| Intervertebral disc degeneration(DDD is one of the most popular diseases undermine people's life quality in modern society. Intervertebral disc (IVD) consist of an outer annulus fibrosus (AF), which surrounds the inner nucleus pulposus (NP),the main function of IVD is connecting the adjacent vertebrae. IVD is considered as a viscoelastic solid material, which shows creep, relaxation, and drag properties during the compression process. Human have 23 IVDs, in vivo, IVD act as shock absorber and protect our spinal cord, while supporting the weight of our body.Nucleus pulposus cells play an important role in maintaining the health of IVD. It's well known that NP cells experienced dramatic changes during aging process, notably, notochord cells disappeared and replaced by the chondrocyte-like cells. Evidence from lots of studies suggests the changing of cell population was highly connected with intervertebral disc degeneration. Proteoglycan and collagen from notochord cells through swelling pressure to resist the mainly compression force. Water is expelled when the disc is loaded, the water is derived back into NP tissue while in the non-load state by the gradient osmotic pressure. The swelling behavior has been previously investigated and is linearly correlated with proteoglycan content, and the osmotic pressure was form by the highly content of negative charge ions attached on the proteoglycan.It was generally accepted that during the DDD, nucleus puplosus exhibited decreased content of water, proteoglycan and type II collagen, and increased content of type I and type III collagen and stiffness of the tissue. The stability of extracellular matrix composition is a critical factor in maintaining the health of intervertebral disc. The intervertebral discs provide free motion of the spine and serve as a central axial structure for cushioning loads. Mechanical stimulation was considered as one of the important factors inducing intervertebral disc degeneration. During human's daily life, the intervertebral disc was beard to multi-type of mechanical loading; usually it is the combination of compression, tensile, and shear stress, while compression is considered the mainly force applied on the IVD. Hydrostatic pressure is formed when the nucleus pulposus is compressed, which transit the loading evenly to the annulus fibrosus. According Newton's third law, the annulus fibrosus produce circumferential compression force onto nucleus pulposus tissue; make the nucleus pulposus tissue experience kind of confined compression. The magnitude of this kind circumferential compression force largely depends on the physical state of annulus fibrosus, such as permeability, health condition, physical structure and geometry et al. Sometimes it is not strictly confined compression, like some kind unconfined compression. In other words, in situ, the loading environment of the NP is neither fully confined nor unconfined. But until now, there is no report about the effects of confined and unconfined compression on nucleus pulposus cells extracellular matrix expression, especially the proteoglycan. In order to explore the effects of confined and unconfined compression on NP cells, we applied dynamic and static compression on the NP cells which from the rat tail. The mainly studies were:â‘ Different effects of confined and unconfined compression on NP cells proteoglycan expression.Mixed population NP cells were primary harvested from rat tail, and seeded into 2% alginate disk, after 24 hour culture, for dynamic compression, we applied 10 kPa peak stress using a sinusoidal waveform at 0.5 Hz; for static compression, displacements were applied at a constant rate and held at 20% strain. By using Real time PCR method detects the expression of proteoglycan gene expression. Aggrecan, laminin, fibronectin, glypican, biglycan, and fibromodulin were generally stimulated, and trends were similar between confined and unconfined compression. On the other hand, lumican expression was differentially regulated between confined and unconfined groups. More interestingly, trends under static loading were opposite that under dynamic loading; lumican was up-regulated in static confined compression and in dynamic unconfined compression. We hypothesis that cell and nuclei deformation may be responsible for this phenomena. By using immunofluorescence confocal microscopy, we investigated the cell/nuclei deformation in alginate disk by applying 20% deformation. Our results showed there is higher cell/nuclei deformation in unconfined condition under both dynamic and static compression. This result is conflict with the lumcian gene expression trends in dynamic and static compression.â‘¡Calculated the pressure inside confined alginate disk under static compression.To identify the potential mechanism responsible for lumican expression, we theorize that different trend of lumican expression is indicative of regulation by fluid flow within loaded alginate disks. In this model, the alginate disk is a biphasic material composed of a solid matrix phase and an interstitial fluid phase. The interstitial fluid was assumed to be intrinsically incompressible and nondissipative, while the solid matrix was assumed to be intrinsically incompressible, linearly elastic and nondissipative. Further, it was assumed that the only dissipation comes from the frictional drag of relative motion between the phases. Calculations using the linear biphasic model suggest that spatial pressure gradients exist in static confined specimens for the majority of the loading duration.â‘¢Fluid shear stress up-regulate lumican expression in nucleus pulposus cells.To verify our hypothesis, nucleus pulposus cells harvested from rat tail were seeded on the matrigel-coated slides, and applied different level of shear stress by using a parallel plate flow chamber. Real time PCR and western blot methods were used to study the lumican gene and protein expression respectively. Our data showed higher fluid shear stress (1 dyn/cm2) can up-regulate lumican both gene and protein expression. To our knowledge, this the first time to report that NP cells are sensitive to fluid shear stress and confirm that lumican expression can be regulated by short durations of exposure to fluid shear.â‘£Fluid shear stress remodels cytoskeleton in nucleus pulposus cells.To further study the potential effects of fluid shear stress on nucleus pulposus cells, we did western bolt and immunofloursence assay on nucleus pulposus cells to review the state of ERK and cytoskeleton respectively after fluid shear stress stimulation. The results showed ERK was activated by shear stress and this pattern was due, in part, to the integrity of the actin network. Also, our data suggested the actin and cytokeratin 8 distributions in nucleus pulposus cells were remolded by fluid shear stress.In summary, we used alginate 3D model to culture NP cells, and then applied dynamic and static compression in confined and unconfined chamber respectively to study the effects of different loading type on NP cells. By using biphasic linear mathematical model, we found fluid shear stress may be responsible for the different trend of lumcian expression in confined and unconfined chamber under dynamic and static compression. Using parallel plate flow chamber, we found both lumican gene and protein expression can be up-regulated by fluid shear stress. In addition, our data suggested ERK signal pathway was activated by shear stress, furthermore, shear stress also result in actin and cytokeratin 8 cytoskeleton remodeling. |
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