Fig 1.
A) An authentic picture of the compression bioreactor is shown. A stepper motor drives the piston downwards and a spring (not visible) covered by a white bellows moves it upward. Parallel to the stepper motor, a digital gauge measures independently the vertical displacements of the piston. The cartridge is the housing for cell cultivation and mechanical application of the strain in the scaffold. It can be disassembled or assembled and hold to the bioreactor chassis by fixation screws. The cartridge cap comprises the embedded piston and the vents for gas exchange covered by 0.22 μm filters to protect from the external environment. B) The technical sketch depicts the inner structures of the bioreactor. The piston compresses simultaneously the elastic ring and the scaffold (not shown) located over the cell reservoir. A non-permeable membrane forms the bottom of the cartridge, isolating the cell cultivation system to protect it from contamination. A force sensor is located underneath the membrane, which is mandatory for reliable measurements of the induced forces. Dimensions of the bioreactor: 120 x 150 x 400 mm (width, depth, height), approximately.
Fig 2.
Assembling of the cartridge components.
A) The sketch shows parts of a disassembled cartridge. The scaffold holder (2) contains the reservoir in the middle where the cells are placed. A scaffold (4) is placed over them, which is held in place by the elastic ring (3) and a mesh above it (5). The mesh is kept in place by a ring anchored to the construction using screws (6). The scaffold holder is placed as an independent movable unit within the cylindrical container (1) and it is covered up by a cap (7) containing the piston and spring. The cartridge is made of sterilizable materials. B) The schematic drawing shows the cross-section of the parts of an assembled scaffold holder without the upper anchored ring. The mesh aims to prevent the scaffold from moving up during the lift maneuvers. An alginate scaffold C) and the elastic ring D) are shown from above and a skew side view in detail.
Fig 3.
The system of the bioreactor is designed as standalone unit.
The bioreactor (1) connected to a power supply (3) is placed inside a CO2 incubator to allow conditions for cell cultivation. The connection wires from the bioreactor are plugged over an electronic box (4) that contains a motion controller, an amplifier of the force sensor and the interpolator for the digital gauge. The motion controller receives the information from the stepper motor, and the signals of the force sensor generated by the loading are processed by the amplifier. The electronic box (4) is plugged to the connector block (5), which communicates with the PC card in the computer (6). The PC card synchronizes the beginning of the experiment with the motion controller software “EasyMotion” (7), where the settings are established for the experiments. Data as force, displacement of the piston and time are visualized and registered at a rate of 50 Hz by the custom-made software “Bioreactor”, programmed in LabView 2011. Finally, the data can be exported (9) and analyzed by statistical software such as Origin 9.0G, which was used in this study.
Fig 4.
Recorded mechanical data after the test.
A) Plot of the overview of a complete run. The force exerted over the system and detected by the force sensor is seen in red. The displacements of the piston detected by the gauge are seen in black. B) Lift maneuver. An unloaded phase of the intermittent dynamic mechanical loading is shown in detail. When the unloaded phase is reached, the piston moves upward (downwards in the depicted record as a black line). During the lift maneuver, the compression on the scaffold is lost and the basal force value corresponds to the offset. Note the elevated force peak when the piston compresses the scaffold again after the lift maneuver. In this case, an offset of about 1.2 N was seen during the lift maneuver.
Fig 5.
Viability of the cells residing in the reservoir.
After 24 hours of mechanical stimulation, the viability of the cells which were not mobilized toward the scaffold was assessed by Trypan blue exclusion assay. The applied intermittent regime comprised 0.3 Hz frequency over 24 hours and interruptions with unloaded phases of 10 seconds after each 180 cycles. For the continuous regime, 0.3 Hz frequency for 24 hours without lift maneuvers loading was applied. The control scaffolds were prepared simultaneously in a separated and identically constructed cartridge, but no mechanical loading was applied. Continuous loading seemed to harm the cells, whereas interruption of the compression increased the cell viability comparable with unloaded cells. The results are shown as triplicates from the same donor; Δ, Ο, □ = replicates.
Fig 6.
A) Representative confocal microscopy images of alginate and alginate-LN521 scaffolds, with or without mechanical stimulation. Calcein-AM is seen in green and represents viable cells, ethidium homodimer-1 is seen in red and represents non-viable cells. There was only a low number of dead cells in the scaffolds. Bar scale = 100 μm. B) Quantification of viable and non-viable cells found in the scaffold after biomechanical stimulation or control. LN521 seems to have improved the alginate scaffold in terms of cell intake. The combination of LN521 in the scaffolds and the intermittent mechanical loading enhanced the number of cells found in the scaffolds. The results for every condition are shown as 3 independent replicates with cells from the same donor. Gray color indicates scaffolds without LN521 and black indicates the use of LN521; Δ, Ο, □ = replicates.