Krull Carly M, Rife Jordan, Klamer Brett, Purmessur Devina, Walter Benjamin A
Department of Biomedical Engineering The Ohio State University Columbus Ohio USA.
Department of Biomedical Informatics, Center for Biostatistics The Ohio State University Columbus Ohio USA.
JOR Spine. 2022 Jun 3;5(2):e1209. doi: 10.1002/jsp2.1209. eCollection 2022 Jun.
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.
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.
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.
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.
日常生理负荷会导致椎间盘（IVD）水合作用的波动；因此，其中的细胞会经历其渗透环境的周期性变化。这些渗透波动被描述为一种将力学与生物学联系起来的机制，并且先前已证明其可促进软骨细胞的生物合成。然而，这一现象在椎间盘内尚未得到充分研究。此外，细胞周围的特殊细胞外基质，即细胞周基质（PCM），可转导细胞最终所经历的生物物理信号。虽然已知在椎间盘退变过程中PCM会发生改变，但它是否会破坏正常的渗透机械转导尚待确定。因此，我们的目标是评估：（1）动态渗透条件是否会刺激髓核细胞的生物合成，以及（2）细胞周硫酸乙酰肝素蛋白聚糖（HSPG）是否会调节对渗透负荷的生物合成反应。
将分离出并保留PCM的牛髓核细胞封装在1.5%的海藻酸钠珠中，并用或不用肝素酶III（一种可降解细胞周HSPG的酶）进行处理。将珠子每天进行1小时的等渗、高渗或低渗负荷处理，持续1、2或4周。在每个时间点，对细胞外和细胞周sGAG/DNA的总量进行定量。此外，通过对硫酸乙酰肝素6-O-硫酸转移酶1（HS6ST1）进行免疫组织化学检测，评估渗透负荷是否会引发HSPG硫酸化的改变。
渗透负荷显著影响sGAG/DNA的积累，与等渗条件相比，高渗变化促进了细胞周区域中最大的sGAG/DNA积累。肝素酶III处理显著降低了细胞外sGAG/DNA，但细胞周sGAG不受影响。HS6ST1的表达不受渗透负荷的影响。
结果表明高渗负荷促进基质合成，并且HSPG的修饰直接影响细胞对渗透波动的代谢反应。总体而言，结果表明与退变相关的细胞周HSPG修饰可能导致疾病中观察到的机械生物学改变。
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