In vitro nucleus pulposus tissue model with physicochemical stresses

Abstract

Intervertebral discs (IVDs) are exposed to changes in physicochemical stresses including hydrostatic and osmotic pressure via diurnal spinal motion. Homeostasis, degeneration, and regeneration in IVDs have been studied using in vitro, ex vivo, and animal models. However, incubation of nucleus pulposus (NP) cells in medium has limited capability to reproduce anabolic turnover and regeneration under physicochemical stresses. We developed a novel pressure/perfusion cell culture system and a semipermeable membrane pouch device for enclosing isolated NP cells for in vitro incubation under physicochemical stresses. We assessed the performance of this system to identify an appropriate stress loading regimen to promote gene expression and consistent accumulation of extracellular matrices by bovine caudal NP cells. Cyclic hydrostatic pressure (HP) for 4 days followed by constant HP for 3 days in high osmolality (HO; 450 mOsm/kg H 2 O) showed a trend towards upregulated aggrecan expression and dense accumulation of keratan sulfate without gaps by the NP cells. Furthermore, a repetitive regimen of cyclic HP for 2 days followed by constant HP for 1 day in HO (repeated twice) significantly upregulated gene expression of aggrecan (P < .05) compared to no pressure and suppressed matrix metalloproteinase-13 expression (P < .05) at 6 days. Our culture system and pouches will be useful to reproduce physicochemical stresses in NP cells for simulating anabolic, catabolic, and homeostatic turnover under diurnal spinal motion.

Augmented Chondroitin Sulfate Proteoglycan Has Therapeutic Potential for Intervertebral Disc Degeneration by Stimulating Anabolic Turnover in Bovine Nucleus Pulposus Cells under Changes in Hydrostatic Pressure

“…The HP at physiological range (0.3–1.0 MPa) increases ECM synthesis in bovine, dog, and rabbit NP cells in vitro [ 28 , 29 , 30 ], while higher HP (>2.5 MPa) induced a catabolic trend, increasing MMP3 activity and decreasing matrix synthesis [ 31 , 32 ]. We developed and applied a repetitive regimen of cyclic HP followed by constant HP at high osmolality to mimic physiological changes in intradiscal pressure and to reproduce the microenvironment in disc homeostasis [ 11 ].…”

“…The OP is generated by water content absorbed within the tissue [ 6 , 7 ] and influences bNP cell metabolism [ 10 , 11 ]. Therefore, to reproduce the microenvironment in IVD homeostasis, we chose CSPG and HA in this study, both of which are capable of absorbing abundant interstitial fluid.…”

“…The NP is also exposed to changes in hydrostatic pressure (HP) in daily cycles owing to weight bearing in the upright position and off-loading in the recumbent position [ 8 , 9 ]. Our latest studies demonstrate that a repetitive regimen of cyclic HP followed by constant HP at high osmolality stimulated anabolic gene upregulation and dense accumulation of ECM in bovine NP (bNP) cells [ 10 , 11 ]. Therefore, the combination of dynamic HP and intradiscal high OP is required in maintaining bNP-cell homeostasis [ 10 , 12 ].…”

“…The concept behind our novel material-based therapy is to implant extracted CSPG into degenerated NP, which prevents progressive degeneration and ultimately promotes regeneration. Based on our previous studies [ 10 , 11 ], we hypothesized that augmentation of CSPG stimulates the anabolic capability of bNP cells under repetitive changes in HP at high osmolality. To test that hypothesis, we incubated isolated bNP cells with CSPG or HA under a repetitive regimen of cyclic HP at 0.2–0.7 MPa, 0.5 Hz for 2 days, followed by constant HP at 0.3 MPa for 1 day at high osmolality (450 mOsm/kg H 2 O) for up to 12 days, and compared the gene expression and immunohistology of metabolic markers in the bNP cells.…”

Improving reproducibility in spine research

“…Of interest, they noted differences in animal samples between the United States and United Kingdom suggesting potential mechanical differences between bovine breeds or maintenance conditions of the animals. Shekouhi et al 5 shared an in silico approach for evaluating the spinal implants of growing rods used for scoliosis correction surgeries. This in silico model enables the simulation of clinical parameters which have not been previously possible. Takeoda et al 6 developed and characterized a novel cell culture system using a semipermeable membrane pouch device to test the effects of culturing bovine nucleus pulposus cells under constant and cyclic hydrostatic pressure under high osmolality. This culture system and pouches have potential use for testing the effects of motion on biochemical changes in the nucleus pulposus cells in vitro. Di Pauli et al 7 compare five different methods to determine neutral zone of rat spinal motion segments using two different load deflection profiles demonstrating poor agreement between methodologies, highlighting the need for further work to identify recommended methods which are most sensitive to disc degeneration and can be used in treatment studies.…”