Glycosaminoglycan accumulation in primary culture of rabbit intervertebral disc cells.
METHODS
With the heterogeneity of the intervertebral disc as the focus, intervertebral discs from normal young rabbits were separated into nucleus pulposus (NP), inner anulus fibrosus (IAF), and outer anulus fibrosus (OAF) zones. Disc cells from each zone were isolated and propagated under monolayer and within agarose gel culture. The metabolism of these cultured disc cells was examined in terms of glycosaminoglycan (GAG) accumulation.


OBJECTIVE
The object was to study the metabolism of disc cells derived from each zone and characterize them on the basis of GAG accumulation and composition.


BACKGROUND
It has been shown that three-dimensional culture systems, such as within-agarose gels or in alginate beads, permit long-term maintenance of the articular chondrocyte phenotype in vitro. However, little has been reported on how the metabolism of intervertebral disc cells, especially GAG accumulation, is affected by different culture conditions.


METHODS
Cells from each zone were subjected to monolayer or three-dimensional culture for up to 12 days. GAG accumulation in the different culture systems was analyzed using chemical, histologic, and immunohistologic methods. Differences of GAG and DNA content among NP, IAF, and OAF cells were statistically evaluated by analysis of variance. The data of keratin sulfate content in three-dimensional culture were compared with that in monolayer culture using nonparametric Mann-Whitney U test.


RESULTS
Monolayer culture revealed that increases in GAG content were significantly higher in IAF cells than in OAF cells. However, in three-dimensional culture GAG content was similar in the two groups. AF cells in three-dimensional cultures showed immunohistochemical localization of chondroitin sulfate and keratan sulfate, suggesting the existence of pericellular matrix. High performance liquid chromatography confirmed the expression of keratan sulfate in cultured cells.


CONCLUSIONS
GAG accumulation in cultures of cells from different zones of the intervertebral disc varied according to the culture conditions used. The importance of choosing the appropriate culture systems to meet the objectives of a study should be emphasized.
Sato
M
M
Department of Orthopaedic Surgery, National Defense Medical College, Tokorozawa, Saitama, Japan. ma-sato@md.phd.com
Kikuchi
T
T
Asazuma
T
T
Yamada
H
H
Maeda
H
H
Fujikawa
K
K
D000704:Analysis of Variance; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D046508:Culture Techniques / Q000379:methods; D004247:DNA / Q000032:analysis; D005109:Extracellular Matrix / Q000378:metabolism; D005260:Female; D005782:Gels; D006025:Glycosaminoglycans / Q000378:metabolism*; D007124:Immunoenzyme Techniques; D007403:Intervertebral Disc / Q000166:cytology* / Q000378:metabolism*; D007632:Keratan Sulfate / Q000032:analysis; D011817:Rabbits; D018709:Statistics, Nonparametric; D014048:Tolonium Chloride
D005782:Gels; D006025:Glycosaminoglycans; D014048:Tolonium Chloride; D004247:DNA; D007632:Keratan Sulfate