Download effect of osmolality and oxygen concentration on glycosaminoglycan

EFFECT OF OSMOLALITY AND OXYGEN CONCENTRATION ON GLYCOSAMINOGLYCAN PRODUCTION AND CELL METABOLISM
+ Takeno, K, +Kobayashi, S, +Uchida, K, +Negoro, K, +Baba, H, *Urban JPG
+University of Fukui, Fukui, Japan. *Physiology Laboratory, Oxford University, Oxford, UK
[email protected]
mg/ml) assuming initial rates were maintained and there was no loss of
GAG, were > 1000 days. Such long culture times are consistent with
results seen in articular cartilage tissue engineering
400
µg/ml
300
200
100
0
2 days
270mOsm
6days
370mOsm
470mOsm
570mOsm
Fig 1. Total GAG in beads /ml beads volume under 21% oxygen
400
300
µg/ml
Introduction. Proteoglycan loss is one of the first signs of disc
degeneration. There is increasing interest in developing biological
methods for its replacement both by in vivo repair and through tissue
engineered constructs. In tissue-engineered constructs, cells are
implanted into a matrix and with time accumulate a matrix. The rate of
proteoglycan accumulation is influenced, among other things, by the
number of cells implanted and the rate of proteoglycan synthesis per
cell. Since the constructs are avascular however, pH and oxygen
gradients develop through them as they do through the disc in vivo and
may influence cellular activity and even viability. In this study, we
examine how physiological levels of extracellular osmolality and
oxygen tension influence the rate of proteoglycan accumulation in
nucleus pulposus cells in a three-dimensional culture system.
Methods. Cells were isolated from the nucleus pulposus of 18-24 month
bovine caudal discs. They were cultured for 6 days in alginate beads1) in
DMEM containing 6% FBS under 0% or 21% O2 at cell densities of 4
million cells/ml. Cells in alginate beads were cultured in DMEM
containing 6% foetal calf serum (FCS) at different osmolalities (270,
370, 470, 570 mOsmol). Medium osmolality was altered by addition of
NaCl and was monitored using a freezing point osmometer (Semi- micro
osmometer, Knauer, Germany ). The cell viability profile was
determined by manual counting using trypan blue staining. Lactate
production and glucose consumption were measured enzymatically as a
marker of energy metabolism 2). GAG accumulation (as a measure of
proteoglycan) was measured using a DMB assay 3). Rate of sulfate GAG
synthesis was measured using a standard 35 S-sulfate radioactive
method4). Profiles across intact beads were determined by manual
counting using fluorescent probes (LIVE/DEAD Viability/Cytotoxicity
Kit, Molecular Probes) and transmission electron microscope (TEM).
Result. The viavility profile is already established by 2 days of culture
since the results after 2 and 6 days are very similar. There was no
difference in the cell viability with or without oxygen. Cell metabolism
increased with time in culture and was higher in the presence of oxygen
than under hypoxia. At hypo-osmolality, cellular metabolism continued
at low level and was similar with and without oxygen present. Glucose
consumption rates fell with time in culture under 21% oxygen but
increased under low oxygen. GAG produced per million cells increased
with time in culture and was lower in the presence of oxygen than under
hypoxia. At hypo osmolality, GAG accumulation/tissue volume fell at
270 mOsmol. It was greatest at 370 mOsmol . After 6 days more GAG
had accumulated in beads cultured at low oxygen and in 370-570 mOsm
than at lower osmolalities or at 21% oxygen. GAG concentration in
beads cultured at 0% oxygen was 0.295 ± 0.010 mg/ml at 370 mOsm
compared to 0.084 ± 0.005?mg/ml at 270 mOsm (Fig.1 and 2). Surfate
incorporation rate were lowest 270 mOsmol. Oxygen concentration in
the medium had no significant effect. In findings of confocal
microscope, cell diameter increased with time in culture at 270 mOsmol
and was similar with and without oxygen presence. At 370, 470 and 570
mOsmol, the cell diameter was already established by 2 days of culture.
The results after 2 and 6 days are similar. Under TEM, all cells appeared
viable at 370 mOsm. However, in the beads cultured at 270 mOsm, the
cells were swelling with cytoplasmic organelles destroyed were visible.
Under 570 mOsm, cells undergoing apoptosis were seen (Fig.3).
Discussion. Loading of tissue leads to osmotic pressure variation. With
short -term applied load, fluid is expressed from disc tissue,
concentrating proteoglycans and thus increasing osmotic pressure.
Conversely, in degenerating disc proteoglycans are lost, which leads to a
fall in osmotic pressure. With long-term applied load, this may lead to
proteoglycan loss following the onset of hypo- and hyper- osmotic
conditions. At cell densities found in vivo (standard conditions) in the
disc viz, 4 million cells/ml and GAG concentration in beads cultures at
0% oxygen was 0.295+0.010 mg/ml at 370 mOsmol in 6 days. With
decrease in osmolality (disc degeneration), rate of lactate and GAG
production per cell decreased significantly and oxygen concentration
limited matrix production. Calculated times to produce a concentration
equal to the in vivo concentrations of 7% GAG per wet weight (viz. 70
200
100
0
2 days
270mOsm
6days
370mOsm
470mOsm
570mOsm
Fig.2. Total GAG in beads /ml beads volume under 0% oxygen
A. 270 mOsm.
B. 370 mOsm.
C. 570 mOsm
Fig.3. Electron micrographs of nucleus pulposus cells under different
osmolality (X5000). arrow: budding
Conclusion. In our model the prevailing osmolality was a powerful
regulator of GAG accumulation by cultured nucleus cells. In vivo
prevailing osmolality is governed by GAG concentration. These results
thus indicate GAG synthesis rates are regulated by GAG concentration,
with implications both for the aetiology of degeneration and for tissue
engineering. The results also suggest that DMEM or other standard
culture mediums do not provide an appropriate ionic and osmotic
environment for nucleus pulposus cells.
References.
1) Guo, J., et al : Connect Tissue Res. 19 : 277-297, 1989.
2) Lee, R.B., et al : Biochem.J. 321 : 95-102, 1997.
3) Enobakhare, B.O., et al : Analytical biochemistry. 243 : 189-191,
1996.
4)Maroudas, A. : In: Studies in Joint Diseases, edited by A. Maroudas
and E. J. Holbrow. London : Pitman Medical, : 59-86, 1980.
51st Annual Meeting of the Orthopaedic Research Society
Poster No: 0905