Intervertebral disc (IVD) degeneration is a major risk factor of low back pain. It is defined by a progressive loss of the IVD structure and functionality, leading to severe impairments with restricted treatment options due to the highly demanding mechanical exposure of the IVD. Degenerative changes in the IVD usually increase with age but at an accelerated rate in some individuals. To understand the initiation and progression of this disease, it is crucial to identify key top-down and bottom-up regulations’ processes, across the cell, tissue, and organ levels, in health and disease. Owing to unremitting investigation of experimental research, the comprehension of detailed cell signaling pathways and their effect on matrix turnover significantly rose. Likewise, in silico research substantially contributed to a holistic understanding of spatiotemporal effects and complex, multifactorial interactions within the IVD. Together with important achievements in the research of biomaterials, manifold promising approaches for regenerative treatment options were presented over the last years. This review provides an integrative analysis of the current knowledge about (1) the multiscale function and regulation of the IVD in health and disease, (2) the possible regenerative strategies, and (3) the in silico models that shall eventually support the development of advanced therapies.
Stem cell–based regenerative strategies are promising for intervertebral disc degeneration. Stimulation of bone-marrow- and adipose-derived multipotent stem cells with recombinant human growth differentiation factor 6 (rhGDF6) promotes anabolic nucleus pulposus like phenotypes. In comparison to mesenchymal stem cells, adipose-derived multipotent stem cells exhibit greater NP-marker gene expression and proteoglycan-rich matrix production. To understand these response differences, we investigated bone morphogenetic protein receptor profiles in donor-matched human mesenchymal stem cells and adipose-derived multipotent stem cells, determined differences in rhGDF6 signalling and their importance in NP-like differentiation between cell populations. Bone morphogenetic protein receptor expression in mesenchymal stem cells and adipose-derived multipotent stem cells revealed elevated and less variable expression of BMPR2 in adipose-derived multipotent stem cells, which corresponded with increased downstream pathway activation (SMAD1/5/8, ERK1/2). Inhibitor studies demonstrated SMAD1/5/8 signalling was required for rhGDF6-induced nucleus-pulposus-like adipose-derived multipotent stem cell differentiation, while ERK1/2 contributed significantly to critical nucleus pulposus gene expression, aggrecan and type II collagen production. These data inform cell regenerative therapeutic choices for intervertebral disc degeneration regeneration and identify further potential optimisation targets.
Purpose: Current clinical trials have attempted the injection of mesenchymal stromal cells (MSCs) into degenerated intervertebral discs (IVDs) to restore height and function. However, retrieval of tissue for intensive characterization of the mechanisms behind any apparent regenerative effect is very limited. In the present research study, a 3D culture system has been developed to act as an ex vivo model for the intervertebral disc that mimics the native environment physically and chemically. The development of a cellularized nucleus pulposus (NP) analogue will help to unravel the cell-cell signaling between MSCs and NP cells in an in vitro setting, and enable identification of specific immunomodulatory targets that are regulated by MSCs. Methods: Alginate co-culture construct creation: Alginate constructs were created in two steps; beads were created by external gelation followed by bead encapsulation within an internally gelling cylindrical geometry. Alginate droplets extruded through a needle into a solution of 102 mM CaCl 2 were left to crosslink for 10 min. The beads were rinsed in PBS and added in a 1:1 volumetric ratio to a solution of alginate with 30 mM CaCO 3 . Glucono-del-lactone (GDL, 60 mM) was added; the combination was mixed to create 3 mm diameter  3 mm height cylindrical constructs with a central bead ( Figure A, B). The addition of cells in UV-sterilized alginate requires little modification to the gelation protocol; cells at greater than 2  10 6 cells/mL concentration are added before crosslinking. Stimulative and degenerative media formulation: Human NP cells (passage 3, ScienCell), were seeded at a concentration of 5000 cells/ cm 2 into poly-l-lysine coated 96 well plates. Base media (DMEM-lg, 10% FBS, 1% ABAM) was combined with variations of pH, osmolarity and growth factors (n ¼ 6) to replicate healthy or degenerative conditions. Cells were cultured for 14 days in 5% O 2 to determine ideal conditions for MSC:NPC co-culture. Cells were assayed and imaged at various timepoints over the 14 day period. Media reserved from the wells was analyzed for ECM and inflammatory factor content via ELISA. Results: Three dimensional constructs of varying concentrations were optimized; overall, 1.2% alginate beads encased in 2% alginate showed 93% bead retention rate ( Figure 1AeC). Encapsulated hNPCs showed no migration between the two constructs over 14 days in culture and full bead separation was achieved over the full time scale, reinforcing the construct as a separatable 3D co-culture method ( Figure 1C, D). The addition of the degenerative growth factors TNFa and IL-1b as well as the adjustment of media pH to degenerative levels (pH 6.8) caused the hNPCs to decrease in size and proliferate significantly higher than control levels. Additions of any combination enhanced proliferation over base media control; brightfield images show an appearance change as well, which may indicate phenotype shift. Stimulative factors such as increased osmolarity with the growth factor FGF-2 caused a reduction in prolife...
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