Glycosaminoglycan accumulation in primary culture of rabbit intervertebral disc cells

Masato Sato, Toshiyuki Kikuchi, Takashi Asazuma, Harumoto Yamada, Hiroshi Maeda, Kyosuke Fujikawa

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

Study Design. 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. Objectives. 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. Summary of Background Data. 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.

Original languageEnglish
Pages (from-to)2653-2660
Number of pages8
JournalSpine
Volume26
Issue number24
DOIs
Publication statusPublished - 15-12-2001

Fingerprint

Intervertebral Disc
Glycosaminoglycans
Rabbits
Keratan Sulfate
Sepharose
Cultured Cells
Gels
Chondroitin Sulfates
Chondrocytes
Nonparametric Statistics
Keratins
Sulfates
Annulus Fibrosus
Analysis of Variance
Cell Culture Techniques
Joints
High Pressure Liquid Chromatography
Phenotype
DNA

All Science Journal Classification (ASJC) codes

  • Orthopedics and Sports Medicine
  • Clinical Neurology

Cite this

Sato, Masato ; Kikuchi, Toshiyuki ; Asazuma, Takashi ; Yamada, Harumoto ; Maeda, Hiroshi ; Fujikawa, Kyosuke. / Glycosaminoglycan accumulation in primary culture of rabbit intervertebral disc cells. In: Spine. 2001 ; Vol. 26, No. 24. pp. 2653-2660.
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abstract = "Study Design. 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. Objectives. 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. Summary of Background Data. 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.",
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Sato, M, Kikuchi, T, Asazuma, T, Yamada, H, Maeda, H & Fujikawa, K 2001, 'Glycosaminoglycan accumulation in primary culture of rabbit intervertebral disc cells', Spine, vol. 26, no. 24, pp. 2653-2660. https://doi.org/10.1097/00007632-200112150-00004

Glycosaminoglycan accumulation in primary culture of rabbit intervertebral disc cells. / Sato, Masato; Kikuchi, Toshiyuki; Asazuma, Takashi; Yamada, Harumoto; Maeda, Hiroshi; Fujikawa, Kyosuke.

In: Spine, Vol. 26, No. 24, 15.12.2001, p. 2653-2660.

Research output: Contribution to journalArticle

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T1 - Glycosaminoglycan accumulation in primary culture of rabbit intervertebral disc cells

AU - Sato, Masato

AU - Kikuchi, Toshiyuki

AU - Asazuma, Takashi

AU - Yamada, Harumoto

AU - Maeda, Hiroshi

AU - Fujikawa, Kyosuke

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