Influence of diurnal hyperosmotic loading on the metabolism and matrix gene expression of a whole‐organ intervertebral disc model

Abstract

It is generally agreed that the mechanical environment of intervertebral disc cells plays an important role in maintaining a balanced matrix metabolism. The precise mechanism by which the signals are transduced into the cells is poorly understood. Osmotic changes in the extracellular matrix (ECM) are thought to be involved. Current in-vitro studies on this topic are mostly short-term and show conflicting data on the reaction of disc cells subjected to osmotic changes which is partially due to the heterogenous and often substantially-reduced culture systems. The aim of the study was therefore to investigate the effects of cyclic osmotic loading for 4 weeks on metabolism and matrix gene expression in a full-organ intervertebral disc culture system. Intervertebral disc/endplate units were isolated from New Zealand White Rabbits and cultured either in iso-osmotic media (335 mosmol/kg) or were diurnally exposed for 8 hours to hyper-osmotic conditions (485 mosmol/kg). Cell viability, metabolic activity, matrix composition and matrix gene expression profile (collagen types I/II and aggrecan) were monitored using Live/Dead cell viability assay, tetrazolium reduction test (WST 8), proteoglycan and DNA quantification assays and quantitative PCR. The results show that diurnal osmotic stimulation did not have significant effects on proteoglycan content, cellularity and disc cell viability after 28 days in culture. However, hyperosmolarity caused increased cell death in the early culture phase and counteracted upregulation of type I collagen gene expression in nucleus and annulus cells. Moreover, the initially decreased cellular dehydrogenase activity recovered with osmotic stimulation after 4 weeks and aggrecan gene down-regulation was delayed, although the latter was not significant according to our statistical criteria. In contrast, collagen type II did not respond to the osmotic changes and was downregulated in both groups. In conclusion, diurnal hyper-osmotic stimulation of a whole-organ disc/ endplate culture partially inhibits a matrix gene expression profile as encountered in degenerative disc disease and counteracts cellular metabolic hypo-activity. ß

Influence of extracellular osmolarity and mechanical stimulation on gene expression of intervertebral disc cells

“…In agreement with our findings (data not illustrated), Ishihara and colleagues observed an increase of sulphated GAG synthesis in bovine NP cells with increase in osmolarity. 6 In an organ culture system, Haschtmann and colleagues found that an increase in osmolarity induced a (tendential) upregulation of aggrecan and downregulation of collagen I expression, 16 similar to our findings. Studies on articular chondrocytes showed that extracellular osmolarity influenced matrix synthesis and regulated aggrecan promoter activity as well as aggrecan expression and sulphated GAG synthesis.…”

The effects of dynamic loading on the intervertebral disc

“…Cell volume changes could affect various membrane ion transporters and alter cell metabolism, resulting in a different biosynthetic activity of the cell [35,116]. Furthermore, studies have shown that osmolality and pH directly affect IVD cell metabolism [12,18,27,37,115,129].…”

Vertebral endplate trauma induces disc cell apoptosis and promotes organ degeneration in vitro

“…Due to the lack of appropriate animal models for endplate trauma, we used an in vitro organ culture approach, which is based on our recent work. There we established a whole-organ rabbit disc/endplate in vitro culture system with the possibility to keep the disc specimens in culture for more then 7 weeks without a significant loss of cell viability and proteoglycans from the extracellular matrix [19,20]. For the current study, we constructed a dropped-weight device for the sterile induction of endplate burst fractures and an accompanying injury of the adjacent intervertebral disc.…”

“…The disc/endplate specimens were dissected as described before [19,20]. After harvest the specimens (n = 10 per animal) were washed with agitation overnight in DMEM/F12 media (Labforce, Nunningen, Switzerland) with 50 lg/ml gentamicin (Labforce) and 10% FCS and 20 mmol sodium citrate.…”

“…We hypothesized that disc trauma initiates biological cascades and the expression of genes which may lead to an ultimate degeneration of the organ. The model is based on our recently developed and validated full-organ disc/endplate system, which has been shown to be suitable for long-term culture [19,20].…”