Introduction: To develop regenerative strategies to treat intervertebral disc (IVD) degeneration, it is necessary to understand the metabolic characteristics of the resident cells in degenerated IVDs. Nucleus pulposus (NP) cells are exposed to changes in hydrostatic pressure (HP) at high osmotic pressure (OP) due to compressive spinal motion and negatively charged extracellular matrix (ECM). Our studies using bovine NP cells indicated that repetitive changes in HP at high OP stimulated the production of NP-specific ECM. We hypothesized that human NP (hNP) cells in moderately degenerated IVDs (defined with Pfirrmann grades) express anabolic turnover under the same regimen of HP in high OP as used for bovine NP cells. We assessed metabolic turnover in hNP cells isolated from tissue with Pfirrmann grades 2-3 or 4 under HP at high osmolality using gene expression assay and immunohistology.

Methods: All experiments were performed under IRB approval. We measured the amounts of DNA, RNA, and sulfated glycosaminoglycan (SGAG) in hNP tissues from subjects who underwent surgery (n=10: age 47.4±16.8 [21⁠–69]) and then evaluated the correlation with Pfirrmann grades. We thereafter divided hNP tissues into 2 groups according to Pfirrmann grade: grade 2–3 (n=13: age 46.7±14.0 [22⁠–71]), and grade 4 (n=13: age 53.0±11.5 [37⁠–75]). NP cells/clusters were isolated and incubated within semi-permeable membrane pouches under 2 different conditions: no HP, in which pouches were placed in culture medium; and HP, in which cyclic HP (0.2–0.7 MPa, 0.5 Hz) was applied for 2 days followed by constant HP (0.3 MPa) for 1 day, repeated twice over 6 days. The OP of medium was set at 450 mOsm/kg H₂O with NaCl. The pouches were harvested at 3 and 6 days. The gene expression of collagen types I (COL1) and II (COL2), aggrecan (ACAN), and matrix metallopeptidase 13 (MMP13) were measured. Immunohistology for MMP13 and TIMP2 were also performed. Three-way repeated-measures ANOVA with the Bonferroni post hoc test was used with significance at P<0.05. Linear regression analysis was also performed to assess the correlation between the expression of MMP13 and TIMP2.

Results: SGAG contents / DNA amounts had a negative correlation with Pfirrmann grade (R=0.69) (Grade 3, 295.8±276.0 mg/mg; Grade 4, 32.3±21.4 mg/mg), whereas RNA amounts had no correlation with Pfirrmann grade. ACAN and COL2 were significantly upregulated under HP in grade 2–3 compared to grade 4 (both, P<0.01). COL1 was significantly lower in both grades under HP than under no HP at 3 and 6 days (all, P<0.01) (Figure A). Linear regression analysis showed a positive correlation between MMP13 and TIMP2 expression in grade 2–3 under HP, but a negative correlation in grade 4 (P=0.04) (Figure B). Immunohistology for MMP13 and TIMP2 showed a similar trend. These results reflect the difference in inhibition capacity for catabolic upregulation under HP between the two groups.

Discussion: Remnant hNP cells of Pfirrmann grade 2–3 under physiological stresses including HP and OP will be capable of to regenerate by reactivating anabolic turnover in hNP cells and suppressing catabolic turnover compared to those of Pfirrmann grade 4.