Establishment of a novel intervertebral disc/endplate culture model: analysis of an ex vivo in vitro whole-organ rabbit culture system.
METHODS
Ex vivo in vitro study evaluating a novel intervertebral disc/endplate culture system.


OBJECTIVE
To establish a whole-organ intervertebral disc culture model for the study of disc degeneration in vitro, including the characterization of basic cell and organ function.


BACKGROUND
With current in vivo models for the study of disc and endplate degeneration, it remains difficult to investigate the complex disc metabolism and signaling cascades. In contrast, more controlled but simplified in vitro systems using isolated cells or disc fragments are difficult to culture due to the unconstrained conditions, with often-observed cell death or cell dedifferentiation. Therefore, there is a demand for a controlled culture model with preserved cell function that offers the possibility to investigate disc and endplate pathologies in a structurally intact organ.


METHODS
Naturally constrained intervertebral disc/endplate units from rabbits were cultured in multi-well plates. Cell viability, metabolic activity, matrix composition, and matrix gene expression profile were monitored using the Live/Dead cell viability test (Invitrogen, Basel, Switzerland), tetrazolium salt reduction (WST-8), proteoglycan and deoxyribonucleic acid quantification assays, and quantitative polymerase chain reaction.


RESULTS
Viability and organ integrity were preserved for at least 4 weeks, while proteoglycan and deoxyribonucleic acid content decreased slightly, and matrix genes exhibited a degenerative profile with up-regulation of type I collagen and suppression of collagen type II and aggrecan genes. Additionally, cell metabolic activity was reduced to one third of the initial value.


CONCLUSIONS
Naturally constrained intervertebral rabbit discs could be cultured for several weeks without losing cell viability. Structural integrity and matrix composition were retained. However, the organ responded to the artificial environment with a degenerative gene expression pattern and decreased metabolic rate. Therefore, the described system serves as a promising in vitro model to study disc degeneration in a whole organ.
Haschtmann
Daniel
D
MEM Research Center for Orthopaedic Surgery, Institute for Surgical Technology and Biomechanics, University of Bern, Stauffacherstrasse 78, CH-3014 Bern, Switzerland. Daniel.Haschtmann@MEMcenter.unibe.ch
Stoyanov
Jivko V
JV
Ettinger
Ladina
L
Nolte
Lutz-P
LP
Ferguson
Stephen J
SJ
D000818:Animals; D002470:Cell Survival / Q000502:physiology; D004247:DNA / Q000378:metabolism; D005260:Female; D007403:Intervertebral Disc / Q000166:cytology* / Q000378:metabolism*; D009924:Organ Culture Techniques / Q000379:methods; D011509:Proteoglycans / Q000378:metabolism; D011817:Rabbits
D011509:Proteoglycans; D004247:DNA