Fig 1.
Customized two degree-of-freedom (i.e., compression and torsion) bioreactor to maintain disc explants alive and to apply repeated mechanical loading for two days.
A. close-up view of single station harboring a bovine coccygeal IVD soaked in culture media. Biocompatible materials are enox aluminium (black), polyoxymethylene (POM, white parts) and glass with a “press-fit” design and silicon rings (black rings) to ensure no leakage between glass and POM. B. 4-unit design arranged in 5% CO2 and 60% humidity incubator. C. Scheme of control of uniaxial compression and axial torsion using fluidic muscle and servo-controlled valve. D. Close-up view of serrated titanium plate surface, which grasps IVD and keeps it in place and ensures nutrition diffusion to the bony endplate.
Fig 2.
Conditions applied to disc explants.
Heat-shock was applied on discs as positive control (A). Compressive loading of different modes was applied at physiological range 0.35 MPa (static loading) and 0.35 ± 0.25 MPa (dynamic loading) for two h (B). For repeated loading, compressive dynamic loading was for two h per day for two days (C). Samples were retrieved for analysis at different time points: 1. Control, 2. DL (right after two h of dynamic loading), 3. DL+Rest (22 h after dynamic loading), 4. DL*2 (right after two h of dynamic loading on Day two), 5. DL+Rest*2 (22 h after dynamic loading on Day two).
Table 1.
Primers used for quantitative real time PCR in organ culture study.
Fig 3.
Cell activity and cytoskeleton were maintained after each condition.
Cell activity was measured by resazurin assay (A-B) and cytoskeleton was visualized by f-actin staining (C-J) for NP and AF after each condition. Values are normalized to control without loading and represented in mean ± SEM. Control: Control discs without loading, HS: Heat-Shock, SL: Static Loading, DL: Dynamic loading, DL+Rest: Dynamic loading with Resting, DL*2: dynamic loading for two days, DL+Rest*2: dynamic loading and resting for two days. NP: Nuclues pulposus, AF: Annulus fibrosus. N = 5. RFU: relative fluorescence unit.
Fig 4.
Relative gene expression after different loading types.
Gene expressions are in log of fold change normalized to control without loading and represented in means ± SEM. SL: Static Loading, DL: Dynamic Loading. * = statistical significant difference with p < 0.05. N = 5 (from 3 animals) compared to control.
Fig 5.
Expressions of HSP70 and HSF1 at the protein level shown by immunofluorescence staining.
Representative images from nucleus pulposus after different conditions are shown. Tissues were immuno-labeled by antibody for HSP72, the inducible form of HSP70 (A-C) and HSF1 (E-G) and nuclei were labeled with DAPI. Signal intensity was quantified and presented as signal intensity per volume for each nucleus. 31 to 47 nuclei were analyzed for HSP70 and 23 to 42 nuclei were analyzed for HSF1 in each group. Control: without loading, SL: Static Loading, DL: Dynamic Loading. * = statistical significant difference with p < 0.05.
Fig 6.
Relative gene expression after repeated dynamic loading and resting.
Gene expressions are presented in log of fold change normalized to control without loading and represented in means ± SEM. DL: after dynamic loading, DL+Rest: after dynamic loading followed by resting, DL*2: after dynamic loading for 2 days, DL+Rest*2: after dynamic loading followed by resting for two days. N = 5. * = statistical significant difference with p < 0.05. N = 5 (from 5 animals) compared to control.