Pericellular heparan sulfate proteoglycans: Role in regulating the biosynthetic response of nucleus pulposus cells to osmotic loading

Abstract

Background: Daily physiologic loading causes fluctuations in hydration of the intervertebral disc (IVD); thus, the embedded cells experience cyclic alterations to their osmotic environment. These osmotic fluctuations have been described as a mechanism linking mechanics and biology, and have previously been shown to promote biosynthesis in chondrocytes. However, this phenomenon has yet to be fully interrogated in the IVD. Additionally, the specialized extracellular matrix surrounding the cells, the pericellular matrix (PCM), transduces the biophysical signals that cells ultimately experience. While it is known that the PCM is altered in disc degeneration, whether it disrupts normal osmotic mechanotransduction has yet to be determined. Thus, our objectives were to assess: (1) whether dynamic osmotic conditions stimulate biosynthesis in nucleus pulposus cells, and (2) whether pericellular heparan sulfate proteoglycans (HSPGs) modulate the biosynthetic response to osmotic loading.

Methods: Bovine nucleus pulposus cells isolated with retained PCM were encapsulated in 1.5% alginate beads and treated with or without heparinase III, an enzyme that degrades the pericellular HSPGs. Beads were subjected to 1 h of daily iso-osmotic, hyper-osmotic, or hypo-osmotic loading for 1, 2, or 4 weeks. At each timepoint the total amount of extracellular and pericellular sGAG/DNA were quantified. Additionally, whether osmotic loading triggered alterations to HSPG sulfation was assessed via immunohistochemistry for the heparan sulfate 6-O-sulfertransferase 1 (HS6ST1) enzyme.

Results: Osmotic loading significantly influenced sGAG/DNA accumulation with a hyper-osmotic change promoting the greatest sGAG/DNA accumulation in the pericellular region compared with iso-osmotic conditions. Heparanase-III treatment significantly reduced extracellular sGAG/DNA but pericellular sGAG was not affected. HS6ST1 expression was not affected by osmotic loading.

Conclusion: Results suggest that hyper-osmotic loading promotes matrix synthesis and that modifications to HSPGs directly influence the metabolic responses of cells to osmotic fluctuations. Collectively, results suggest degeneration-associated modifications to pericellular HSPGs may contribute to the altered mechanobiology observed in disease.

Keywords: heparan sulfate proteoglycan; intervertebral disc; mechanotransduction; nucleus pulposus; osmotic; pericellular matrix.

FIGURE 1

Degradation of pericellular sGAG by heparinase III: (A) Toluidine Blue staining demonstrating a reduction in pericellular staining with increased heparinase III concentration. (B) 3G10 heparinase III neo‐epitope and Hoescht stain for untreated and 0.1 U/ml heparinase treated cells. (C) Pericellular sGAG measured via modified DMMB assay after 4 h degradation with heparinase III. (D) Quantification of 3G10 stain intensity normalized to Hoescht and untreated cells. Bar denotes a significant difference. Scale bars: 10 μm.

FIGURE 2

Effect of osmotic loading on sGAG synthesis: sGAG/DNA accumulation is shown for beads that were cultured in static osmotic conditions (400→400) or experienced a daily hypo‐osmotic (400→200) or hyper‐osmotic (400→600) change for 1, 2, or 4 weeks. Beads were either left intact (untreated) (A,B) or treated with heparninase III (C,D) prior to culture. The amount of sGAG/DNA is broken down into the extracellular (A, C) and pericellular (B, D) regions for each osmotic loading condition. The global effect of osmotic loading on sGAG/DNA content was significant for both ECM (p = 0.037) and PCM (p < 0.001). Averaged across time and treatment, each pairwise difference between osmotic loading levels in the PCM were significant while the ECM showed significant differences only between the hypo‐ and hyper‐osmotic groups.

FIGURE 3

Effect of heparinase treatment on sGAG synthesis: sGAG/DNA accumulation is shown for beads that were treated with or without Heparinase III in each osmotic condition. Beads were cultured in static osmotic conditions (C,D) or experienced a daily hypo‐osmotic (A,B) or hyper‐osmotic (E,F) change for 1, 2, or 4 weeks. The amount of total sGAG/DNA is broken down into the extracellular (A,C,E) and pericellular (B,D,F) regions for each osmotic loading condition. Averaged across time and osmotic condition, mean sGAG/DNA was found to be significantly lower in heparinase treated ECM beads, however, no significant difference by heparinase treatment was observed in the PCM.

FIGURE 4

HS6ST1 Staining: Representative images of immunohistochemistry staining for the heparan sulfate 6‐O‐sulfotransferase (HS6ST1) enzyme. Each row represents one experimental condition (e.g., cells treated with or without heparinase III enzyme and cultured in on of the three osmotic culture conditions (hypo‐, hyper‐, or static). Each column represents a different timepoint of 1, 2, or 4 weeks in culture. Scale bar: 100μm. Inserts: 25μm