Physical limitations to tissue engineering of intervertebral disc cells: effect of extracellular osmotic change on glycosaminoglycan production and cell metabolism. Laboratory investigation.
OBJECTIVE
In this study, the authors examined how physiological levels of extracellular osmolality influence proteoglycan accumulation in nucleus pulposus cells in a 3D culture system.


METHODS
Cells were isolated from the nucleus pulposus of caudal discs obtained from 18- to 24-month-old bovines. They were cultured for 6 days in alginate beads at 4 million cells/ml in Dulbecco modified Eagle medium containing 6% fetal bovine serum under 21% O2. Medium osmolality was altered by NaCl addition between 270 and 570 mOsm and monitored using a freezing point osmometer. The cell viability profile was determined by manual counting after trypan blue staining. Profiles across intact beads were determined by manual counting by using fluorescent probes and a transmission electron microscope. Lactate production was measured enzymatically, and glycosaminoglycan (GAG) accumulation was measured using a dimethylmethylene blue assay. Rate of sulfate GAG synthesis was measured using a standard [35S]sulfate radioactive method.


RESULTS
The cell viability was similar for the high- and low-osmolality cultures. However, confocal microscopy showed that the cells were the largest at 270 mOsm and became smaller with increasing osmotic pressure. The GAG production was largest at 370 mOsm, the capacity for GAG production and cell metabolism (lactate production) was low under hypoosmolality and hyperosmolality, and cell death was observed on electron microscopy.


CONCLUSIONS
In the authors' model, the prevailing osmolality was a powerful regulator of GAG accumulation by cultured nucleus cells. Thus, these results indicate that GAG synthesis rates are regulated by GAG concentration, with implications both for the cause of degeneration and for tissue engineering.
Takeno
Kenichi
K
Department of Orthopaedics and Rehabilitation Medicine, Faculty of Medical Science, The University of Fukui, Fukui, Japan.
Kobayashi
Shigeru
S
Negoro
Kohei
K
Uchida
Kenzo
K
Miyazaki
Tsuyoshi
T
Yayama
Takafumi
T
Shimada
Seiichiro
S
Baba
Hisatoshi
H
D000818:Animals; D002417:Cattle; D048429:Cell Size; D002470:Cell Survival; D045604:Extracellular Fluid / Q000378:metabolism*; D006025:Glycosaminoglycans / Q000096:biosynthesis*; D007403:Intervertebral Disc / Q000166:cytology* / Q000378:metabolism* / Q000502:physiology; D007773:Lactates / Q000378:metabolism; D008297:Male; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D009994:Osmolar Concentration; D009997:Osmotic Pressure; D018874:Spheroids, Cellular; D013997:Time Factors; D023822:Tissue Engineering
D006025:Glycosaminoglycans; D007773:Lactates