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PLoS One ; 9(12): e112454, 2014.
Article in English | MEDLINE | ID: mdl-25474689

ABSTRACT

Intervertebral disc (IVD) homeostasis is mediated through a combination of micro-environmental and biomechanical factors, all of which are subject to genetic influences. The aim of this study is to develop and characterize a genetically tractable, ex vivo organ culture model that can be used to further elucidate mechanisms of intervertebral disc disease. Specifically, we demonstrate that IVD disc explants (1) maintain their native phenotype in prolonged culture, (2) are responsive to exogenous stimuli, and (3) that relevant homeostatic regulatory mechanisms can be modulated through ex-vivo genetic recombination. We present a novel technique for isolation of murine IVD explants with demonstration of explant viability (CMFDA/propidium iodide staining), disc anatomy (H&E), maintenance of extracellular matrix (ECM) (Alcian Blue staining), and native expression profile (qRT-PCR) as well as ex vivo genetic recombination (mT/mG reporter mice; AdCre) following 14 days of culture in DMEM media containing 10% fetal bovine serum, 1% L-glutamine, and 1% penicillin/streptomycin. IVD explants maintained their micro-anatomic integrity, ECM proteoglycan content, viability, and gene expression profile consistent with a homeostatic drive in culture. Treatment of genetically engineered explants with cre-expressing adenovirus efficaciously induced ex vivo genetic recombination in a variety of genetically engineered mouse models. Exogenous administration of IL-1ß and TGF-ß3 resulted in predicted catabolic and anabolic responses, respectively. Genetic recombination of TGFBR1fl/fl explants resulted in constitutively active TGF-ß signaling that matched that of exogenously administered TGF-ß3. Our results illustrate the utility of the murine intervertebral disc explant to investigate mechanisms of intervertebral disc degeneration.


Subject(s)
Intervertebral Disc Degeneration/genetics , Intervertebral Disc Displacement/pathology , Intervertebral Disc/pathology , Organ Culture Techniques , Animals , Cell Survival/drug effects , Disease Models, Animal , Humans , Interleukin-1beta/administration & dosage , Interleukin-1beta/metabolism , Intervertebral Disc Degeneration/metabolism , Intervertebral Disc Degeneration/pathology , Intervertebral Disc Displacement/genetics , Intervertebral Disc Displacement/metabolism , Mice , Microscopy, Fluorescence , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Receptor, Transforming Growth Factor-beta Type I , Receptors, Transforming Growth Factor beta/genetics , Receptors, Transforming Growth Factor beta/metabolism , Signal Transduction/genetics , Transcriptome
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