Introduction
Chondroitin sulfate proteoglycan (CSPG), a major component of the intervertebral disc matrix, increases the anabolic turnover in nucleus pulposus (NP) cells incubated under repetitive cyclic hydrostatic pressure (HP) followed by constant hydrostatic pressure in high osmolality medium.# However, CS glycosaminoglycan diminished Its concentration must be optimized. We hypothesize that the anabolic turnover of NP cells increases in a CSPG concentration-dependent manner.
Methods
Bovine NP (bNP) tissues were collected from tails (n=6) and digested in collagenase. Isolated bNP cells were pre-incubated on 1.5% cell culture grade agarose-coated 6-well plates for 2 days. After carefully collecting the NP cells/clusters, 1.0×10⁵ NP cells were packed in a pouch made of semipermeable membrane with 0, 4, 16 mg/mL CSPG (extracted from bovine NP tissue with guanidine. The pouches were incubated under two different culture conditions:(1) no HP: Pouches were incubated at atmospheric pressure; and (2) HP: 2 days of cyclic HP (0.2-0.7 MPa, 0.5 Hz) followed by 1 day of constant HP (0.3 MPa) were repeated 4 times over 12 days. Both conditions stimulated a physiological hypoxic environment at 37°C, 5% CO2, 3% O2. Cells were collected and subjected to gene expression assay by RT-PCR. The probes included typical ECM related molecules, aggrecan core protein, chondroitin sulfate N-acetylgalactosaminyltransferase 1, hyaluronan synthetase 2, collagen type-I, collagen type-II; a catabolic molecule, matrix metalloproteinase-13; and cellular molecules, tissue inhibitor of metalloproteinases-2.
Immunohistochemical staining was also performed to confirm the accumulation of Keratan sulfate (KS) and the inflammatory cytokine MMP13 in the bNP cells/cluster. Counterstaining was performed with Harris’s hematoxylin for KS and with Contrast Red for MMP13. RT-PCR and immunohistochemical staining were performed on days 3 and 12, and One-way ANOVA was used to identify statistical differences (p<0.05).
Results
There was no obvious increase in gene expression in each group by 3 days after the start of HP loading in the NP cells. There was no obvious increase in gene expression in the HP group with 0 mg of CSPG, even after 12 days of HP loading, compared to the No HP group. NP cells showed significantly increased expression of aggrecan core protein, collagen type II and chondroitin sulfate N-acetylgalactosaminyltransferase-1 (P < 0.05) by 12 days after HP loading under CSPG treatment (0, 4, and 16 mg/ml). On the other hand, there was no increase in MMP13, a catabolic factor. No clear differences in gene expression were observed between CSPG concentrations. Immunohistological staining showed KS accumulation in bNP cells/clusters. There was no significant difference in KS accumulation between CSPG concentrations and with or without HP, but all groups accumulated more intensely on day 12 than on day 3. On the other hand, MMP13 tended to decrease in the presence of HP, although no significant difference was observed between day3 and day12, or between CSPG concentrations.
Conclusions
CSPG promotes anabolic turnover without promoting catabolic turnover of intervertebral disc tissue, but its ability to do so is limited. HP also enhances anabolic turnover in intervertebral disc tissue, but it is particularly effective in the presence of CSPG.