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 &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 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 &deg;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 &deg;C before mixing with cells. Warm up the cell suspension to 37 &deg;C. Once the agarose hits 43 &deg;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 &deg;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="Equation 1" fo:content-width="3.26in" fo:src="/files/ftp_upload/50387/50387eq1highres.jpg" src="/files/ftp_upload/50387/50387eq1.jpg" />
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>&nbsp;</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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+movement+in+embryonic+joint+development.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+compression+modulates+matrix+biosynthesis+in+chondrocyte/agarose+culture.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioreactor+Cultivation+Conditions+Modulate+the+Composition+and+Mechanical+Properties+of+Tissue-Engineered+Cartilage.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Mixing+Intensity+on+Tissue-Engineered+Cartilage.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increasing+extracellular+matrix+production+in+regenerating+cartilage+with+intermittent+physiological+pressure.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+versatile+shear+and+compression+apparatus+for+mechanical+stimulation+of+tissue+culture+explants.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrostatic+pressure+enhances+chondrogenic+differentiation+of+human+bone+marrow+stromal+cells+in+osteochondrogenic+medium.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+Tissue+Engineering:+The+Role+of+Biomechanics.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+Tissue+Engineering.+The+role+of+biomechanics+in+articular+cartilage+repair.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+cartilaginous+ECM+gene+transcription+by+chondrocytes+and+MSCs+in+3D+culture+in+response+to+dynamic+loading.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+anabolic+activity+of+bone+tissue,+suppressed+by+disuse,+is+normalized+by+brief+exposure+to+extremely+low-magnitude+mechanical+stimuli.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tribology+approach+to+the+engineering+and+study+of+articular+cartilage.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+cartilaginous+matrix+accumulation+on+viscoelastic+response+of+chondrocyte/agarose+constructs+under+dynamic+compressive+and+shear+loading.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cartilage+responses+to+a+novel+triaxial+mechanostimulatory+culture+system.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-axial+mechanical+stimulation+of+tissue+engineered+cartilage:+review.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioreactor+for+biaxial+mechanical+stimulation+to+tissue+engineered+constructs.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+mechanical+loading+enhances+functional+properties+of+tissue-engineered+cartilage+using+mature+canine+chondrocytes.">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>Bilgen, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+of+a+Biaxial+Loading+Device+for+Cartilage+Tissue+Engineering.">Design of a Biaxial Loading Device for Cartilage Tissue Engineering.</a> , 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+cell+seeding+density+and+nutrient+supply+for+articular+cartilage+tissue+engineering+with+deformational+loading.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+tissue+engineering+of+articular+cartilage+through+dynamic+loading+of+chondrocyte-seeded+agarose+gels.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+validation+of+a+bioreactor+for+physical+stimulation+of+engineered+cartilage.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+motion+upregulates+superficial+zone+protein+and+hyaluronan+production+in+chondrocyte-seeded+three-dimensional+scaffolds.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+combination+of+shear+and+dynamic+compression+leads+to+mechanically+induced+chondrogenesis+of+human+mesenchymal+stem+cells.">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 border="1">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>REAGENTS</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 &beta;1</td>
			<td>R&amp;D Systems</td>
			<td>240-B-010</td>
			<td></td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>EQUIPMENT</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

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 &mu;g/ml streptomycin, 2 mM L-glutamine, 2.5 &mu;g/ml amphotericin B,...

Watch this Scientific Journal Video about Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering at JoVE.com

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

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  ...

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

Research

Education

JoVE Core

JoVE Journal

Mechanical Engineering

Bioengineering

Unit 1

Analysis and Design of Beams for Bending

SCIENTISTS IN ACTION

14.3K 次观看.  The Warren Alpert Brown Medical School of Brown University and the Rhode Island Hospital. 我们设计了一种新型的机械负荷生物反应器可应用于单轴或双轴机械应变移植到关节软骨缺损的软骨生物复合材料之前。

视频: Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

Engineering Biological-Based Vascular Grafts Using a Pulsatile Bioreactor

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological environment, both mechanical and chemical stimulation are required to maintain the proper development and functionality of arterial vessels1,2.
Bioreactor culture systems developed by our group are designed to support vessel regeneration within a precisely controlled chemo-mechanical environment mimicking that of native vessels. Our bioreactor assembly and maintenance procedures are fairly simple and highly repeatable3,4. Smooth muscle cells (SMCs) are seeded onto a tubular polyglycolic acid (PGA) mesh that is threaded over compliant silicone tubing and cultured in the bioreactor with or without pulsatile stimulation for up to 12 weeks. There are four main attributes that distinguish our bioreactor from some predecessors. 1) Unlike other culture systems that simulate only the biochemical surrounding of native blood vessels, our bioreactor also creates a physiological pulsatile environment by applying cyclic radial strain to the vessels in culture. 2) Multiple engineered vessels can be cultured simultaneously under different mechanical conditions within a controlled chemical environment. 3) The bioreactor allows a mono layer of endothelial cells (EC) to be easily coated onto the luminal side of engineered vessels for animal implantation models. 4) Our bioreactor can also culture engineered vessels with different diameter size ranged from 1 mm to 3 mm, saving the effort to tailor each individual bioreactor to fit a specific diameter size. 
The engineered vessels cultured in our bioreactor resemble native blood vessels histologically to some degree. Cells in the vessel walls express mature SMC contractile markers such as smooth muscle myosin heavy chain (SMMHC)3. A substantial amount of collagen is deposited within the extracellular matrix, which is responsible for ultimate mechanical strength of the engineered vessels5. Biochemical analysis also indicates that collagen content of engineered vessels is comparable to that of native arteries6. Importantly, the pulsatile bioreactor has consistently regenerated vessels that exhibit mechanical properties that permit successful implantation experiments in animal models3,7. Additionally, this bioreactor can be further modified to allow real-time assessment and tracking of collagen remodeling over time, non-invasively, using a non-linear optical microscopy (NLOM)8. To conclude, this bioreactor should serve as an excellent platform to study the fundamental mechanisms that regulate the regeneration of functional small-diameter vascular grafts.

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.

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological ...

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

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

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

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.

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

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.

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

Operation of a Benchtop Bioreactor

Fermentation systems are used to provide an optimal growth environment for many different types of cell cultures. The ability afforded by fermentors to carefully control temperature, pH, and dissolved oxygen concentrations in particular makes them essential to efficient large scale growth and expression of fermentation products. This video will briefly describe the advantages of the fermentor over the shake flask. It will also identify key components of a typical benchtop fermentation system and give basic instruction on setup of the vessel and calibration of its probes. The viewer will be familiarized with the sterilization process and shown how to inoculate the growth medium in the vessel with culture. Basic concepts of operation, sampling, and harvesting will also be demonstrated. Simple data analysis and system cleanup will also be discussed.

Fermentors are used to increase culture yield and productivity of bioengineered cells. After screening multiple microbial or animal cell culture candidates in shake flasks, the next logical step is to increase the selected culture&#x2019;s biomass with the fermentor. This video demonstrates the setup and operation of a typical benchtop bioreactor system.

Fermentation systems are used to provide an optimal growth environment for many different types of cell cultures. The ability afforded by fermentors to ...

Design and Construction of Artificial Extracellular Matrix (aECM) Proteins from Escherichia coli for Skin Tissue Engineering

Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins that have incorporated functional domains derived from nature (or created de novo) have been reported. In particular, artificial extracellular matrix (aECM) proteins have been developed; these aECM proteins consist of biological domains taken from fibronectin, laminins and collagens and are combined with structural domains including elastin-like repeats, silk and collagen repeats. To date, aECM proteins have been widely investigated for applications in tissue engineering and wound repair. Recently, Tjin and coworkers developed integrin-specific aECM proteins designed for promoting human skin keratinocyte attachment and propagation. In their work, the aECM proteins incorporate cell binding domains taken from fibronectin, laminin-5 and collagen IV, as well as flanking elastin-like repeats. They demonstrated that the aECM proteins developed in their work were promising candidates for use as substrates in artificial skin. Here, we outline the design and construction of such aECM proteins as well as their purification process using the thermo-responsive characteristics of elastin.

Design and Construction of Artificial Extracellular Matrix aECM Proteins from Escherichia coli for Skin Tissue Engineering

Recombinant technologies have enabled material designers to create novel artificial proteins with customized functionalities for tissue engineering applications. For example, artificial extracellular matrix proteins can be designed to incorporate structural and biological domains derived from native ECMs. Here, we describe the construction and purification of aECM proteins containing elastin-like repeats.

Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation purposes. The process to generate such tissues is complex, and includes an appropriate combination of specific cell types, scaffolds, and physical or biochemical stimuli to guide cell growth and differentiation. Microcarriers represent an appealing tool to expand cells in a three-dimensional (3D) microenvironment, since they provide higher surface-to volume ratios and mimic more closely the in vivo situation compared to traditional two-dimensional methods. The vascular system, supplying oxygen and nutrients to the cells and ensuring waste removal, constitutes an important building block when generating engineered tissues. In fact, most constructs fail after being implanted due to lacking vascular support. In this study, we present a protocol for endothelial cell expansion on recombinant collagen-based microcarriers under dynamic conditions in spinner flask and bioreactors, and we explain how to determine in this setting cell viability and functionality. In addition, we propose a method for cell delivery for vascularization purposes without additional detachment steps necessary. Furthermore, we provide a strategy to evaluate the cell vascularization potential in a perfusion bioreactor on a decellularized&#160;biological matrix. We believe that the use of the presented methods could lead to the development of new cell-based therapies for a large range of tissue engineering applications in the clinical practice.

We propose a cell expansion protocol on macroporous microcarriers and their use as delivery system in a perfusion bioreactor to seed a decellularized tissue matrix. We also include different techniques to determine cell proliferation and viability of cells cultured on microcarriers. Furthermore, we demonstrate functionality of cells after bioreactor cultures.

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation ...

Agarose-based Tissue Mimicking Optical Phantoms for Diffuse Reflectance Spectroscopy

This protocol describes how to make agarose-based tissue-mimicking phantoms and demonstrates how to determine their optical properties using a conventional optical system with an integrating sphere. Measuring systems for the acquisition of the diffuse reflectance and total transmittance spectra are constructed with a broadband white light source, a light guide, an achromatic lens, an integrating sphere, a sample holder, an optical fiber probe, and a multi-channel spectrometer. An acrylic mold consisting of two rectangular acrylic pieces and a U-shaped acrylic piece is constructed to create an epidermal phantom and a dermal phantom with whole blood. The application of a sodium dithionite (Na2S2O4) solution to the dermal phantom enables the researcher to deoxygenate hemoglobin in red blood cells distributed in the dermal phantom. The inverse Monte Carlo simulation with the diffuse reflectance and total transmittance spectra measured by a spectrometer with an integrating sphere is performed to determine the absorption coefficient spectrum &#181;a(&#955;) and the reduced scattering coefficient spectrum &#181;s&#39;(&#955;) of each layer phantom. A two-layered phantom mimicking the diffuse reflectance of human skin tissue is also demonstrated by piling up the epidermal phantom on the dermal phantom.

Here, we demonstrate how agarose-based tissue-mimicking optical phantoms are made and how their optical properties are determined using a conventional optical system with an integrating sphere.

This protocol describes how to make agarose-based tissue-mimicking phantoms and demonstrates how to determine their optical properties using a ...

Biaxial Mechanical Characterizations of Atrioventricular Heart Valves

Extensive biaxial mechanical testing of the atrioventricular heart valve leaflets can be utilized to derive&#160;optimal parameters used in constitutive models, which provide a mathematical representation of the mechanical function of those structures. This presented biaxial mechanical testing protocol involves (i) tissue acquisition, (ii) the preparation of tissue specimens, (iii) biaxial mechanical testing, and (iv) postprocessing of the acquired data. First, tissue acquisition requires obtaining porcine or ovine hearts from a local Food and Drug Administration-approved abattoir for later dissection to retrieve the valve leaflets. Second, tissue preparation requires using tissue specimen cutters on the leaflet tissue to extract a clear zone for testing. Third, biaxial mechanical testing of the leaflet specimen requires the use of a commercial biaxial mechanical tester, which consists of force-controlled, displacement-controlled, and stress-relaxation testing protocols to characterize the leaflet tissue&#39;s mechanical properties. Finally, post-processing requires the use of data image correlation techniques and force and displacement readings to summarize the tissue&#39;s mechanical behaviors in response to external loading. In general, results from biaxial testing demonstrate that the leaflet tissues yield a nonlinear, anisotropic mechanical response. The presented biaxial testing procedure is advantageous to other methods since the method presented here allows for a more comprehensive characterization of the valve leaflet tissue under one unified testing scheme, as opposed to separate testing protocols on different tissue specimens. The proposed testing method has its limitations in that shear stress is potentially present in the tissue sample. However, any potential shear is presumed negligible.

This protocol involves characterizations of atrioventricular valve leaflets with force-controlled, displacement-controlled, and stress-relaxation biaxial mechanical testing procedures. Results acquired with this protocol can be used for constitutive model development to simulate the mechanical behavior of functioning valves under a finite element simulation framework.

Extensive biaxial mechanical testing of the atrioventricular heart valve leaflets can be utilized to derive&#160;optimal parameters used in constitutive ...

3D Bioprinting of Murine Cortical Astrocytes for Engineering Neural-Like Tissue

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also respond to neural injuries and act to protect the tissue from degenerative events. In vitro studies of astrocytes&#39; functionality are important to elucidate the mechanisms involved in such events and contribute to developing therapies to treat neurological disorders. This protocol describes a method to biofabricate a neural-like tissue structure rich in astrocytes by 3D bioprinting astrocytes-laden bioink. An extrusion-based 3D bioprinter was used in this work, and astrocytes were extracted from C57Bl/6 mice pups&#39; brain cortices. The bioink was prepared by mixing cortical astrocytes from up to passage 3 to a biomaterial solution composed of gelatin, gelatin-methacryloyl (GelMA), and fibrinogen, supplemented with laminin, which presented optimal bioprinting conditions. The 3D bioprinting conditions minimized cell stress, contributing to the high viability of the astrocytes during the process, in which 74.08% &#177; 1.33% of cells were viable right after bioprinting. After 1 week of incubation, the viability of astrocytes significantly increased to 83.54% &#177; 3.00%, indicating that the 3D construct represents a suitable microenvironment for cell growth. The biomaterial composition allowed cell attachment and stimulated astrocytic behavior, with cells expressing the specific astrocytes marker glial fibrillary acidic protein (GFAP) and possessing typical astrocytic morphology. This&#160;reproducible protocol provides a valuable method to biofabricate 3D neural-like tissue rich in astrocytes that resembles cells&#39; native microenvironment, useful to researchers that aim to understand astrocytes&#39; functionality and their relation to the mechanisms involved in neurological diseases.

Here we report a method of 3D bioprinting murine cortical astrocytes for biofabricating neural-like tissues to study the functionality of astrocytes in the central nervous system and the mechanisms involving glial cells in neurological diseases and treatments.

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also ...