Long-term culture of bovine nucleus pulposus explants in a native environment

B.G.M. Dijk, van, E. Potier, K. Ito

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Abstract

BACKGROUND 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 models. PURPOSE: To develop a physiological in vitro explant model that incorporates the native environment 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 isotonic 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% O2 and 4.5 g/L glucose) or physiological (5% O2 and 1 g/L glucose) levels. Samples were analyzed at Day 0, 3, and 42 for tissue composition (water, sulfated glycosaminoglycans, DNA, and hydroxyproline contents 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 O2 and 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. All rights reserved.
Original languageEnglish
Pages (from-to)454-463
JournalThe Spine Journal
Volume13
Issue number4
DOIs
Publication statusPublished - 2013

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Metalloproteases
Osmolar Concentration
A73025
Glucose
Aggrecans
Intervertebral Disc Degeneration
Intervertebral Disc
Hydroxyproline
Collagen Type I
Low Back Pain
Nucleus Pulposus
Cell Survival
Histology
Down-Regulation
Oxygen
Pressure
Water
DNA
Therapeutics

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Dijk, van, B.G.M. ; Potier, E. ; Ito, K. / Long-term culture of bovine nucleus pulposus explants in a native environment. In: The Spine Journal. 2013 ; Vol. 13, No. 4. pp. 454-463.
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abstract = "BACKGROUND 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 models. PURPOSE: To develop a physiological in vitro explant model that incorporates the native environment 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 isotonic 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{\%} O2 and 4.5 g/L glucose) or physiological (5{\%} O2 and 1 g/L glucose) levels. Samples were analyzed at Day 0, 3, and 42 for tissue composition (water, sulfated glycosaminoglycans, DNA, and hydroxyproline contents 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 O2 and 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. All rights reserved.",
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Long-term culture of bovine nucleus pulposus explants in a native environment. / Dijk, van, B.G.M.; Potier, E.; Ito, K.

In: The Spine Journal, Vol. 13, No. 4, 2013, p. 454-463.

Research output: Contribution to journalArticleAcademicpeer-review

TY - JOUR

T1 - Long-term culture of bovine nucleus pulposus explants in a native environment

AU - Dijk, van, B.G.M.

AU - Potier, E.

AU - Ito, K.

PY - 2013

Y1 - 2013

N2 - BACKGROUND 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 models. PURPOSE: To develop a physiological in vitro explant model that incorporates the native environment 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 isotonic 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% O2 and 4.5 g/L glucose) or physiological (5% O2 and 1 g/L glucose) levels. Samples were analyzed at Day 0, 3, and 42 for tissue composition (water, sulfated glycosaminoglycans, DNA, and hydroxyproline contents 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 O2 and 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. All rights reserved.

AB - BACKGROUND 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 models. PURPOSE: To develop a physiological in vitro explant model that incorporates the native environment 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 isotonic 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% O2 and 4.5 g/L glucose) or physiological (5% O2 and 1 g/L glucose) levels. Samples were analyzed at Day 0, 3, and 42 for tissue composition (water, sulfated glycosaminoglycans, DNA, and hydroxyproline contents 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 O2 and 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. All rights reserved.

U2 - 10.1016/j.spinee.2012.12.006

DO - 10.1016/j.spinee.2012.12.006

M3 - Article

C2 - 23340344

VL - 13

SP - 454

EP - 463

JO - The Spine Journal

JF - The Spine Journal

SN - 1529-9430

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