Takeoka Yoshiki, Kang James D, Mizuno Shuichi
Department of Orthopaedic Surgery Brigham and Women's Hospital and Harvard Medical School Boston Massachusetts USA.
JOR Spine. 2020 Jul 1;3(3):e1105. doi: 10.1002/jsp2.1105. eCollection 2020 Sep.
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 HO) 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 ( < .05) compared to no pressure and suppressed matrix metalloproteinase-13 expression ( < .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.
椎间盘（IVD）通过脊柱的日常运动承受包括静水压力和渗透压在内的物理化学应力变化。人们已使用体外、离体和动物模型研究了椎间盘的稳态、退变和再生。然而，在培养基中培养髓核（NP）细胞在物理化学应力下再现合成代谢周转和再生的能力有限。我们开发了一种新型的压力/灌注细胞培养系统和一种半透膜袋装置，用于封闭分离的NP细胞，以便在物理化学应力下进行体外培养。我们评估了该系统的性能，以确定一种合适的应力加载方案，以促进牛尾NP细胞的基因表达和细胞外基质的持续积累。在高渗（HO；450 mOsm/kg HO）条件下，先进行4天的循环静水压力（HP），然后进行3天的恒定HP，显示出NP细胞中聚集蛋白聚糖表达上调以及硫酸角质素密集积累且无间隙的趋势。此外，在HO中，先进行2天的循环HP，然后进行1天的恒定HP（重复两次）的重复方案，与无压力相比，在第6天时显著上调了聚集蛋白聚糖的基因表达（<0.05），并抑制了基质金属蛋白酶-13的表达（<0.05）。我们的培养系统和袋子将有助于在NP细胞中再现物理化学应力，以模拟脊柱日常运动下的合成代谢、分解代谢和稳态周转。
