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

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Abstract

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.

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