The overall goal of the following experiment is to fabricate functional tissue engineered bio composites for transplantation. This is achieved by applying precise uni axial or bio axial mechanical strain to provide biophysical stimuli to cells in three dimensional biomaterial composites. As a second step, the bioreactor is outfitted with a load cell, which provides force feedback and mechanical testing of the bio composites during loading.
Next bio composites are cultured over a period of weeks in order to induce macroscale changes in construct biochemical content and strength results are obtained that show the effect of different types of loading based on the significant differences in glycosaminoglycan and collagen content, scaffold, thickness, and equilibrium modulus. Though this method can provide insight into the effects of biaxial loading on engineered cartilage tissue, it can also be applied to other low bearing tissues such as bone and tendon, demonstrating the procedure will be Amy May and Rob Stefani from our laboratory. To begin this procedure, set up a bio axial bioreactor as shown here.
The base of the bioreactor is composed of an aluminum plate measuring 25 centimeters by 30 centimeters by 12.5 millimeters. The variable positioning options available allows for flexible mounting of tissue culture plates of different shapes and sizes. Next, ensure that the stages are mounted on the base so that the horizontal stage provides dynamic shear by oscillating only along the x axis.
And the vertical stage provides dynamic compression by oscillating only along the Z axis. The next step is to fabricate a custom polysulfone loading plate. The polysulfone material is ideal because of its biocompatibility, ease of machining and ease of sterilization.
Couple the plate, the biaxial moving platform via a precision machined right angle bracket. The kinematic stops attached to the base plate, have fine adjustment screws to allow for precise alignment of the culture plates not achievable by hand. Next, attach the stepper motor actuator to each platform, which will acquire positional feedback for the software.
The stepper motor actuators shown here have bidirectional repeatability of 0.1 micrometer and a resolution of 20 nanometers translating to an accuracy of less than three micrometers Over 50 millimeters of travel control the movements of the stages using Thor Lab's advanced positioning technology software. The software controls the stepper motor in order to adjust the load parameters of frequency and amplitude of both sheer and compression independently and simultaneously. Next position a miniature five pound load cell between the loading platinum and the moving platform to provide the force feedback necessary for detecting contact between the platinum and constructs, as well as to evaluate loading responses.
Ensure that the load cell has high accuracies of 0.15%to 0.25%full scale, and that the display unit for the load cell has an RS 2 32 port to allow for data collection on a computer. In order to immobilize the samples within the wells of a 24 well plate prepare a mold for a 1.5 millimeter thick aros gel using a gel casting system with 1.5 millimeter spacers. Once assembled, pour a solution of 4%aros between the two glass plates and allow the mixture to cool.
Then remove the top plate and punch a 16 millimeter diameter disc from the agro slab. For each, well sample in each disc, punch a hole for the sample to be placed into. Place the completed discs in the wells of a 24 well plate.
Then press 2.25 millimeter thick, five millimeter diameter tissue constructs into each of the gel holders. The sample should protrude from the top of each aeros holder. Next, add 1.5 milliliters of culture medium to each well and replace the cover.
Then place the plate back into the incubator until it is time for sample loading. To begin mechanical loading of tissue constructs, secure the aluminum plate to the load cell and attach a sterile platin assembly to the vertical stage at all times. The incubator should be run in low humidity conditions to prevent instrument failure.
Next, turn on the stepper motor controller and the pc. Open the advanced positioning technology user program and go to the graphical control tab on both screens. Press the home zero button to send both stepper motors to the zero position, which is defined as the top and rightmost position.
Prepare the samples for loading by removing some media from each well to prevent overflow during mechanical loading. Next place the 24 well plate into the bioreactor and carefully line up the plate with the platin using four adjustable kinematic locators, make sure to line the plate up. Flush with the front of the bioreactor base in the graphical control tab.
Manually fine. Tune the alignment by clicking on the position box to move it horizontally. When the plate is aligned with the wells of the plate, begin to lower the plate until it first makes contact with the samples.
Once the starting position is reached, go to the move sequencer tab and load the desired move sequence by pressing load or create your own program as described in the text protocol accompanying this video. Next press run To begin the program. The software will direct the motors to apply the desired conditioning regimen to the samples.
When the program has finished, manually raise the plate so that it clears the top of the tissue culture plate. Then remove the 24 wheel plate from the bioreactor and replace the media. Finally, carefully remove the platinum from the load cell and then turn off the instruments.
The use of the biaxial bioreactor described in this video had a significant effect on the amount of glycosaminoglycans and collagen secreted by cells within tissue engineered constructs. Conditioning took place five days a week for three hours per day with 10%compression strain and 5%shear strain at one hertz. After 30 days in culture, the amount of glyco Amin, glycans and collagen secreted by chondrocytes exposed to by loading, was significantly higher than control samples Uniaxial loading only significantly increased the amount of glycosemia glycans.
Additionally, biaxial loading created thicker constructs, but showed no effect on the elasticity of the constructs. Using this dosing protocol call, following 30 days of conditioning and culture, cross sections of the tissue constructs were stained with typical markers of cartilage, including blue saffron and o and type two collagen. The three different groups include no conditioning, uniaxial loading, and biaxial loading.
While all the groups show positive staining for alium Blue and Saffron and O, the bixi loaded samples show increased amounts of type two collagen compared to the no load and uni axially loaded samples. After watching this video, you should have a good understanding of how to apply uni axial or bi axial loads on developing tissue engineered constructs.
