The responses of nucleus pulposus cells to pressure and ultrasound stimulation

Abstract

A cellular stimulation device with a pressurized chamber is developed to investigate the effect of ultrasound and pressure fluctuation on nucleus pulposus (NP) cells. The pressurized chamber is designed to emulate the in vivo environment of intervertebral discs, which are under dynamic pressure, and to emulate impact during sports and exercise. Both hydrostatic pressure and ultrasound stimulation increase phosphorylation of ERK (pERK) in NP cells, and promote its translocation into nucleus. This increase in pERK levels might be activated through calcium signaling pathways as intracellular calcium in NP cells was strongly elevated by pressure changes.

Dynamic Pressure Stimulation Upregulates Collagen II and Aggrecan in Nucleus Pulposus Cells Through Calcium Signaling

“…It has been demonstrated previously by our lab that pressure fluctuation, either increase or decrease, can lead to elevation of intracellular calcium in NP cells. 14 In this work, we extended our previous study to examine possible mechanor-eceptors like focal adhesion related integrins and mechano-sensitive ion channels. During the calcium imaging recording, fluorescent images with Fura-2 staining before, during, and after pressurization (upper panel in Figure 3A) showed a clear elevation of intracellular calcium by pressure increase ( t = 25 seconds) and also by pressure decrease ( t = 75 seconds).…”

“…ERK phosphorylation is an indication of cellular activation caused by mechanosensitive responses of many cells, 23 including ultrasound stimulation of NP cells. 14 Therefore, we tested whether ERK was phosphorylated upon pressure loading. We found p-ERK was responsive to pressure stimulation and consistent with SOX9 where there was an enhanced level in the dynamic pressure samples (Figure 2D).…”

“…The second is a live imaging chamber with a pressurized enclosure (Figure 1B) developed previously by our lab. 14 The chamber space was pressurized within two coverslips of 0.4 mm thick and 24 mm in diameter. Cells were cultured on the bottom coverslip.…”

“…Based on our previous studies, 13,14,16,17 mechanosensitive ion channels and focal adhesion receptors like integrins are presumably involved in mediating the cell signaling triggered by pressure stimulations. For ion channels, we used ruthenium red (a TRP channels inhibitor, 18 30 μM, Sigma Aldrich, St. Louis, MO), an extracellular calcium chelator EGTA (5mM, Sigma Aldrich, St. Louis, MO), and gadolinium (a Piezo inhibitor, 19 30 μM, Sigma Aldrich, St. Louis, MO).…”

“…12 While ECM synthesis by NP cells upon physical stimulation is the desirable outcome in this study, reliable measurements of ECM proteins often require days of stimulation. Thus, we establish a baseline of dynamic pressure stimulation using the following cellular assays: (1) an immediate response by live cell imaging of intracellular calcium, 13,14 (2) cell signaling markers represented by phosphorylation of ERK (p-ERK), and (3) a chondrogenic lineage differentiation marker SOX9 Western analysis. We demonstrated that these cellular responses can be upregulated in NP cells by pressure stimulation and investigated which response can be related to the synthesis of Collagen II and aggrecan.…”