Toward an Optimum System for Intervertebral Disc Organ Culture: TGF-β3 Enhances Nucleus Pulposus and Anulus Fibrosus Survival and Function Through Modulation of TGF-β-R Expression and ERK Signaling

Study Design. Rat lumbar discs comprising nucleus pulposus, annulus fibrosus, and cartilaginous endplates were cultured for 1 week in a specialized media containing either TGF-β1 or TGF-β3. Role of TGF-β isoforms on cell function was evaluated. Objective. To develop an in vitro organ culture of rat intervertebral disc and evaluate effects of TGF-β3 on disc cell function. Summary of Background Data. 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. Materials and Methods. Rat lumbar intervertebral discs were maintained in organ culture in media supplemented with TGF-β3 or TGF-β1 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-β3 and TGF-β1, 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-β1, TGF-β3 elevated the expression of critical matrix genes, enhanced [35S] incorporation into proteoglycans, preserved the expression of TGF-β 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-β3, resulted in suppression of collagen Type II, aggrecan, TGF-β-RI, TGF-β-RII and TGF-β-RIII mRNA expression. Conclusions. TGF-β3 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-β-RI and TGF-β-RII.

[1]  T. Albert,et al.  Differentiation of Mesenchymal Stem Cells Towards a Nucleus Pulposus-like Phenotype In Vitro: Implications for Cell-Based Transplantation Therapy , 2004, Spine.

[2]  Junzo Tanaka,et al.  Chondrogenic differentiation of human mesenchymal stem cells cultured in a cobweb-like biodegradable scaffold. , 2004, Biochemical and biophysical research communications.

[3]  Junzo Tanaka,et al.  Growth factor combination for chondrogenic induction from human mesenchymal stem cell. , 2004, Biochemical and biophysical research communications.

[4]  Su-Hyang Kim,et al.  Chondrogenic differentiation of mesenchymal stem cells and its clinical applications. , 2004, Yonsei medical journal.

[5]  J. Lotz,et al.  In Vivo Growth Factor Treatment of Degenerated Intervertebral Discs , 2004, Spine.

[6]  T. Albert,et al.  An Organ Culture System for the Study of the Nucleus Pulposus: Description of the System and Evaluation of the Cells , 2003, Spine.

[7]  J. Massagué,et al.  Mechanisms of TGF-β Signaling from Cell Membrane to the Nucleus , 2003, Cell.

[8]  M. Aebi,et al.  The Potential and Limitations of a Cell-Seeded Collagen/Hyaluronan Scaffold to Engineer an Intervertebral Disc-Like Matrix , 2003, Spine.

[9]  K. Miyazono,et al.  Two major Smad pathways in TGF‐β superfamily signalling , 2002, Genes to cells : devoted to molecular & cellular mechanisms.

[10]  K. Budde,et al.  Cyclosporine A up-regulates the expression of TGF-beta1 and its receptors type I and type II in rat mesangial cells. , 2002, Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association.

[11]  T. Sanchez-Elsner,et al.  Extracellular and Cytoplasmic Domains of Endoglin Interact with the Transforming Growth Factor-β Receptors I and II* , 2002, The Journal of Biological Chemistry.

[12]  Jeffrey L. Wrana,et al.  Signal Transduction by the TGF-β Superfamily , 2002, Science.

[13]  Hiroshi Mizuta,et al.  The spatiotemporal expression of TGF‐β1 and its receptors during periosteal chondrogenesis in vitro , 2002, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[14]  I. Otterness,et al.  Matrix metalloproteinases are involved in C-terminal and interglobular domain processing of cartilage aggrecan in late stage cartilage degradation. , 2002, Matrix biology : journal of the International Society for Matrix Biology.

[15]  Darwin J. Prockop,et al.  In vitro cartilage formation by human adult stem cells from bone marrow stroma defines the sequence of cellular and molecular events during chondrogenesis , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[16]  F. Verrecchia,et al.  Transforming Growth Factor-β Signaling Through the Smad Pathway: Role in Extracellular Matrix Gene Expression and Regulation , 2002 .

[17]  M. D. de Caestecker,et al.  Transcriptional Cross-talk between Smad, ERK1/2, and p38 Mitogen-activated Protein Kinase Pathways Regulates Transforming Growth Factor-β-induced Aggrecan Gene Expression in Chondrogenic ATDC5 Cells* , 2001, The Journal of Biological Chemistry.

[18]  C. Little,et al.  Mechanisms involved in cartilage proteoglycan catabolism. , 2000, Matrix biology : journal of the International Society for Matrix Biology.

[19]  J. Massagué,et al.  Controlling TGF-β signaling , 2000, Genes & Development.

[20]  L G Gilbertson,et al.  Modulation of the biologic activity of the rabbit intervertebral disc by gene therapy: an in vivo study of adenovirus-mediated transfer of the human transforming growth factor beta 1 encoding gene. , 1999, Spine.

[21]  G. Adler,et al.  Transforming growth factor-β-induced upregulation of transforming growth factor-β receptor expression in pancreatic regeneration , 1999 .

[22]  A. Roberts Molecular and Cell Biology of TGF-β , 1998, Mineral and Electrolyte Metabolism.

[23]  H E Gruber,et al.  Human intervertebral disc cells from the annulus: three-dimensional culture in agarose or alginate and responsiveness to TGF-beta1. , 1997, Experimental cell research.

[24]  S. Roberts,et al.  Proteoglycan synthesis in the intervertebral disk nucleus: the role of extracellular osmolality. , 1997, The American journal of physiology.

[25]  J. Massagué,et al.  Mechanisms of TGF-beta signaling from cell membrane to the nucleus. , 2003, Cell.

[26]  J. Wrana,et al.  Signal transduction by the TGF-beta superfamily. , 2002, Science.

[27]  J. Massagué,et al.  Controlling TGF-beta signaling. , 2000, Genes & development.

[28]  G. Adler,et al.  Transforming growth factor-beta-induced upregulation of transforming growth factor-beta receptor expression in pancreatic regeneration. , 1999, Biochimica et biophysica acta.

[29]  A. Roberts Molecular and cell biology of TGF-beta. , 1998, Mineral and electrolyte metabolism.

[30]  J. Urban,et al.  Swelling pressure of the inervertebral disc: influence of proteoglycan and collagen contents. , 1985, Biorheology.