1. Isolation of Primary Articular Chondrocytes
Harvest 10-15 full thickness cartilage slices from the articular surfaces of animal joints (e.g. the metacarpal-phalangeal joint of skeletally mature cows obtained from a local abbatoir).
<ol>
 <li>Place cartilage slices in a 100 mm Petri dish and Incubate in 20 ml of 0.5% protease in Ham's F-12 (w/v) for 2 hr at 37 &deg;C. Rinse three times in Ham's F-12 culture media, and incubate with 20 ml of 0.15% Collagenase A in Ham's F-12 cultur...

<ol>
	<li>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=Cartilage+tissue+remodeling+in+response+to+mechanical+forces.">Cartilage tissue remodeling in response to mechanical forces.</a> Annual Review of Biomedical Engineering. 2, 691-713 (2000).</li><li>Kuettner, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemistry+of+articular+cartilage+in+health+and+disease.">Biochemistry of articular cartilage in health and disease.</a> Clinical Biochemistry. 25, 155-163 (1992).</li><li>Neu, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interface+of+functional+biotribology+and+regenerative+medicine+in+synovial+joints.">The interface of functional biotribology and regenerative medicine in synovial joints.</a> Tissue Engineering Part B: Reviews. 14, 235-247 (2008).</li><li>Demarteau, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+compression+of+cartilage+constructs+engineered+from+expanded+human+articular+chondrocytes.">Dynamic compression of cartilage constructs engineered from expanded human articular chondrocytes.</a> Biochemical and Biophysical Research Communications. 310, 580-588 (2003).</li><li>Waldman, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+intermittent+compressive+stimulation+improves+the+composition+and+mechanical+properties+of+tissue-engineered+cartilage.">Long-term intermittent compressive stimulation improves the composition and mechanical properties of tissue-engineered cartilage.</a> Tissue Engineering. 10, 1323-1331 (2004).</li><li>Hunter, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+compression+of+chondrocyte-seeded+fibrin+gels:+effects+on+matrix+accumulation+and+mechanical+stiffness.">Dynamic compression of chondrocyte-seeded fibrin gels: effects on matrix accumulation and mechanical stiffness.</a> Osteoarthritis and Cartilage. 12, 117-130 (2004).</li><li>Buschmann, M. D. <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> Journal of Cell Science. 108 (Pt 4), 1497-1508 (1995).</li><li>Quinn, T. M. <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+alters+proteoglycan+deposition+and+matrix+deformation+around+individual+cells+in+cartilage+explants.">Mechanical compression alters proteoglycan deposition and matrix deformation around individual cells in cartilage explants.</a> Journal of Cell Science. 111 (Pt 5), 573-583 (1998).</li><li>Kuettner, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+cartilage+matrix+by+mammalian+chondrocytes+in+vitro.+I.+Isolation,+culture+characteristics,+and+morphology.">Synthesis of cartilage matrix by mammalian chondrocytes in vitro. I. Isolation, culture characteristics, and morphology.</a> The Journal of Cell Biology. 93, 743-750 (1982).</li><li>Lee, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+loading+of+chondrocytes+embedded+in+3D+constructs:+in+vitro+methods+for+assessment+of+morphological+and+metabolic+response+to+compressive+strain.">Mechanical loading of chondrocytes embedded in 3D constructs: in vitro methods for assessment of morphological and metabolic response to compressive strain.</a> Methods in Molecular Medicine. 100, 307-324 (2004).</li><li>McGowan, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+quantification+of+DNA+in+human+articular+and+septal+cartilage+using+PicoGreen+and+Hoechst+33258.">Biochemical quantification of DNA in human articular and septal cartilage using PicoGreen and Hoechst 33258.</a> Osteoarthritis and Cartilage. 10, 580-587 (2002).</li><li>Fan, J. C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+intermittent+static+biaxial+tensile+strains+on+tissue+engineered+cartilage.">The effect of intermittent static biaxial tensile strains on tissue engineered cartilage.</a> Annals of Biomedical Engineering. 38, 1672-1682 (2010).</li><li>Kaupp, 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=Mechanical+vibrations+increase+the+proliferation+of+articular+chondrocytes+in+high-density+culture.">Mechanical vibrations increase the proliferation of articular chondrocytes in high-density culture.</a> Proceedings of the Institution of Mechanical Engineers, Part H: Journal of Engineering in Medicine. 222, 695-703 (2008).</li><li>Waldman, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+intermittent+shear+deformation+improves+the+quality+of+cartilaginous+tissue+formed+in+vitro.">Long-term intermittent shear deformation improves the quality of cartilaginous tissue formed in vitro.</a> Journal of Orthopaedic Research. 21, 590-596 (2003).</li><li>Waldman, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single+application+of+cyclic+loading+can+accelerate+matrix+deposition+and+enhance+the+properties+of+tissue-engineered+cartilage.">A single application of cyclic loading can accelerate matrix deposition and enhance the properties of tissue-engineered cartilage.</a> Osteoarthritis and Cartilage. 14, 323-330 (2006).</li><li>Kisiday, J. D. <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+dynamic+compressive+loading+on+chondrocyte+biosynthesis+in+self-assembling+peptide+scaffolds.">Effects of dynamic compressive loading on chondrocyte biosynthesis in self-assembling peptide scaffolds.</a> Journal of Biomechanics. 37, 595-604 (2004).</li><li>Chowdhury, T. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+regulation+of+chondrocyte+metabolism+in+agarose+constructs+subjected+to+dynamic+compression.">Temporal regulation of chondrocyte metabolism in agarose constructs subjected to dynamic compression.</a> Archives of Biochemistry and Biophysics. 417, 105-111 (2003).</li></ol>

No conflicts of interest declared.

The described method for applying controlled mechanical stimuli to cell-seeded agarose hydrogels allows for the direct investigation into the effects of dynamic compressive forces on chondrocyte metabolism. The use of the custom-testing rig in conjunction with the retaining rings provided lateral constraint for the constructs to avoid potential problems of sample tipping. The use of dead-weighted loading platens secured by sets crews ensures direct contact with constructs despite potential differences in sample height. R...

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent or equipment</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Ham's F-12</td>
 <td>Thermo Fisher Scientific</td>
 <td>SH3001002</td>
 <td></td>
 </tr>
 <tr>
 <td>Collagenase A</td>
 <td>Sigma Aldrich Ltd.</td>
 <td>C0130</td>
 <td></td>
 </tr>
 <tr>
 <td>Protease</td>
 <td>Sigma Aldrich Ltd.</td>
 <td>P5147</td>
 <td></td>
 </tr>
 <tr>
 <td>Fetal Bovine Serum</td>
 <td>Sigma Aldrich Ltd.</td>
 <td>F1051</td>
 <td></td>
 </tr>
 <tr>
 <td>Ascorbate</td>
 <td>Sigma Aldrich Ltd.</td>
 <td>A4034</td>
 <td></td>
 </tr>
 <tr>
 <td>Antibiotics/antimycotics</td>
 <td>Sigma Aldrich Ltd.</td>
 <td>A5955</td>
 <td></td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>Bioshop Canada Ltd.</td>
 <td>HEP001</td>
 <td></td>
 </tr>
 <tr>
 <td>Trypan blue</td>
 <td>Sigma Aldrich Ltd.</td>
 <td>93595</td>
 <td></td>
 </tr>
 <tr>
 <td>Reichert Bright-Line Hemacytometer</td>
 <td>Hausser Scientific</td>
 <td>1490</td>
 <td></td>
 </tr>
 <tr>
 <td>Quant-iT PicoGreen</td>
 <td>Invitrogen</td>
 <td>P7589</td>
 <td></td>
 </tr>
 <tr>
 <td>Papain from papaya latex</td>
 <td>Sigma Aldrich Ltd.</td>
 <td>P3125</td>
 <td></td>
 </tr>
 <tr>
 <td>Ammonium Acetate </td>
 <td>Sigma Aldrich Ltd.</td>
 <td>A1542</td>
 <td></td>
 </tr>
 <tr>
 <td>Ethyldiaminetetraacetic Acid </td>
 <td>Sigma Aldrich Ltd.</td>
 <td>E9884</td>
 <td></td>
 </tr>
 <tr>
 <td>DL-Dithiothreitol </td>
 <td>Sigma Aldrich Ltd.</td>
 <td>43819</td>
 <td></td>
 </tr>
 <tr>
 <td>Low Melting Point Agarose, Type VII</td>
 <td>Sigma Aldrich Ltd.</td>
 <td>A9045</td>
 <td></td>
 </tr>
 <tr>
 <td>Mesh Screen (200) Filter</td>
 <td>Sigma Aldrich Ltd.</td>
 <td>S4145</td>
 <td></td>
 </tr>
 <tr>
 <td>Mach-1 Micromechanical Tester</td>
 <td>Biomomentum Inc.</td>
 <td>V500cs</td>
 <td></td>
 </tr>
 <tr>
 <td>Compression Loading Jig</td>
 <td>Custom-built</td>
 <td></td>
 <td>Similar product could be supplied by Biomomentum Inc.</td>
 </tr>
 <tr>
 <td>Falcon 24 Well Culture Plate</td>
 <td>Thermo Fisher Scientific</td>
 <td>B353047</td>
 <td></td>
 </tr>
 <tr>
 <td>&beta;-Liquid Scintillation Counter</td>
 <td>Beckman Coulter</td>
 <td>LS6500</td>
 <td></td>
 </tr>
 <tr>
 <td>[3H] Proline</td>
 <td>Perkin-Elmer</td>
 <td>NET323005MC </td>
 <td></td>
 </tr>
 <tr>
 <td>[35S] Sulfur</td>
 <td>Perkin-Elmer</td>
 <td>NEX041005MC </td>
 <td></td>
 </tr>
 </tbody>
</table>

mechanical stimulation of chondrocyte-agarose hydrogels

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy this deficiency, several medical interventions have been developed. One such method is to resurface the damaged area with tissue-engineered cartilage; however, the engineered tissue typically lacks the biochemical properties and durability of native cartilage, questioning its long-term survivability. This limits the application of cartilage tissue engineering to the repair of small focal defects, relying on the surrounding tissue to protect the implanted material. To improve the properties of the developed tissue, mechanical stimulation is a popular method utilized to enhance the synthesis of cartilaginous extracellular matrix as well as the resultant mechanical properties of the engineered tissue. Mechanical stimulation applies forces to the tissue constructs analogous to those experienced in vivo. This is based on the premise that the mechanical environment, in part, regulates the development and maintenance of native tissue1,2. The most commonly applied form of mechanical stimulation in cartilage tissue engineering is dynamic compression at physiologic strains of approximately 5-20% at a frequency of 1 Hz1,3. Several studies have investigated the effects of dynamic compression and have shown it to have a positive effect on chondrocyte metabolism and biosynthesis, ultimately affecting the functional properties of the developed tissue4-8. In this paper, we illustrate the method to mechanically stimulate chondrocyte-agarose hydrogel constructs under dynamic compression and analyze changes in biosynthesis through biochemical and radioisotope assays. This method can also be readily modified to assess any potentially induced changes in cellular response as a result of mechanical stimuli.

Watch this Scientific Journal Video about Mechanical Stimulation of Chondrocyte-agarose Hydrogels at JoVE.com

Mechanical Stimulation of Chondrocyte-agarose Hydrogels

The biosynthesis of cartilaginous extracellular matrix by chondrocytes can be affected by application of mechanical stimuli. This method describes the technique of applying dynamic compressive strains to chondrocytes encapsulated in 3D constructs and the evaluation of induced changes in chondrocyte metabolism.

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy ...

mechanical-stimulation-of-chondrocyte-agarose-hydrogels

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Biology

11.5K vistas.  Queen's University. La biosíntesis de la matriz extracelular cartilaginosa por los condrocitos pueden ser afectados por la aplicación de estímulos mecánicos. Este método describe la técnica de aplicación de dinámicas tensiones compresivas a los condrocitos encapsulados en construcciones en 3D y la evaluación de los cambios inducidos en el metabolismo de los condrocitos.