Figure 1.
Constructs were press-fitted into custom made PTFE wells such that the bottom 2 mm of the construct thickness was confined.
Figure 2.
Oxygen tension is modulated by the cell seeding density and radial confinement.
MSCs encapsulated in agarose at 20×106 cells/mL were cultured for 4 days, at which point constructs were confined and allowed to equilibrate for 24 hours. (A) Oxygen concentration through the media and along the construct axis was measured for unconfined and confined constructs with representative samples of each presented (Exp). Model predictions were fit to experimental data obtained from the surface of the culture media through the depth of the construct. For clarity, only the fit through the construct is shown (Model). (B) Predicted gradients in oxygen volume fraction for unconfined and confined constructs with seeding densities of 20×106 and 50×106 cells/mL. Oxygen volume fraction corresponds to molar fraction (×10 pmol/mm3).
Figure 3.
Partial radial confinement alters pore pressure and strain distribution under dynamic compression.
Theoretical predictions of the maximum principle strain and pore pressure [MPa] for both unconfined and confined configurations during steady state dynamic compression at day 0.
Figure 4.
Radial confinement enhances sGAG accumulation in the bottom of engineered cartilaginous constructs.
MSCs encapsulated in agarose at 20×106 cells/mL were cultured for 21 days in unconfined or confined conditions. (A) The top and bottom regions of unconfined and confined free-swelling constructs were analysed for DNA, sGAG and collagen contents. (n = 4) (B) Total sGAG and collagen accumulated in the constructs (sum of top and bottom) and secreted to the media. Media data is presented as µg accumulated and secreted (n = 4) (C) Unconfined and confined constructs were stained with alcian blue for sulphated mucins, picro-sirius red for collagen and immunohistochemically for collagen type II. Representative full-depth half construct sections are shown as indicated. (n = 2) Scale bar 500 µm.
Figure 5.
Radial confinement coupled with dynamic compression enhances collagen accumulation in the top of the construct.
Agarose constructs containing MSCs at 20×106 cells/mL were confined from day 21 to day 42 of culture while 10% dynamic compression was applied. The top and bottom regions of constructs were analysed for DNA, sGAG and collagen contents. Top and bottom regions of constructs were also mechanically tested for both the equilibrium modulus and the dynamic modulus at 1 Hz. FS: free-swelling; DC: dynamic compression. (n = 4).
Figure 6.
Radial confinement coupled with dynamic compression suppresses endochondral progression.
Agarose constructs containing MSCs at 20×106 cells/mL were confined from day 21 to day 42 of culture while 10% dynamic compression was applied. Constructs at day 42 were stained with alcian blue for sulphated mucins, picro-sirius red for total collagen, alizarin red for calcific deposition and immunohistochemically for collagen type I, collagen type II and collagen type X. Representative full-depth half construct sections are shown for all but collagen type X where a representative quarter section is shown in immunofluorescence. Collagen type I and collagen type II staining is indicated in brown, with calcific deposits evident in black. FS: free-swelling; DC: dynamic compression. (n = 2) Scale bar 500 µm. Inset scale bar 100 µm.
Figure 7.
MSC response to extrinsic signals is dependent on the cell seeding density.
Agarose constructs containing MSCs at 50×106 cells/mL were confined from day 21 to day 42 of culture while 10% dynamic compression was applied. (A) The top and bottom regions of constructs were analysed for DNA, sGAG and collagen contents. Top and bottom regions of constructs were also mechanically tested for both the equilibrium modulus and the dynamic modulus at 1 Hz. (n = 4) (B) Constructs at day 42 were stained immunohistochemically for proteoglycan-4 (PRG4). FS: free-swelling; DC: dynamic compression. (n = 2) Scale bar 100 µm.