Hyperosmotic Condition Reduces Nucleus Pulposus Growth in Monolayer Culture and 3D Alginate Beads Culture

Abstract

Introduction The intervertebral disc (IVD) is a specialized structure composed of mainly collagens and proteoglycans to hold water and to accommodate applied mechanical forces. The high glycosaminoglycans side chains of the aggrecan raise the osmolarity of the nucleus pulposus, therefore, nucleus pulposus cells (NPC) are under hyperosmotic condition inside the disc. Recent studies raised contradictory results in the NPC responses to isosmotic and hyperosmotic culture conditions, while one proposed hyperosmotic culture condition could preserve the matrix production activity of NPC. However, another study suggested that a hyperosmotic condition reduced NPC viability and matrix production. This study aimed to monitor cell growth and changes of the NPC phenotype in isosmotic and hyperosmotic culture condition by monitoring a selection of their important NPC surface antigens by flow cytometry. Material and Methods Five human NPC were isolated from human nucleus pulposus tissue (aged 54–65 years) by enzymatic digestion with hyaluronidase and liberase (Roche). Primary cells were expanded in monolayer culture to passage 1 and then seeded at 10,000 cells per 6—well in duplicates and cultured in—MEM containing 10% FSC either in isosmotic medium (300 Osm/L), or in hyperosmotic medium (400 Osm/L) adjusted with 1% 5 M NaCl and 0.4 M KCl. Cells were trypsinized after 7 days culture and stained with the following three NPC surface markers: antihuman disialoganglioside GD2 (GD2) (BD Pharmingen; 14; G2a) mAb and FITC-conjugated antimouse Ig goat (BD Biosciences), allophycocyanin-conjugated antihuman Tie2 (R&D Systems, clone 83,715) mAb and PE-conjugated antihuman CD24 (BD Biosciences; clone ML5) mAb for flow cytometry characterization for percentage of cells expressing NPC surface markers by FACS Calibur. Cumulative population doubling and changes of NPC cell surface antigens were monitored at days 0, 7, 14, 21 and 28. To culture the cells in alginate beads, NPC were mixed with 1.2% alginate solution in a density of 10,000 cells/mL. Alginate beads were formed by adding the alginate solution into 102 mM CaCl2 solution through a 23G needle. Beads were cultured in isosmotic or hyperosmotic medium as described earlier. After 28 days culture, cells were released from the alginate by incubating in 55 mM sodium citrate, 0.9% NaCl2 for 10 minutes followed by trypsinization for 10 minutes. Two-way ANOVA analysis was performed to analyze the difference between isosmotic and hyperosmotic culture conditions. Results Cumulative population doubling time indicated that NP cell growth was inhibited in hyperosmotic condition over the 28-day culture, which was 50% lower in hyperosmotic condition than isosmotic condition. Hyperosmotic medium reduced NPC growth even stronger in 3D alginate beads culture. Total cell number recovered from the beads was four times reduced in hyperosmotic medium (71,000 ± 48,432) than in isosmotic medium (15,000 ± 7,906).