Name: Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering (Video) | JoVE Journal
Uploaded: 2013-04-25T13:00:00.000+00:00
Duration: 8 min 4 s
Description: Learn to design a biaxial mechanical loading bioreactor for tissue engineering applications. This method applies precise strain to enhance biochemical content and strength in cartilage biocomposites for transplantation.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

This work was supported by the Office of Research and Development, RR&amp;D Service, U.S. Department of Veterans Affairs, NIH COBRE 1P20RR024484, NIH K24 AR02128 and Department of Defense W81XWH-10-1-0643.

1. Biaxial Loading Bioreactor Design
<ol>
 <li>The bioreactor employs two stages manufactured by Thor-labs (Newton, MA) for precision-guided laser applications for applying uniaxial or biaxial mechanical strain to engineered tissues, with great precision of loading dose (amplitude and frequency) and application to a wide variety of tissue culture conditions from single to 24 well plates (Figure 1).</li>
 <li>Bi-axial loading is accomplished by two TravelMax stages (LNR50SE). These stages are mounted orthogonally in an XZ configuration. The horizontal stage provides dynamic shearing motions by oscillating along the X-axis. The vertical stage provides dynamic compressive loading by oscillating along the Z-axis. These stages have a 50 mm travel range and are driven independently by stepper motor actuators (DRV014), controlled by a closed-loop stepper motor driver (BSC102) that features micro-stepping capabilities, enabling step sizes of less than 50 nm.</li>
 <li>The device is mounted on a rigid 25 cm x 30 cm x 12.5 mm aluminum base plate that is used as a platform for assembly of machine components and for mounting of tissue culture plates. Adjustable kinematic stops are used to lock tissue culture plates in place on the aluminum base plate. These kinematic stops have fine adjustment screws to allow for precise alignment that is otherwise not achievable by hand. The modular design of the base plate allows for flexible placement of these kinematic stops to accommodate plates of different sizes and shapes (Petri dishes vs. multi-well plates).</li>
 <li>A custom-machined polysulfone loading platen is coupled to the bi-axial moving platform via a precision-machined right angle bracket. Polysulfone material was chosen due to its biocompatibility, ease of machining, and ease of sterilization.</li>
 <li>Movements of the stages are controlled by Thor-labs' Advanced Positioning Technology (APT) software. The stepper motor driver is used in combination with the software that permits the adjustment of load parameters of frequency and amplitude of both shear and compression independently and simultaneously.</li>
 <li>Positional feedback is provided by linear optical encoders that are attached to each moving platform and are integrated with the software. The encoder system has a bidirectional repeatability of 0.1 μm and a resolution of 20 nm, translating to a positional accuracy of less than 3 μm over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities and a direct readout of the absolute position.</li>
 <li>In order to provide the force feedback necessary for detecting contact between platen and samples and evaluate loading responses, a precision miniature load cell (Honeywell Model 31) is positioned between the loading platen and the moving platform (Figure 2). The load cell has high accuracies of 0.15% to 0.25% full scale. The display unit (SC500) for the load cell can also provide load measurements of up to 5 decimal places. Additionally, it has an RS-232 port to allow for data collection on a computer.</li>
</ol>
2. Cell-Seeded Agarose Constructs
<ol>
 <li>Prepare 4% agarose: Add 0.8 g agarose to 20 ml DMEM (no additives) in a 50 ml flask, boil, and then keep in 70 °C oven until use.</li>
 <li>Adjust volume of cell suspension for double amount of the desired cell seeding density. This suspension is mixed with equal volume of 4% w/v agarose to create a 2% agarose gel at the desired seeding density.</li>
 <li>Place both the cell suspension and one 10 ml pipette in the incubator.</li>
 <li>Set up gel casting system. One 1.5 mm and one 0.75 mm spacer plate should be put together to create a 2.25 mm thick gel. Other size spacer plates may be used to create different gel thicknesses. The gel casting system is not large enough to hold these plates together, so they must be securely taped to prevent leaking.</li>
 <li>The next step is to quickly mix the cell suspension with the previously prepared agarose and pipette into the gel mold before the agarose solidifies. Remove the liquid agarose from the oven and place a sterile thermometer in it. The agarose must cool to 42-43 °C before mixing with cells. Warm up the cell suspension to 37 °C. Once the agarose hits 43 °C, quickly pipette up the desired amount and then immediately pipette the cell suspension up and down a few times to mix. Then immediately pipette the entire mixture into the gel mold.</li>
 <li>Allow the gel to solidify for 10-15 min and then carefully tilt it to a horizontal position.</li>
 <li>Remove the top glass plate and punch disks with the biopsy punch. Disks can be picked up with a small sterile spatula. In our experience, a 9 ml gel was large enough to make more than a hundred 5-mm-diameter discs.</li>
</ol>
3. Culture the Disks
<ol>
 <li>Place one disk in each well of a 24 well non-tissue culture treated plate.</li>
 <li>Add 2 ml of serum-free chondrogenic differentiation medium to each well.</li>
 <li>Put plates in incubator (37 °C, 5% CO2).</li>
 <li>For media changes, replace 1 ml per well every 2-3 days.</li>
</ol>
4. Immobilization of Samples for Mechanical Loading
<ol>
 <li>Prepare 4% agarose (no cell suspension added) and the gel should be thinner than the samples themselves (to prevent interference during loading). The recommended thickness is 1.5-1.9 mm (for 2.25 mm thick samples).</li>
 <li>Once gelled, punch 16 mm diameter disks for 24 well plates. In each disk, punch one 5 mm hole for the sample to be placed in. If desired, punch another 5 mm hole on the edge of the disk for the pipette to be placed during media changes.</li>
 <li>Once agarose wells have been made, place in 24 well plate, as shown in Figure 3.</li>
 <li>Once the agarose wells are in the 24 well plate, press fit samples in each well. The sample should protrude from the top of the agarose well.</li>
</ol>
5. Mechanical Loading
<ol>
 <li>Sterilize platen (Figure 2).</li>
 <li>Secure aluminum plate to load cell. Secure loading platen/load cell/aluminum plate assembly to the stage.</li>
 <li>Turn on stepper motor controller (switch in back).</li>
 <li>Turn on PC and open "APT User" program (Figure 4).
 <ol>
 <li>Left screen controls horizontal stepper motor. Right screen controls vertical stepper motor. In each screen, "Graphical Control" tab allows for manual positioning and "Move Sequencer" tab allows for automation. All units are mm.</li>
 </ol>
 </li>
 <li>Go to "Graphical Control" tab on both screens and press "Home/Zero" button. Both stepper motors have a range of 50 mm. Pressing "Home/Zero" will send both stepper motors to the zero position (top- and right- most positions).</li>
 <li>Prepare the samples in the 24 well plate for loading by removing some media from each well. No more than 1 ml of media should be left in each well to prevent overflow during loading. Be sure that enough media is left in the well to keep the sample covered.
 <ol>
 <li>Note that the incubator is kept in low-humidity conditions to prevent instrument failure.</li>
 </ol>
 </li>
 <li>Place 24 well plate in bioreactor and carefully line up with the platen.
 <ol>
 <li>The plate is secured to the bioreactor using four adjustable kinematic locators. To make it easier to line up the platen, the two left locators have been pre-positioned. Tighten the two right locators so that the plate is secure. Make sure to line the plate up flush with the front of the bioreactor base.</li>
 <li>In "Graphical Control" tab, a specific stepper motor position can be manually entered by clicking on the position box. Use this capability to slowly lower the platen and move it horizontally to line up with plate.</li>
 </ol>
 </li>
 <li>Once the platen is close to coming in contact with samples, start to bring the platen down in very slow increments (0.1 mm) until you reach the predetermined starting position (see part 6).</li>
 <li>Once the starting position is reached, go to "Move Sequencer" tab and load the desired move sequence by pressing "Load." Then press "Run" to start. (Figure 5) See part 7. Writing a Dosing Protocol.</li>
 <li>When finished loading, manually raise the platen. If any samples are stuck to the platen, carefully put them back into the appropriate well using a sterile spatula.</li>
 <li>Remove 24 well plate from bioreactor and replace the media.</li>
 <li>Carefully remove the platen from the load cell and then turn off instruments.</li>
</ol>
6. Calibrating Loading Platen
To ensure that the proper strains are applied to samples, each platen must be carefully calibrated prior to starting an experiment.
<ol>
 <li>Perform steps 5.1 to 5.8.</li>
 <li>Manually put horizontal stepper motor at the 25 mm position.</li>
 <li>Carefully lower platen until it just barely comes in contact with the base of the bioreactor. Load cell will show increased loads at this point. Take note of the exact position of the vertical stepper motor (be as precise as possible, as all compression strain measurements will be computed from this value).</li>
 <li>Record the position. This value will be used to write a dosing protocol based on sample dimensions and desired strains.</li>
</ol>
7. Writing a Dosing Protocol
<ol>
 <li>The bioreactor is capable of applying both compressive and shear strain, either simultaneously or individually. Three main parameters must be decided: tare compressive strain, dynamic strain amplitude, and loading frequency.</li>
 <li>A tare strain is applied in order to prevent liftoff of the platen from the sample.</li>
 <li>Sample dynamic strain amplitude and loading frequency are chosen.</li>
</ol>
In this study we define compressive and shear strain as follows: 
<img alt="Strain calculation formulas for compression and shear in material science, showing equations." fo:content-width="3.26in" fo:src="/files/ftp_upload/50387/50387eq1highres.jpg" src="/files/ftp_upload/50387/50387eq1.jpg" data-altupdatedat="1787630408000" data-alt="Equation 1">
Example Biaxial Dosing Protocol 
Sample thickness: 2.25 mm 
Tare Strain (Compression): 10% of the sample thickness (0.225 mm) 
Dynamic Strain Amplitude (Compression): 10% (+/- 5% of the sample thickness) 
Frequency (Compression): 1 Hz 
Dynamic Strain Amplitude (Shear): 25% of the sample thickness (0.5625 mm): Shear stress is 
applied to the sample by the platen moving horizontally. 
Frequency (Shear): 0.5 Hz 
Typical dosing protocol is 3 hr of loading per day.
In this example, dynamic and shear loading is applied simultaneously rather than sequentially. We believe this pattern better mimics the complex loading environment in the human knee.
<ol start="4">
 <li>Once a dosing protocol has been selected, a compression move sequence program must be written.</li>
 <li>The move sequence is exactly what it sounds like, a list of positions that the stepper motor will move to at a specified acceleration and maximum velocity.</li>
 <li>Compute desired vertical positions based on dosing protocol and platen calibration value (from part 6).</li>
 <li>Example computations for Platen 1 are provided below:</li>
</ol>
<table border="1" fo:keep-together.within-page="always">
 <tbody>
 <tr>
 <td> </td>
 <td>Difference from Calibration Value</td>
 <td>Vertical Position</td>
 </tr>
 <tr>
 <td>Platen Calibration Value (touches bottom of bioreactor)</td>
 <td>0 mm</td>
 <td>29.7700 mm</td>
 </tr>
 <tr>
 <td>Platen makes contact with Sample (2.25 mm sample)</td>
 <td>4.4140 mm</td>
 <td>25.3560 mm</td>
 </tr>
 <tr>
 <td>Strain (5% Thickness )</td>
 <td>4.3015 mm</td>
 <td>25.4705 mm</td>
 </tr>
 <tr>
 <td>Strain (10% Thickness )</td>
 <td>4.1890 mm</td>
 <td>25.5810 mm</td>
 </tr>
 <tr>
 <td>Strain (15% Thickness )</td>
 <td>4.0765 mm</td>
 <td>25.6955 mm</td>
 </tr>
 </tbody>
</table>
<ol start="8">
 <li>Once positions are computed, experiment with acceleration and maximum velocity values to get the correct frequency. The number of cycles should be chosen, accordingly (e.g. 10,800 cycles for 3 hr at 1 Hz).</li>
 <li>Example Dynamic Compression Move Sequence Program (10% tare compression, 10% dynamic strain amplitude, 1 Hz) (Figure 5)</li>
 <li>Dynamic shear move sequence program: The number of cycles should be chosen according to the desired frequency and duration (e.g. 5,400 cycles for 3 hr at 0.5 Hz).</li>
 <li>Example Dynamic Shear Move Sequence Program (10% tare compression, 0.5625 mm (25% of the thickness) dynamic shear strain amplitude, 0.5 Hz) (Figure 5).</li>
</ol>

<ol><li>Drachman, D. B., Sokoloff, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+role+of+movement+in+embryonic+joint+development%5BTitle%5D)+AND+%22Devl.+Biol%22%5BJournal%5D)">The role of movement in embryonic joint development.</a> Devl. Biol. 14, 401-420 (1966).</li>
<li>Buschmann, M. D., Gluzband, Y. A., Grodzinsky, A. J., Hunziker, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanical+compression+modulates+matrix+biosynthesis+in+chondrocyte%2Fagarose+culture%5BTitle%5D)+AND+%22J.+Cell+Sci%22%5BJournal%5D)">Mechanical compression modulates matrix biosynthesis in chondrocyte/agarose culture.</a> J. Cell Sci. 108, 1497-1508 (1995).</li>
<li>Vunjak-Novakovic, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bioreactor+Cultivation+Conditions+Modulate+the+Composition+and+Mechanical+Properties+of+Tissue-Engineered+Cartilage%5BTitle%5D)+AND+%22Journal+of+Orthopaedic+Research%22%5BJournal%5D)">Bioreactor Cultivation Conditions Modulate the Composition and Mechanical Properties of Tissue-Engineered Cartilage.</a> Journal of Orthopaedic Research. 17, 130-138 (1999).</li>
<li>Gooch, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+Mixing+Intensity+on+Tissue-Engineered+Cartilage%5BTitle%5D)+AND+%22Biotechnology+and+Bioengineering%22%5BJournal%5D)">Effects of Mixing Intensity on Tissue-Engineered Cartilage.</a> Biotechnology and Bioengineering. 72, 402-407 (2001).</li>
<li>Carver, S. E., Heath, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Increasing+extracellular+matrix+production+in+regenerating+cartilage+with+intermittent+physiological+pressure%5BTitle%5D)+AND+%22Biotechnology+and+Bioengineering%22%5BJournal%5D)">Increasing extracellular matrix production in regenerating cartilage with intermittent physiological pressure.</a> Biotechnology and Bioengineering. 62, 166-174 (1999).</li>
<li>Frank, E. H., Jin, M., Loening, A. M., Levenston, M. E., Grodzinsky, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+versatile+shear+and+compression+apparatus+for+mechanical+stimulation+of+tissue+culture+explants%5BTitle%5D)+AND+%22J.+Biomech%22%5BJournal%5D)">A versatile shear and compression apparatus for mechanical stimulation of tissue culture explants.</a> J. Biomech. 33, 1523-1527 (2000).</li>
<li>Wagner, D. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hydrostatic+pressure+enhances+chondrogenic+differentiation+of+human+bone+marrow+stromal+cells+in+osteochondrogenic+medium%5BTitle%5D)+AND+%22Ann.+Biomed.+Eng%22%5BJournal%5D)">Hydrostatic pressure enhances chondrogenic differentiation of human bone marrow stromal cells in osteochondrogenic medium.</a> Ann. Biomed. Eng. 36, 813-820 (2008).</li>
<li>Butler, D. L., Goldstein, S. A., Guilak, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+Tissue+Engineering%3A+The+Role+of+Biomechanics%5BTitle%5D)+AND+%22J.+Biomech.+Eng%22%5BJournal%5D)">Functional Tissue Engineering: The Role of Biomechanics.</a> J. Biomech. Eng. 122, 570-575 (2000).</li>
<li>Guilak, F., Butler, D. L., Goldstein, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+Tissue+Engineering.+The+role+of+biomechanics+in+articular+cartilage+repair%5BTitle%5D)+AND+%22Clin.+Orthop%22%5BJournal%5D)">Functional Tissue Engineering. The role of biomechanics in articular cartilage repair.</a> Clin. Orthop. 391S, S295-S305 (2001).</li>
<li>Mauck, R. L., Byers, B. A., Yuan, X., Tuan, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulation+of+cartilaginous+ECM+gene+transcription+by+chondrocytes+and+MSCs+in+3D+culture+in+response+to+dynamic+loading%5BTitle%5D)+AND+%22Biomech.+Model+Mechanobiol%22%5BJournal%5D)">Regulation of cartilaginous ECM gene transcription by chondrocytes and MSCs in 3D culture in response to dynamic loading.</a> Biomech. Model Mechanobiol. 6, 113-125 (2007).</li>
<li>Rubin, C., Xu, G., Judex, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+anabolic+activity+of+bone+tissue%2C+suppressed+by+disuse%2C+is+normalized+by+brief+exposure+to+extremely+low-magnitude+mechanical+stimuli%5BTitle%5D)+AND+%22FASEB+J%22%5BJournal%5D)">The anabolic activity of bone tissue, suppressed by disuse, is normalized by brief exposure to extremely low-magnitude mechanical stimuli.</a> FASEB J. 15, 2225-2229 (2001).</li>
<li>Wimmer, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tribology+approach+to+the+engineering+and+study+of+articular+cartilage%5BTitle%5D)+AND+%22Tissue+Eng%22%5BJournal%5D)">Tribology approach to the engineering and study of articular cartilage.</a> Tissue Eng. 10, 1436-1445 (2004).</li>
<li>Miyata, S., Tateishi, T., Ushida, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Influence+of+cartilaginous+matrix+accumulation+on+viscoelastic+response+of+chondrocyte%2Fagarose+constructs+under+dynamic+compressive+and+shear+loading%5BTitle%5D)+AND+%22J.+Biomech.+Eng%22%5BJournal%5D)">Influence of cartilaginous matrix accumulation on viscoelastic response of chondrocyte/agarose constructs under dynamic compressive and shear loading.</a> J. Biomech. Eng. 130, 051016(2008).</li>
<li>Heiner, A. D., Martin, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cartilage+responses+to+a+novel+triaxial+mechanostimulatory+culture+system%5BTitle%5D)+AND+%22J.+Biomech%22%5BJournal%5D)">Cartilage responses to a novel triaxial mechanostimulatory culture system.</a> J. Biomech. 37, 689-695 (2004).</li>
<li>Waldman, S. D., Couto, D. C., Grynpas, M. D., Pilliar, R. M., Kandel, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multi-axial+mechanical+stimulation+of+tissue+engineered+cartilage%3A+review%5BTitle%5D)+AND+%22Eur.+Cell+Mater%22%5BJournal%5D)">Multi-axial mechanical stimulation of tissue engineered cartilage: review.</a> Eur. Cell Mater. 13, 66-73 (2007).</li>
<li>Wartella, K. A., Wayne, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bioreactor+for+biaxial+mechanical+stimulation+to+tissue+engineered+constructs%5BTitle%5D)+AND+%22J.+Biomech.+Eng%22%5BJournal%5D)">Bioreactor for biaxial mechanical stimulation to tissue engineered constructs.</a> J. Biomech. Eng. 131, 044501(2009).</li>
<li>Bian, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dynamic+mechanical+loading+enhances+functional+properties+of+tissue-engineered+cartilage+using+mature+canine+chondrocytes%5BTitle%5D)+AND+%22Tissue+Eng.+Part+A%22%5BJournal%5D)">Dynamic mechanical loading enhances functional properties of tissue-engineered cartilage using mature canine chondrocytes.</a> Tissue Eng. Part A. 16, 1781-1790 (2010).</li>
<li>Design of a Biaxial Loading Device for Cartilage Tissue Engineering. Bilgen, B., et al. 57th Annual Meeting of the Orthopaedic Research Society (ORS), , 1815(2011).</li>
<li>Mauck, R. L., Wang, C. C., Oswald, E. S., Ateshian, G. A., Hung, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+role+of+cell+seeding+density+and+nutrient+supply+for+articular+cartilage+tissue+engineering+with+deformational+loading%5BTitle%5D)+AND+%22Osteoarthritis+Cartilage%22%5BJournal%5D)">The role of cell seeding density and nutrient supply for articular cartilage tissue engineering with deformational loading.</a> Osteoarthritis Cartilage. 11, 879-890 (2003).</li>
<li>Mauck, R. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+tissue+engineering+of+articular+cartilage+through+dynamic+loading+of+chondrocyte-seeded+agarose+gels%5BTitle%5D)+AND+%22J.+Biomech.+Eng%22%5BJournal%5D)">Functional tissue engineering of articular cartilage through dynamic loading of chondrocyte-seeded agarose gels.</a> J. Biomech. Eng. 122, 252-260 (2000).</li>
<li>Demarteau, O., Jakob, M., Schafer, D., Heberer, M., Martin, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+and+validation+of+a+bioreactor+for+physical+stimulation+of+engineered+cartilage%5BTitle%5D)+AND+%22Biorheology%22%5BJournal%5D)">Development and validation of a bioreactor for physical stimulation of engineered cartilage.</a> Biorheology. 40, 331-336 (2003).</li>
<li>Grad, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Surface+motion+upregulates+superficial+zone+protein+and+hyaluronan+production+in+chondrocyte-seeded+three-dimensional+scaffolds%5BTitle%5D)+AND+%22Tissue+Eng%22%5BJournal%5D)">Surface motion upregulates superficial zone protein and hyaluronan production in chondrocyte-seeded three-dimensional scaffolds.</a> Tissue Eng. 11, 249-256 (2005).</li>
<li>Schatti, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+combination+of+shear+and+dynamic+compression+leads+to+mechanically+induced+chondrogenesis+of+human+mesenchymal+stem+cells%5BTitle%5D)+AND+%22Eur.+Cell+Mater%22%5BJournal%5D)">A combination of shear and dynamic compression leads to mechanically induced chondrogenesis of human mesenchymal stem cells.</a> Eur. Cell Mater. 22, 214-225 (2011).</li>
</ol>

The authors declare that they have no competing financial interests.

We have designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to tissue engineered constructs fabricated for transplantation. The device can be used as a bioreactor for in vitro cultivation of engineered biocomposites or as a testing device to describe the mechanical characteristics of the native tissue or after other treatments prior to. The device subjects engineered tissue constructs to biaxial mechanical loading with great precision of loading dose (amplitude and ...

We have designed a loading bioreactor that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. This device is primarily designed as a bioreactor for engineered replacements for articular cartilage; it could also be used for other load-bearing tissues in the human body. Our motivation in this bioreactor design stems from Drachman and Sokoloff 1, who made the seminal observation of abnormal formation of articular cartilage in paralyzed chick embryos due to absence of motion. Similarly, physical exercise is essential for development of normal muscle and bone. In keeping with this concept, many research groups have investigated how different modes of physical stimuli during in vitro cultivation modulates the biochemical and mechanical properties of cell-biomaterial biocomposites and tissue explants 2-7. The concept of functional tissue engineering involves the in vitro use of mechanical stimuli to enhance the functional properties of tissues, i.e. the mechanical properties that enable the tissue to withstand the expected in vivo stress and strain 8,9. Numerous studies report the use mechanical loading in terms of shear and compression to stimulate engineered cartilage constructs for articular joints. Mauck et al. 10 suggest that mechanical loading alone can induce chondrogenesis of mesenchymal stem cells even in the absence of growth factors that are considered vital. Application of intermittent mechanical loading such as compression or shear during tissue cultivation has been shown to modulate cartilage and bone formation, however the optimum dosimetry of loading differs with cell and tissue properties 11.
	The most important function of articular cartilage is the ability to withstand compressive and shear forces within the joint, therefore it has to have high compressive and shear moduli. The lack of functional mechanical strength and physiological ultrastructure in engineered cartilage has resulted in the breakdown on neo-cartilage in vivo and the failure of cartilage replacement strategies in joints. Although compression and shear have been commonly demonstrated to modulate and improve mechanical strength of articular cartilage biocomposites, a combination approach is rare 6,12-15. Wartella and Wayne 16 designed a bioreactor that applied tension and compression to produce meniscal cartilage replacements. Waldman et al. 15 designed a device to apply compression and shear to chondrocytes cultured in a porous calcium polyphosphate substrate. Bian et al. 17 demonstrated mechanical properties matching native cartilage with the in vitro cultivation of adult canine chondrocytes in gels and application of biaxial mechanical loading (compressive deformational loading and sliding contact loading). 
	The biaxial mechanical loading bioreactor was originally designed by Danielle Chu in our laboratory with the overall goal to induce morphological adaptations in tissue engineered cartilage constructs resulting in higher compressive and shear moduli than currently available 18. We believe this research will significantly increase our broader understanding of how mechanotransduction can be modulated to engineer clinically relevant tissues.

<table><tbody><tr><td colspan="4">&lt;strong&gt;REAGENTS&lt;/strong&gt;</td></tr><tr><td>DMEM, High glucose, pyruvate</td><td>Invitrogen</td><td>11995</td><td></td></tr><tr><td>Agarose Type II</td><td>Sigma</td><td>CAS 39346-81-1</td><td></td></tr><tr><td>Penicillin Streptomycin Glutamine 100X</td><td>Invitrogen</td><td>10378-016</td><td></td></tr><tr><td>ITS+ Premix</td><td>BD Biosciences</td><td>354352</td><td></td></tr><tr><td>Pen Strep Glutamine</td><td>Invitrogen</td><td>10378-016</td><td></td></tr><tr><td>Amphotericin B</td><td>Invitrogen</td><td>041-95780</td><td></td></tr><tr><td>Ascorbic Acid</td><td>Sigma</td><td>A-2218</td><td></td></tr><tr><td>Nonessential Amino Acid Solution 100x</td><td>Sigma</td><td>M-7145</td><td></td></tr><tr><td>L-proline</td><td>Sigma</td><td>P-5607</td><td></td></tr><tr><td>Dexamethasone</td><td>Sigma</td><td>D-2915</td><td></td></tr><tr><td>Recombinant Human Transforming Growth Factor &amp;#946;1</td><td>R&amp;amp;D Systems</td><td>240-B-010</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;EQUIPMENT&lt;/strong&gt;</td></tr><tr><td>Model 31 Load Cell (1000 g)</td><td>Honeywell</td><td>AL311</td><td></td></tr><tr><td>Model 31 Load Cell (1000 g)</td><td>Honeywell</td><td>AL311</td><td></td></tr><tr><td>Single Channel Display</td><td>Honeywell</td><td>SC500</td><td></td></tr><tr><td>50 mm Linear Encoded Travelmax Stage with Stepper Actuator</td><td>Thorlabs</td><td>LNR50SE/M</td><td></td></tr><tr><td>Two Channel Stepper Motor Controller</td><td>Thorlabs</td><td>BSC102</td><td></td></tr><tr><td>50 mm Trapezoidal Stepper Motor Drive (2)</td><td>Thorlabs</td><td>DRV014</td><td></td></tr><tr><td>Adjustable Kinematic Locator (4)</td><td>Thorlabs</td><td>KL02</td><td></td></tr><tr><td>Precision Right Angle Plate</td><td>Thorlabs</td><td>AP90/M</td><td></td></tr><tr><td>Vertical Mounting Bracket</td><td>Thorlabs</td><td>LNR50P2/M</td><td></td></tr><tr><td>Solid Aluminum Breadboard</td><td>Thorlabs</td><td>MB3030/M</td><td></td></tr><tr><td>Gel Casting System with 1.5 mm and 0.75 mm spacer plates </td><td>BioRad</td><td>#1653312 and #1653310</td><td></td></tr><tr><td>Disposable Biopsy Punch, 5 mm</td><td>Miltex, Inc.</td><td>33-35</td><td></td></tr><tr><td>16 mm hollow punch</td><td>Neiko Tools</td><td></td><td></td></tr><tr><td>Non-Tissue Culture Treated Plates, 24 Well, Flat Bottom</td><td>BD Biosciences</td><td>351147</td><td></td></tr><tr><td>Ultra-Moisture-Resistant Polysulfone sheet for loading platens</td><td>McMaster-Carr</td><td>86735k19</td><td>Custom-machined</td></tr></tbody></table>

design of a biaxial mechanical loading bioreactor for tissue engineering

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

The device was tested by using agarose gels seeded with 20 million cells/ml chondrocytes and cultivated in the presence of uniaxial (compression) or biaxial (compression and shear) mechanical loading. Primary porcine chondrocytes were isolated from the articular cartilage of 2-4 month old pigs. 5 mm diameter and 1.5 mm thick samples were cultured in 2 ml of defined chondrogenic culture medium (High glucose DMEM, 1% ITS+ Premix, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine, 2.5 μg/ml amphotericin B, 50 μg...

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

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.

design-biaxial-mechanical-loading-bioreactor-for-tissue

Research

JoVE Journal

Bioengineering

Our group has developed a bioreactor culture system that mimics the physiological pulsatile stresses of the cardiovascular system to regenerate implantable small-diameter vascular grafts.

Engineering Biological-Based Vascular Grafts Using a Pulsatile Bioreactor

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

Postproduction Processing of Electrospun Fibres for Tissue Engineering

We present in this article a novel stretching platform that can be used to investigate single cell responses to complex anisotropic biaxial mechanical deformation and quantify the mechanical properties of biological tissues.

A Novel Stretching Platform for Applications in Cell and Tissue Mechanobiology

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Here we present a protocol to develop a pure uniaxial loading machine. Critical design aspects are employed to ensure accurate and reproducible testing results.

Application of Design Aspects in Uniaxial Loading Machine Development

A three-dimensional uniaxial mechanical stimulation bioreactor system is an ideal bioreactor for tenogenic-specific differentiation of tendon-derived stem cells and neo-tendon formation.

Applying a Three-dimensional Uniaxial Mechanical Stimulation Bioreactor System to Induce Tenogenic Differentiation of Tendon-Derived Stem Cells

The goal of this protocol is to execute a dynamic co-culture of human macrophages and myofibroblasts in tubular electrospun scaffolds to investigate material-driven tissue regeneration, using a bioreactor which enables the decoupling of shear stress and cyclic stretch.

A Multi-Cue Bioreactor to Evaluate the Inflammatory and Regenerative Capacity of Biomaterials under Flow and Stretch

Three-dimensional cardiac tissues bioengineered using stem-cell-derived cardiomyocytes have emerged as promising models for studying healthy and diseased human myocardium in vitro while recapitulating key aspects of the native cardiac niche. This manuscript describes a protocol for fabricating and analyzing high-content engineered cardiac tissues generated from human induced pluripotent stem-cell-derived cardiomyocytes.

Designing a Bioreactor to Improve Data Acquisition and Model Throughput of Engineered Cardiac Tissues

This protocol presents the setup and operation of a newly developed, 3D printed bioreactor for the ex vivo culture of blood vessels in perfusion. The system is designed to be easily adopted by other users, practical, affordable, and adaptable to different experimental applications, such as basic biology and pharmacological studies.

Ex Vivo Perfusion Culture of Large Blood Vessels in a 3D Printed Bioreactor

Learn to design a biaxial mechanical loading bioreactor for tissue engineering applications. This method applies precise strain to enhance biochemical content and strength in cartilage biocomposites for transplantation.

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

Explore More Videos