The mechano-response of murine annulus fibrosus cells to cyclic tensile strain is frequency dependent

This study quantified how annulus fibrosus (AF) cells from healthy murine intervertebral discs respond to cyclic tensile strain (CTS) as a function of loading frequency, and identified mechanosensitive signaling and gene-expression programs. Using a cell-type-specific reporter mouse to distinguish NP vs AF cells, primary AF cells were isolated, expanded, and cultured on deformable silicone membranes. The bioreactor strain field was validated by motion tracking, demonstrating that a programmed 3.5 mm displacement produced uniform ~6% biaxial strain across the culture membrane. AF cells maintained AF marker expression in culture and responded to CTS by frequency-dependent cytoskeletal remodeling: higher frequencies (1.0–2.0 Hz) significantly increased stress fiber formation. Signaling analysis showed that 2.0 Hz CTS induced a significant but transient ERK1/2 phosphorylation increase at 15 minutes (not sustained at 30 minutes), while p38 activation showed non-significant trends. Gene expression responses were also frequency-dependent: 0.1 Hz increased ECM/anabolic and remodeling genes (Acan,Prg4,Col1a1,Mmp3), 1.0 Hz increased Acan and the mechanosensitive/proliferation-linked gene Myc and strongly increased Tnfα at later timepoints, and 2.0 Hz produced the most robust response with increased Acan,Prg4,pro-inflammatory and mechanosensitive genes (Tnfα,Cox2,Myc,Fos) and increased expression of mechanoreceptor candidates (Itgα5,Itgβ1,Trpv4). Overall, the findings support a “window” of loading parameters for IVD homeostasis and suggest that altered loading frequency can directly drive matrix remodeling and inflammatory signaling in AF cells.