An organ culture system for the study of the nucleus pulposus: description of the system and evaluation of the cells.
METHODS
The goal of this study was to develop a methodology to maintain intervertebral discs in organ culture, thereby preserving tissue architecture and metabolic function in a three-dimensional environment.


METHODS
Using a microdissection technique, intervertebral discs were removed from rat lumbar vertebrae. The discs were maintained in organ culture, and cell viability was evaluated histochemically and using probes that measured mitochondrial function and thiol status. The biosynthetic activity of the cells was evaluated by Western blot and RT-PCR analysis.


RESULTS
The in vitro organ culture system maintained the vitality of the nucleus pulposus cells. Cells exhibited a high membrane potential for 1 week. When cells were exposed to carbonyl cyanide 4-trifluoromethoxy phenylhydrazone, a known protonophore, the fluorescence was lost, indicating that the staining was specific for viable cells. In many cells, Celltracker Green, probe for reduced thiols, colocalized with the membrane potential. Histologic studies revealed that in culture for 1 week, normal nucleus pulposus structure was maintained; after this time period, alterations were observed. We evaluated the two tissues for characteristic phenotypic markers HIF-1alpha and MMP-2. We noted that the nucleus pulposus expressed these proteins. The RT-PCR profile at 7 days indicated that the cells also expressed collagen type II, aggrecan, and decorin.


CONCLUSIONS
Three factors contributed to success in maintaining the vitality of the nucleus pulposus in vitro. First, the cells were confined within the disc itself; second, the medium was hyperosmotic; third, the medium was supplemented with transforming growth factor-beta. The fluorescence measurement provided a rapid method for evaluation of the status of nucleus pulposus cells. Histologic analysis confirmed that the cells remained viable for at least 1 week. Viability in terms of biosynthetic activity was further confirmed using RT-PCR and Western blot analysis. We conclude that short-term intervertebral disc organ culture can be used as a suitable in vitro model to study effects of environmental factors linked to disc degeneration and/or regeneration.
Risbud
Makarand V
MV
Department of Orthopaedic Surgery, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA. makarand.risbud@mail.tju.edu
Izzo
Marc W
MW
Adams
Christopher S
CS
Arnold
William W
WW
Hillibrand
Alan S
AS
Vresilovic
Edward J
EJ
Vaccaro
Alexander R
AR
Albert
Todd J
TJ
Shapiro
Irving M
IM
D000818:Animals; D015153:Blotting, Western; D002470:Cell Survival; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007403:Intervertebral Disc / Q000033:anatomy & histology / Q000166:cytology* / Q000378:metabolism; D008297:Male; D009924:Organ Culture Techniques / Q000379:methods*; D009930:Organic Chemicals; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction
D005452:Fluoresceins; D005456:Fluorescent Dyes; D009930:Organic Chemicals; C107472:red dye CMXRos; C069306:5-chloromethylfluorescein