Identification of differential growth differentiation factor 6 signalling responses in subpopulations of human bone marrow and adipose-derived stem cells: implications for intervertebral disc regeneration

Abstract

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...

In vitro evaluation of 3D printed polycaprolactone scaffolds with angle-ply architecture for annulus fibrosus tissue engineering

“…These can include cartilage oligomatrix matrix protein [38], glypican 3 [64], integrin-binding sialoprotein [29], and fibulin 1 [65], Collagen V [66], CD146+ [67]. Lastly, adipose and bone derived MSCs have been demonstrated to have potential in AF repair [11, 68]. MSCs can be obtained with less morbidity than somatic cell types [69] and thus have more clinical significance, and should be evaluated in further studies.…”