Long-term culture of bovine nucleus pulposus explants
in a native environment
Bart G.M. van Dijk, MSc, Esther Potier, PhD, Keita Ito, MD, ScD*
Orthopaedic Biomechanics, Department of Biomedical Engineering, Eindhoven University of Technology, PO. Box 513,
GEM-Z 4.115, 5600 MB, Eindhoven, The Netherlands
Received 8 February 2012; revised 7 September 2012; accepted 9 December 2012
AbstractBACKGROUND CONTEXT: Chronic low back pain is a disease with tremendous financial
and social implications, and it is often caused by intervertebral disc degeneration. Regenerative
therapies for disc repair are promising treatments, but they need to be tested in physiological
PURPOSE: To develop a physiological in vitro explant model that incorporates the native environ-
ment of the intervertebral disc, for example, hypoxia, low glucose, and high tissue osmolarity.
STUDY DESIGN: Bovine nucleus pulposus (NP) explants were cultured for 42 days in conditions
mimicking the native physiological environment. Two different approaches were used to balance
the swelling pressure of the NP: raised medium osmolarity or an artificial annulus.
METHODS: Bovine NP explants were either cultured in media with osmolarity balanced at iso-
tonic and hypertonic levels compared with the native tissue or cultured inside a fiber jacket used as
an artificial annulus. Oxygen and glucose levels were set at either standard (21% O2and 4.5 g/L
glucose) or physiological (5% O2and 1 g/L glucose) levels. Samples were analyzed at Day 0, 3,
and 42 for tissue composition (water, sulfated glycosaminoglycans, DNA, and hydroxyproline con-
tents and fixed charge density), tissue histology, cell viability, and cellular behavior with messenger
RNA (mRNA) expression.
RESULTS: Both the hypertonic culture and the artificial annulus approach maintained the tissue
matrix composition for 42 days. At Day 3, mRNA expressions of aggrecan, collagen Type I, and
collagen Type II in both hypertonic and artificial annulus cultures were not different from Day
0; however, at Day 42, the artificial annulus preserved the mRNA expression closer to Day 0. Gene
expressions of matrix metalloprotease 13, tissue inhibitor of matrix metalloprotease 1, and tissue
inhibitor of matrix metalloprotease 2 were downregulated under physiological O2and glucose
levels, whereas the other parameters analyzed were not affected.
CONCLUSIONS: Although the hypertonic culture and the artificial annulus approach are both
promising models to test regenerative therapies, the artificial annulus was better able to maintain
a cellular behavior closer to the native tissue in longer term cultures.
? 2013 Elsevier Inc.
Open access under the Elsevier OA license.
Keywords:Regenerative therapy; Disc degeneration; Nucleus pulposus; Explant culture; Osmolarity
Up to 80% of the population will suffer at least once from
low back pain [1,2], and when this disease becomes chronic,
its financial and social implications are tremendous. Low
back pain can be caused by different mechanisms but is also
heavily associated with disc degeneration . During this
process, there is a shift from an anabolic to a catabolic envi-
crease their production of the main matrix proteins  and
increase their production of degrading enzymes [5–7]. As
FDA drug/device status: Not applicable.
Author disclosures: BGMvD: Nothing to disclose. EP: Nothing to dis-
close. KI: Grants: BMM (F); Board of Directors: AOSpine (E); Research
Support (Staff/Materials): DSM (amount unknown, paid directly to
The disclosure key can be found on the Table of Contents and at www.
* Corresponding author. Department of Biomedical Engineering, Eind-
hoven University of Technology, PO Box 513, GEM-Z 4.115, 5600 MB,
Eindhoven, The Netherlands. Tel.: (31) 40-2474350; fax: (31) 40-2473744.
E-mail address: K.Ito@tue.nl (K. Ito)
1529-9430 ? 2013 Elsevier Inc.
The Spine Journal 13 (2013) 454–463
Open access under the Elsevier OA license.
a result, the NP loses proteoglycans (PGs) and water  and
over time changes from a gel-like to a fibrous-like structure
. This leads to decreased disc height and loss of function,
which ultimately can cause pain [2,10].
therapy and spinal fusion, do not treat this degeneration and
have a limited long-term success. Regeneration of the disc is
an alternative long-term approach to treat the disc degenera-
tion at an early stage and prevent low back pain from occur-
ring [11,12]. A number of regenerative therapies have been
shown to be promising in animal models [13–16], as they
could delay the onset of degeneration [15,16] but still need
to be further developed because they could not fully restore
the disc to its original healthy state [15–17].
Before these therapies can be used, they need to be tested
ally involve induced degeneration by needle aspiration 
or enzymatic digestion , which is different from the hu-
put, cost intensive, and have ethical considerations [20,21].
Hence, using invitromodelsisappealing.Cellculture isless
costly and high-volume throughput; however, the native tis-
sueenvironment is lostduringcellisolation, which mightaf-
posus explant culture is another suitable model because the
earliest detectable changes during degeneration occur in
the NP  and the native tissue environment can be main-
tained  in such a model.
Previously we have successfully cultured bovine NP ex-
plants for 21 days . In that study we used polyethylene
glycol (PEG) to raise the medium osmolarity to the native
level and showed that we were able to prevent the swelling
and PG loss. However, the cellular behavior was different
from the fresh tissue at the gene level. We hypothesized that
although balancing the osmolarity is important to prevent
PG loss at the tissue level, other factors in the native envi-
ronment (eg, hypoxia, low glucose levels, and low pH )
might be important for the cellular behavior. Indeed, it has
been shown that glycosaminoglycan (GAG) production is
maximal at 5% O2for NP explants cultured for 24 hours
 and also at a pH of 7.1 when cultured for 4 hours
. Therefore, it appears important to culture NP explants
under physiological O2, glucose, and pH .
A second possible explanation for the difference in the
cellular behavior we previously observed is that a balanced
medium osmolarity is different from the physiological situ-
ation in which the annulus fibrosus contains NP swelling.
Our second hypothesis is that an artificial annulus approach
is more physiological than a balanced medium osmolarity
approach and might benefit the cellular behavior in NP
To see the effects of regenerative therapies on the tissue
level, we need a model that is stable for even longer than 21
days of culture; therefore, the culture duration was ex-
tended to 42 days. Furthermore, we analyzed the explants
at Day 3 to assess if the changes in the cellular behavior
were a direct effect of the harvesting and culture initiation
stress or occurred over time in culture.
The aim of this study was to improve our NP explant
model by incorporating more factors of the native physio-
logical environment and to determine the importance of
the separate factors. Therefore, we cultured NP explants
for 6 weeks under physiological O2and glucose levels in
a swelling balanced environment using either an osmotic
balance or an artificial annulus approach. We analyzed
the tissue composition with biochemical assays and histol-
ogy and the cellular behavior with gene expression.
Materials and methods
NP explants were harvested from fresh caudal discs of
24-month-old cows. These were obtained from the abattoir
according to the local regulations, and a total of 70 discs
(CC2–CC5) were harvested from 18 donors. The different
levels were distributed equally and randomly among the
different conditions. The discs were opened transversally
directly underneath the end plate, and NP explants were
punched out with an 8-mm diameter biopsy punch (Kruuse,
Sherburn, UK) from the center of the NP.
Standard medium was prepared from basic Dulbecco’s
Modified Eagle Medium powder (Gibco; Invitrogen, Carls-
bad, CA, USA) in milli-Q filtered water (8.3 g/L), supple-
mented with 15.9 mg/L phenol red (Sigma, Zwijndrecht,
The Netherlands), 2% L-glutamine (Lonza, Basel, Switzer-
land), 1% pyruvate (Gibco), 1% penicillin/streptomycin
(Lonza), 3.7 g/L sodium bicarbonate (Sigma), 50 mg/L as-
corbic acid (Sigma), and 10% fetal bovine serum (Gibco).
Glucose was added to the media for a final concentration of
ical conditions. All media were filter sterilized and the pH
was adjusted to 7.1, the pH of a healthy human disc .
In PEG culture, the medium osmolarity was adjusted to
two levels based on the previous study 
? Isotonic to native in situ NP (430 mOsm/kg H2O):
standard mediumþ8.2% w/v PEG (20kD, Sigma)
? Hypertonic to native in situ NP (570 mOsm/kg H2O):
standard mediumþ13.3% w/v PEG.
NPexplantswere placed inside the dialysistubing (15 kD
molecular weight cut-off, Spectra-Por, Rancho Dominguez,
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