Steroid treatment can inhibit nuclear localization of members of the NF-κB pathway in human disc cells stimulated with TNF-α.

Steroid applications are able to repress inflammatory activity in various conditions, including herniation of the nucleus pulposus (HNP), by inhibiting tumour necrosis factor (TNF)-α, but the effects of long-term use are unknown. Here, we investigated the effect of dexamethasone (DEXA) on TNF-α-stimulated intervertebral disc cells by monitoring the expression and localization of NF-κB in the cytoplasm and nucleus. Cultured human intervertebral disc cells were left untreated or treated with only TNF-α, only DEXA, or with TNF-α and DEXA simultaneously. Cytoplasmic and nuclear proteins were extracted and Western blotted after 10 min, 1 or 2 h, to evaluate the expression of p50, p65, p52, and p100 (components of NF-κB). Immunofluorescence analysis was used to determine the subcellular localization of the proteins at 1 h. DEXA had limited effects on NF-κB expression in TNF-α-stimulated disc cells within the first 10 min. At 1 h, DEXA prevented the TNF-α-stimulated translocation of p50, p52, and p65. After 2 h, DEXA reduced the nuclear expression of p50, p65, and p52. Thus, DEXA resulted in delayed expression of NF-κB components and inhibited the translocation of p50, p52, and p65 to the nucleus, which would prevent expression of the corresponding genes. Therefore, following stimulation with TNF-α, transcriptional regulation of NF-κB in disc cells is mainly mediated via the classical pathway, but also to some extent via the alternative pathway. Hence, blockade of sub-acute inflammatory changes in HNP can be achieved by early injection of steroids, whereas long-term injection of a steroid may initiate NF-κB autophosphorylation.

Induction of chondrogenic differentiation in cultured fibroblasts isolated from the inferior turbinate.

OBJECTIVES: To explore options of cell sources for regeneration of cartilage, turbinate fibroblasts were tested for their differentiation potential when cultured in three-dimensional scaffolds with growth factors and co-cultured with septal chondrocytes. METHODS: Fibroblasts and chondrocytes were seeded on to an alginate sponge in different proportions and were stimulated with TGF-beta1 and IGF-I. Chondrogenic differentiation of fibroblasts was assessed by histology including immunohistochemical staining, which was examined by reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: Chondroid cells with extracellular matrices occupied the porous structures of alginate sponge and immunolocalizations for type II collagen became prominent at four weeks in culture. Expression of type II collagen and aggrecan mRNA were detected by RT-PCR. Co-culture of fibroblasts and chondrocytes showed comparable expansion of cells and extracellular matrix to culture of chondrocytes only. CONCLUSION: These findings suggest that human turbinate fibroblasts are apparently redirected toward chondrogenic phenotype by in vitro culture system under specific conditions. Co-culture of turbinate fibroblasts with septal chondrocytes would be an effective method to expand cartilage specific extracellular matrix.