LSCS is of interest to spinal physicians due to its increasing prevalence and clinical significance. Prior reports have indicated that LFH and LSCS are significantly associated with various clinical symptoms including back pain and radiculopathy with or without neurogenic claudication.9,12−16 The LF is a ligamentous structure lying over the dorsal aspect of the central spinal canal, LFH directly results in physical narrowing of the spinal canal leading to clinical LSCS. To discover a novel therapeutic candidate targeting LFH, it is important to understand the pathomechanism of LFH. Elucidating the role of mechanical stress on the LF is critical, and inflammation/angiogenesis of the LF following mechanical stress are hallmarks of LFH.
We previously reported that inflammation and subsequent angiogenesis are involved in the pathomechanism of LFH in vitro, indicative of close relationships among inflammation, angiogenesis, and LFH.6 In a follow-up study of the association between in vitro and clinical data, we discovered links among mechanical stress, angiogenesis, and LFH.7 However, these studies were limited in that mechanical stress was not loaded directly onto the LR cells. Instead, the effects of mechanical stress were evaluated indirectly based on radiological findings. In this study, we developed a novel mechanical stress loading device with multidirectional torsion that mimics the mechanical load on LF tissue in vivo. Rather than inducing inflammation by transforming growth factor-β1 (TGF- β1) or interleukin-1β (IL-1β), we used mechanical stress on the LF and believe it reflects the effects of mechanical stress on LFH.
Our results demonstrated that multi-torsional MSS load for 24 h under 5% stretch force stimulation resulted in an increase in IL-6 and VEGF levels. IL-6 activates neutrophils, whose adhesion and fibrosis are promoted by increased expression of ECM-regulating molecules or cytokines.17 A similar response leads to LFH after triggering inflammation in LF cells.6 An increase in IL-6 can also upregulate mRNA expression and DNA synthesis of LF cells, resulting in ossification or fibrosis.18 Our finding of a significant increase in IL-6 expression confirms that MSS loading induced inflammation in LF cells, mimicking the initial inflammatory phase of LFH. Likewise, VEGF initiates and stimulates the angiogenic cascade of LFH, and its concentration in degenerated or hypertrophied LF is significantly higher than that in healthy ligaments.6,7,19,20 MSS stimulation for 24 h resulted in marked elevation of VEGF expression in LF cells, indicating that MSS loading mimics the angiogenic cascade that occurs after an inflammatory reaction.
As well as inflammation and angiogenesis, the resultant changes in ECM-modulating factors (such as the elastin to collagen ratio) are important. Our data on ECM-regulating enzymes provide insight into the response of LF cells to mechanical stress. MMPs are endopeptidases involved in ECM homeostasis and in cell–cell interactions and angiogenesis. Significant changes or dysregulation of MMPs occur in cells during inflammation,21,22 as well as in LF fibroblasts.8,23 MMP-1 is a collagenase for all collagen subtypes, and MMP-3 a broad-spectrum proteinase that regulates activation of other MMPs.24 Elevated MMP-1 and MMP-3 levels after MSS stimulation by our novel multi-torsional stress loading device are compatible with prior reports confirming an association with LFH.25,26 This suggests the key role of mechanical stress in LFH as a result of altered ECM regulation in LF cells, indicating the dysregulation of regenerative potential and vulnerability to mechanical stress. However, Kim et al. reported increased expression of MMP-9 after inflammatory stimulation of LF cells in vitro,8 and Lakemeier et al. indicated that MMP-9 expression is higher in LFH tissue.19 TIMPs also regulate ECM homeostasis, and TIMP-1 and TIMP-2 play key roles in fibrosis in various cell types by increasing proliferation. Park et al. hypothesized that TIMP-1 and TIMP-2 influence LFH by increasing ECM density and promoting hypertrophy by suppressing MMP activities.4 This hypothesis was confirmed by the significant association between elevated TIMP-1 and TIMP-2 expression in LF fibroblasts and spinal stenosis, a reproducible finding of several different experiments of various methods.27 This is compatible with our TIMP-1 and TIMP-2 expression data.
Mechanical stress is a key factor in LFH, as confirmed by in vitro28–32, in vivo10,11, and clinical studies7. Chao et al. developed an in vitro method of loading stress on LF cells by centrifuging them in a horizontal microplate rotor.32 Nakamura et al. loaded a cyclic uniaxial load to LF cells by attaching the cell culture chamber to a stretching apparatus,28 and Nakatani et al. loaded mechanical stress using a vacuum unit to pull a flexible cell culture plate from the center.30 It is meaningful that centrifugal and cyclic one-dimensional mechanical forces on LF fibroblasts affected the mechanostress pathway. However, because one- and two-dimensional forces are unlike that on LF tissue in vivo, the accuracy of the model is unknown. Therefore, it is significant that we developed a reproducible repetitive mechanical stress loading device that recapitulates the mechanical stress on LF cells. The device will be used to provide insight into the role of direct mechanical stress on LFH in vitro and the cells’ fate after mechanical stress loading.