Dados do Trabalho

Título

HYDROSTATIC PRESSURE MIMICKING DIURNAL SPINAL MOVEMENTS MAINTAINS ANABOLIC TURNOVER IN BOVINE NUCLEUS PULPOSUS CELLS IN VITRO

Objetivo

With the goal of developing strategies to promote the regeneration of the nucleus pulposus (NP), the present study tried to identify the biological effects of hydrostatic (HP) and osmotic pressures on NP cells. The study hypothesis was that a repetitive regimen of cyclic HP followed by constant HP in high-osmolality medium would increase anabolic molecules in NP cells.

Metodologia

Bovine nucleus pulposus cells/clusters were enclosed within semi-permeable membrane pouches and incubated under 6 different regimens of hydrostatic pressure and osmotic pressure (fig.2). 1- LOsm/No HP group, low-osmolality medium at 320 mmol/kg H2O for 18 d; 2-HOsm/No HP group, high-osmolality medium at 450 mmol/kg H2O for 18 d; 3- HOsm/HP group, cyHP at 0.2-0.7 MPa, 0.5 Hz for 2 d in HOsm followed by coHP at 0.3MPa for 1 in HOsm, repeated 6 times for 18 d; 4- HOsm/cyHP group, cyHP at 0.2-0.7 MPa, 0.5 Hz in HOsm for 18 d; 5- HOsm/coHP group, coHP at 0.3 MPa in HOsm for 18 d; 6- H-LOsm/HP group: cyHP at 0.2-0.7 MPa, 0.5 Hz in HOsm for 2 d followed by coHP at 0.3 MPa in LOsm for 1 d, repeated 6 times for 18 d. The pouches were placed in a pressure chamber, which was installed in a pressure/perfusion culture system.The pouches were harvested for RNA extraction, immunohistology as well as sGAG and DNA assays at 3, 12, and 18 d (fig.1).

Resultados

NP cells showed a significantly increased expression of anabolic genes over time (fig.4): aggrecan, chondroitin sulfate N-acetylgalactosaminyltransferase 1, hyaluronan synthase 2, collagen type 2 (p < 0.05). In addition, the expression of catabolic or degenerative genes (matrix metalloproteinase 13, collagen type 1) and cellular characteristic genes (proliferating cell nucleic antigen, E-cadherin) was suppressed. The amount of sulfated glycosaminoglycan increased significantly at day 18 compared to day 3 (p < 0.01). Immunostaining revealed deposition of extracellular-matrix molecules and localization of other specific molecules corresponding to their genetic expression.

Conclusões

The present study demonstrated that a repetitive regimen of HOsm/HP in NP cells increased gene expression and accumulation of ECM molecules, increased or maintained the proliferation capability and phenotypes of NP cells as well as suppressed the expression of degenerative/catabolic molecules over 18 d. An improved understanding of how cells respond to physicochemical stresses will help to better treat the degenerating disc using either cell- or gene-based therapies as well as other potential matrix-enhancing therapies. Efforts to apply these tissue-engineering and regenerative-medicine strategies will need to consider these important physicochemical stresses that may have a major impact on the survivability of such treatments.

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