Poster Presentation 50th International Society for the Study of the Lumbar Spine Annual Meeting 2024

Introduction

We previously reported that chondroitin sulfate proteoglycan (CSPG), a major component of the intervertebral disc matrix, stimulates anabolic turnover in nucleus pulposus (NP) cells incubated under repetitive cyclic hydrostatic pressure (HP) followed by constant hydrostatic pressure in high-osmolality medium in vitro. However, fragmented chondroitin sulfate glycosaminoglycan (CS) diminished anabolic turnover. This difference becomes critical when addressing therapeutic strategies for degeneration in intervertebral discs. We hypothesize that the large molecules of CSPG stimulate anabolic turnover in NP cells in a dose-dependent manner. We tested this hypothesis by comparing CS and CSPG extracted using bovine NP tissues under the aforementioned culture conditions.

Methods

Bovine NP (bNP) tissues were harvested from tails (n=6), bNP cells were isolated with collagenase, then pre-incubated on 1.5% cell culture–grade agarose-coated 6-well plates in DMEM/F12 for 2 days. After careful collection of the NP cells/clusters, 1.0×10⁵ NP cells were enclosed in a pouch made of semipermeable membrane with 0, 4, or 16 mg/mL CSPG (extracted from bovine NP tissue with guanidine and validated with SDS-PAGE). The pouches were incubated under two culture conditions: 1) Control (no HP): atmospheric pressure; and 2) HP: cyclic HP (0.2-0.7 MPa, 0.5 Hz) for 2 days followed by constant HP (0.3 MPa) for 1 day, repeated 4 times over 12 days. The pouches were suspended in DMEM/F12 medium with 10% FBS and antibiotics at 37°C, 5% CO2, and 3% O2. Cells were collected at 3, 6, and 12 days and subjected to gene expression assay by RT-PCR and immunohistological evaluation. The probes included typical ECM molecules, aggrecan core protein (Acan), chondroitin sulfate N-acetylgalactosaminyltransferase 1 (Csgalnact1), hyaluronan synthetase 2, collagen type-I (Col-1), collagen type-II (Col-2); a catabolic molecule, matrix metalloproteinase-13 (Mmp13); and cellular molecules, tissue inhibitor of metalloproteinases-2 (Timp2). Immunohistochemical staining and lectin staining indicates hyaluronan and CS were performed at 12 days to confirm the accumulation of keratan sulfate (KS) and MMP13 in the bNP cells/cluster. One-way ANOVA was used to identify statistical differences (p<0.05).

Results

Extracted CSPG maintained high molecular weight in acellular pouches for 12 days and retained within the pouch validated with SDS-PAGE and dimethyl-methylene blue assay. Anabolic molecules: Acan, Col-2, and Csgalnact1 were upregulated under HP by 12 days compared to control (P < 0.01, P < 0.05, P < 0.05, Fig. 1), whereas the catabolic molecule Mmp13 was suppressed defined as low intensity of MMP13 molecule (Fig. 2). With CSPG, more anabolic molecules were upregulated than without CSPG illuminated with Wisteria Floribunda lectin-Fluorescein and Wheat germ agglutinin lectin. However, there was no distinct differences between 4 and 16 mg/ml of CSPG. Under HP and with/out CSPG, intense MMP-13 staining was not seen at 12 days (Fig. 2). KS in bNP cells/clusters under HP showed more intense staining at 12 days compared to 3 days.

Conclusions

High molecular weight CSPG extracted from bovine NP promotes anabolic turnover and suppresses catabolic turnover in bNP cells under HP. High molecular weight CSPG or its analogue have the potential to promote regeneration of NP cells under physiological conditions.