Introduction: We recently demonstrated that bovine nucleus pulposus (bNP) cells produced more cartilaginous extracellular matrices (ECMs) under repetitive cyclic hydrostatic pressure (HP) followed by constant HP in high-osmolality culture medium compared to no HP.# These findings indicate that NP cells need to be under intradiscal pressure, which must be held by the annulus fibrosus (AF) to maintain homeostasis and to promote intervertebral disc regeneration. We hypothesized that anabolic turnover in AF cells is maintained by HP as seen in NP cells. We assessed the effects of HP on production of ECMs by AF cells with qPCR using bAF cells. Since AF is exposed to compressive and tensile stresses, we evaluated the effects of HP in bAF cells isolated from inner or outer AF.

Methods: Fresh bovine tails were purchased from a local slaughterhouse (USDA certified). Inner AF and outer AF were harvested from the caudal IVDs (Fig. 1A), followed by digestion in 0.15% collagenase. The AF cells were rinsed, and 5 x 10⁵ cells/25 µl were enclosed in a semipermeable membrane pouch (1 x 35 mm hollow fiber, 500 kD molecular cut-off)(Fig. 1B). The pouches were incubated with cyclic HP at 0.2 to 0.7 MPa, 0.5 Hz for 2 days followed by constant HP at 0.3 MPa for 1 day at 3% O₂ and 5% CO₂ repeated twice over 6 days using a pressure culture system (Fig. 2). For the no-HP control, pouches were suspended in culture medium with a stirrer. These pouches were incubated in Dulbecco minimum essential medium/Ham’s F12 media with 10% fetal bovine serum and antibiotics at high osmolality (450 mOsm/kg H₂O) adjusted with NaCl. The cells were harvested on days 3 and 6, and gene expression of typical ECM molecules: Acan, Col-1, Col-2, Eln, and the catabolic molecule Mmmp13 were evaluated with RT-PCR (n=3).

Results: In inner AF cells, the expression of Acan and Col-2 were higher under HP and high OP conditions compared to no HP (Fig. 3A). On the other hand, in outer AF cells, the expression of Acan, Col-1, and Col-2 were downregulated with HP compared to no HP (Fig. 3B). In inner and outer AF cells, Eln expression was suppressed with HP, and the expression of Mmp13 was higher with HP than no HP on day 3.

Discussion: Inner and outer AF cells showed opposite gene expression profiles on ECM molecules in response to HP. Since inner AF connects to NP, inner AF may contain more NP-like cells compared to outer AF. On the other hand, outer AF expressed more fibrous molecules e.g., Col-1, Eln compared to inner AF cells. Physicochemical stresses in this study did not include deviatoric stress, which alters cell shape. Since outer AF cells are exposed mainly to deviatoric stress due to less hydration, we expect that combined HP and deviatoric stress will modify profiles obtained from current regimen of HP and high OP. Opposite characteristics in ECM production by inner and outer AF cells suggest multiple approaches (combined or steps) to repair the wound/incision.