Toward an optimum system for intervertebral disc organ culture: TGF-beta 3 enhances nucleus pulposus and anulus fibrosus survival and function through modulation of TGF-beta-R expression and ERK signaling.
METHODS
Rat lumbar discs comprising nucleus pulposus, annulus fibrosus, and cartilaginous endplates were cultured for 1 week in a specialized media containing either TGF-beta1 or TGF-beta3. Role of TGF-beta isoforms on cell function was evaluated.


OBJECTIVE
To develop an in vitro organ culture of rat intervertebral disc and evaluate effects of TGF-beta3 on disc cell function.


BACKGROUND
An in vitro model system is of considerable value in understanding the cell biology of the intervertebral disc. Development of a useful organ culture model would enhance understanding of disc function in health and disease.


METHODS
Rat lumbar intervertebral discs were maintained in organ culture in media supplemented with TGF-beta3 or TGF-beta1 for 1 week. Tissue morphology was studied using routine histologic, histochemical and immunohistochemical techniques. Cell function was assessed by gene expression, sulfate incorporation, and Western blot analysis.


RESULTS
After 1 week in culture with TGF-beta3 and TGF-beta1, the gross morphology and tissue architecture of the disc were preserved. TUNEL analysis indicated that there was no evidence of cell death in the nucleus pulposus or the anulus fibrosus. The level of Alcian blue staining in the nucleus pulposus was similar to that of the freshly isolated disc. However, when compared with TGF-beta1, TGF-beta3 elevated the expression of critical matrix genes, enhanced [S] incorporation into proteoglycans, preserved the expression of TGF-beta receptors, and decreased aggrecan turnover. There was also increased activation (phosphorylation) of ERK, a critical signaling protein. Moreover, inhibition of ERK activity, in the presence TGF-beta3, resulted in suppression of collagen Type II, aggrecan, TGF-beta-RI, TGF-beta-RII and TGF-beta-RIII mRNA expression.


CONCLUSIONS
TGF-beta3 maintains the phenotype of disc cells in organ culture. It exerts this effect, in part, by elevating the levels of activated ERK1/2, which in turn regulates the expression of TGF-beta-RI and TGF-beta-RII.
Risbud
Makarand V
MV
Department of Orthopaedic Surgery and Graduate Program in Tissue Engineering and Regenerative Medicine, Thomas Jefferson University, Philadelphia, PA 19107, USA. makarand.risbud@jefferson.edu
Di Martino
Alberto
A
Guttapalli
Asha
A
Seghatoleslami
Reza
R
Denaro
Vincenzo
V
Vaccaro
Alexander R
AR
Albert
Todd J
TJ
Shapiro
Irving M
IM
D030201:Activin Receptors, Type I / Q000096:biosynthesis; D000818:Animals; D048049:Extracellular Signal-Regulated MAP Kinases / Q000096:biosynthesis / Q000502:physiology*; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D007403:Intervertebral Disc / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology*; D008297:Male; D009924:Organ Culture Techniques / Q000379:methods; D017346:Protein Serine-Threonine Kinases; D011509:Proteoglycans / Q000096:biosynthesis*; D051381:Rats; D017208:Rats, Wistar; D000077293:Receptor, Transforming Growth Factor-beta Type I; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018125:Receptors, Transforming Growth Factor beta / Q000096:biosynthesis*; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D053782:Transforming Growth Factor beta3
D011509:Proteoglycans; D018125:Receptors, Transforming Growth Factor beta; C509046:Tgfb3 protein, rat; D016212:Transforming Growth Factor beta; D053782:Transforming Growth Factor beta3; C071736:betaglycan; D017346:Protein Serine-Threonine Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; D030201:Activin Receptors, Type I; D000077293:Receptor, Transforming Growth Factor-beta Type I; D000077294:Receptor, Transforming Growth Factor-beta Type II; C000625843:Tgfbr1 protein, rat