the objective of
the current study was to measure the effects of time in culture on the
viability of IVD cells and endplate chondrocytes.
MATERIALS AND METHODS:
Tissue Culture Method: Lumbar spines were harvested from Sprague
Dawley (SD) rats (~2.5 months old) under sterile conditions
immediately after sacrifice with CO2 asphyxiation. IVD motion
segments were dissected from consecutive levels (L1-L6). Soft tissues e. f.
and posterior elements were removed and discs were rinsed in saline
solution before being placed in 6 well culture plates. The discs were Figure1. NP regions of the IVD showing NBT (left panels) and DAPI
assigned to one of four groups- 0 day (control), 0 day (negative control), (right panels) signals in 0 day control (a, b), 14 Day (c, d) and negative
7 days, and 14 days. Each of the groups was comprised of 3 discs. All control (e, f). Arrows indicate corresponding cell locations with DAPI
specimens were cultured in complete medium (DMEM medium and NBT stains
containing 12 % FBS, 25 mM HEPES, 50 g/ml L-ascorbate, 50 g/ml NS NS NS
gentamicin and 2.5 g/ml fungizone). Discs of the 0 day negative 100

control group were frozen and thawed in liquid N2 thrice before placing
in complete medium. The complete medium was replaced daily for 80
groups of 7 day and 14 day cultures. At the end of the culture period,
Percentage Viability (%)

adjacent vertebral bodies of IVDs were grossly dissected close to the

endplates and the discs were treated with 0.75mg/ml of Nitroblue
Tetrazolium (NBT) in a fresh complete medium and incubated for 18
hours at 37 C before histological processing.
Histological Analyses: Discs were removed after 0, 7 and 14 days and
fixed in 10% neutral buffered formalin. After fixation, discs were
processed and embedded in paraffin for sectioning. Mid-sagittal sections
(5 m thick) from each paraffin embedded disc were mounted with
nuclear material labeling DAPI (4, 6- Diamidino-2-phenylindole)-
containing mount. The sections were imaged at visible wavelength for
NBT stains and at near UV range (~359 nm) for DAPI fluorescence.
Cells possessing both DAPI and NBT stains were determined viable and
IVD Regions
cells with DAPI signals alone were registered dead. Areas with neither
NBT nor DAPI stains were registered as empty lacunae. Sections were
analyzed qualitatively and also through cell counts for LIVE/DEAD
cells ratio at distinct regions of the IVD [(Nucleus Pulposus (NP),
Endplates (EP), Annulus Fibrosus (AF)] at 20X magnification. The
Viable cell ratio was determined by the ratio of cells with NBT stains
and cells with DAPI signals.
Statistical Analyses: Cell counts and percentage viability of cells at
various regions [(Endplate (EP), Nucleus Pulposus (NP) and Annulus
Fibrosus (AF)] at each time point were determined at 3 sections from
each specimen of each group (n=3/group). Statistical analysis was
performed using Kruskal-Wallis One-Way ANOVA on ranks with
Dunns multiple comparisons.
RESULTS:
Histological analyses showed that staining of discs of the 14 day group
(Figure1 c, d) for viable cells (NBT and DAPI) were comparable to
controls (day 0) (Figure 1 a, b). The negative controls showed no NBT
staining, confirming the reliability of the viability stain (Figure 1 e). Cell
density of 14 day discs was comparable to that of the control discs.
Statistical analyses of cell viability percentages showed no significant
difference among the control, 7 day and 14 day discs in the endplate,
nucleus and annulus regions (Figure 2).
60
40
20
0
Conditions + - 7 14 + - 7 14 + - 7 14
EP NP AF
Figure 2.Percentage viability of cells at three distinct regions of the IVD
[+: Day 0 (control), -: Day 0 (Negative control), 7: 7 Day, 14: 14 Day]
[EP: Endplate region; NP: Nucleus Pulposus: AF: Annulus Fibrosus].
NS: No significant difference compared to control (p>0.05)
DISCUSSION:
Current opinion is that whole motion segment culture is not feasible
because IVD cells would not survive due to lack of perfusion and
nutrient supply. However, this study showed a high degree of cell
viability in motion segments cultured for up to 14 days. Even NP cells
and EP cells deep in the tissue were > 90% viable after 14 days.
Although the metabolic function of these cells has yet to be determined,
our initial results suggest that long-term motion segment culture is
practical. The inclusion of vertebral bodies will facilitate anchoring
during biomechanical stimulation. Thus we expect the culture system to
provide us with an excellent model for studying the pathomechanics of
IVD degeneration and the effects of mechanical stimulation on the
biology of IVD cells.
REFERENCES:
1. Takegami K, M.K., An H, et.al, Orthopedic Research society, 2002.
2. Risbud, M.V., et al., Spine, 2003. 28(24): p. 2652-8;
3. Chiba, K., et al., Spine, 1998. 23(17): p. 1821-7.

51st Annual Meeting of the Orthopaedic Research Society

Paper No: 0110