Name: biotribological testing and analysis of articular cartilage sliding against metal for implants
Uploaded: 2020-05-14T15:00:00
Description: Watch this Scientific Journal Video about Biotribological Testing and Analysis of Articular Cartilage Sliding against Metal for Implants at JoVE.com

This research was funded by N&#214; Forschungs- und Bildungsges.m.b.H. and the provincial government of Lower Austria through the Life Science Calls (Project ID: LSC15-019) and by the Austrian COMET Program (Project K2 XTribology, Grant No. 849109).

1. Preparation of metal cylinders
<ol>
	<li>Analyze cylindrical cobalt-chromium-molybdenum (CoCrMo) rods fulfilling the standard specifications for surgical implants for their chemical composition using scanning electron microscopy (SEM) with energy dispersive x-ray spectroscopy per manufacturer&#8217;s protocol to confirm provided values. 
	NOTE: The elemental composition of the CoCrMo alloy used for this experiment is 65% Co, 28% Cr, 5% Mo and 2% others.</li>
	<li>Wet grind the samples with silicon carbide grind...

<ol>
	<li>Zengerink, M., Struijs, P. A. A. A., Tol, J. L., van Dijk, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+osteochondral+lesions+of+the+talus:+a+systematic+review.">Treatment of osteochondral lesions of the talus: a systematic review.</a> Knee Surgery, Sports Traumatology, Arthroscopy. 18 (2), 238-246 (2009).</li><li>Aurich, M., 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=Behandlung+osteochondraler+L&#228;sionen+des+Sprunggelenks:+Empfehlungen+der+Arbeitsgemeinschaft+Klinische+Geweberegeneration+der+DGOU.">Behandlung osteochondraler L&#228;sionen des Sprunggelenks: Empfehlungen der Arbeitsgemeinschaft Klinische Geweberegeneration der DGOU.</a> Zeitschrift fur Orthopadie und Unfallchirurgie. 155 (1), 92-99 (2017).</li><li>Van Bergen, C. J. A., Zengerink, M., Blankevoort, L., Van Sterkenburg, M. N., Van Oldenrijk, J., Van Dijk, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+metallic+implantation+technique+for+osteochondral+defects+of+the+medial+talar+dome.">Novel metallic implantation technique for osteochondral defects of the medial talar dome.</a> Acta Orthopaedica. 81 (4), 495-502 (2010).</li><li>Sweet, S. J., Takara, T., Ho, L., Tibone, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+Partial+Humeral+Head+Resurfacing.">Primary Partial Humeral Head Resurfacing.</a> The American Journal of Sports Medicine. 43 (3), 579-587 (2015).</li><li>Becher, C., 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=Minimum+5-year+results+of+focal+articular+prosthetic+resurfacing+for+the+treatment+of+full-thickness+articular+cartilage+defects+in+the+knee.">Minimum 5-year results of focal articular prosthetic resurfacing for the treatment of full-thickness articular cartilage defects in the knee.</a> Archives of Orthopaedic and Trauma Surgery. 131 (8), 1135-1143 (2011).</li><li>Lea, M. A., Barkatali, B., Porter, M. L., Board, T. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteochondral+Lesion+of+the+Hip+Treated+with+Partial+Femoral+Head+Resurfacing.+Case+Report+and+Six-Year+Follow-up.">Osteochondral Lesion of the Hip Treated with Partial Femoral Head Resurfacing. Case Report and Six-Year Follow-up.</a> HIP International. 24 (4), 417-420 (2018).</li><li>Becher, C., Huber, R., Thermann, H., Paessler, H. H., Skrbensky, G. <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+a+contoured+articular+prosthetic+device+on+tibiofemoral+peak+contact+pressure:+a+biomechanical+study.">Effects of a contoured articular prosthetic device on tibiofemoral peak contact pressure: a biomechanical study.</a> Knee Surgery, Sports Traumatology, Arthroscopy. 16 (1), 56-63 (2007).</li><li>Malahias, M. -. 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N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+osteochondral+defects+of+the+talus+with+a+metal+resurfacing+inlay+implant+after+failed+previous+surgery.">Treatment of osteochondral defects of the talus with a metal resurfacing inlay implant after failed previous surgery.</a> Bone and Joint Journal. 95 (12), 1650-1655 (2013).</li><li>Kim, Y. S. Y. -. H. H. Y. -. S., Kim, Y. S. Y. -. H. H. Y. -. S., Hwang, K. -. T. T., Choi, I. -. Y. 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+cartilage+degeneration+and+joint+motion+of+bipolar+hemiarthroplasty.">The cartilage degeneration and joint motion of bipolar hemiarthroplasty.</a> International Orthopaedics. 36 (10), 2015-2020 (2012).</li><li>Moon, K. H., 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=Degeneration+of+Acetabular+Articular+Cartilage+to+Bipolar+Hemiarthroplasty.">Degeneration of Acetabular Articular Cartilage to Bipolar Hemiarthroplasty.</a> Yonsei Medical Journal. 49 (5), 716-719 (2008).</li><li>Wimmer, M. A., Pacione, C., Laurent, M. P., Chubinskaya, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+wear+testing+of+living+cartilage+articulating+against+alumina.">In vitro wear testing of living cartilage articulating against alumina.</a> Journal of Orthopaedic Research. , (2016).</li><li>Bowland, P., Ingham, E., Fisher, J., Jennings, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+geometry+tribological+study+of+osteochondral+graft+implantation+in+the+knee.">Simple geometry tribological study of osteochondral graft implantation in the knee.</a> Proceedings of the Institution of Mechanical Engineers, Part H: Journal of Engineering in Medicine. 232 (3), 249-256 (2018).</li><li>Bowland, P., Ingham, E., Fisher, J., Jennings, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+preclinical+natural+porcine+knee+simulation+model+for+the+tribological+assessment+of+osteochondral+grafts+in+vitro.">Development of a preclinical natural porcine knee simulation model for the tribological assessment of osteochondral grafts in vitro.</a> Journal of Biomechanics. 77, 91-98 (2018).</li><li>Trevino, 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=Establishing+a+live+cartilage-on-cartilage+interface+for+tribological+testing.">Establishing a live cartilage-on-cartilage interface for tribological testing.</a> Biotribology. 9, 1-11 (2017).</li><li>Oungoulian, S. 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=Wear+and+damage+of+articular+cartilage+with+friction+against+orthopedic+implant+materials.">Wear and damage of articular cartilage with friction against orthopedic implant materials.</a> Journal of Biomechanics. 48 (10), 1957-1964 (2015).</li><li>Stotter, C., 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+Loading+Conditions+on+Articular+Cartilage+in+a+Metal-on-Cartilage+Pairing.">Effects of Loading Conditions on Articular Cartilage in a Metal-on-Cartilage Pairing.</a> Journal of Orthopaedic Research. 37 (12), 2531-2539 (2019).</li><li>Becher, C., Huber, R., Thermann, H., Tibesku, C. O., von Skrbensky, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tibiofemoral+contact+mechanics+with+a+femoral+resurfacing+prosthesis+and+a+non-functional+meniscus.">Tibiofemoral contact mechanics with a femoral resurfacing prosthesis and a non-functional meniscus.</a> Clinical biomechanics. 24 (8), 648-654 (2009).</li><li>Temple, D. K., Cederlund, A. A., Lawless, B. M., Aspden, R. M., Espino, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viscoelastic+properties+of+human+and+bovine+articular+cartilage:+a+comparison+of+frequency-dependent+trends.">Viscoelastic properties of human and bovine articular cartilage: a comparison of frequency-dependent trends.</a> BMC Musculoskeletal Disorders. , 1-8 (2016).</li><li>Caligaris, M., Ateshian, G. 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Focal metallic implants represent a salvage procedure for osteochondral defects, especially in middle-aged patients and after failed primary treatment. Although clinical studies demonstrated promising short-term results, one observed complication is damage to the opposing, native cartilage10. Cadaver and biomechanical studies show clear evidence that proper implantation with flat or slightly recessed positioning maintains natural contact pressures19. Tribological experiment...

The treatment of osteochondral defects is demanding and requires surgery in many cases. For focal osteochondral lesions in middle-aged patients, focal metallic implants are a viable option, especially after the failure of primary treatment, like bone marrow stimulation (BMS) or autologous chondrocyte implantation (ACI)1. Partial surface replacements can be considered salvage procedures that can reduce pain and improve the range of motion2. These implants are typically composed of a CoCrMo alloy and are available in different sizes and offset configurations to match the normal anatomy3. While initially developed for defects on the medial femoral condyle in the knee, such implants are now available and in use for the hip, ankle, shoulder, and elbow4,5,6. For a satisfactory outcome, it is crucial to assess the mechanical joint alignment and condition of the opposing cartilage. Furthermore, correct implantation without protrusion of the implant has been shown to be fundamental7.
Clinical studies demonstrated excellent short-term results in terms of pain reduction and improvement of function in middle-aged patients for various locations5,6,8. Compared with allograft implantation, focal metal implants allow early weight bearing. However, the opposing articular cartilage showed accelerated wear in a considerable number of patients9,10. Hence, even with proper placement, in many cases degeneration of the native cartilage seems inevitable, while the underlying mechanisms remain unclear. Similar degenerative changes have been observed after bipolar hemiarthroplasty of the hip11 and are increased with activity and loading12.
Tribological experiments provide the possibility to study such pairings in vitro and simulate different loading situations occurring under physiological conditions13. The use of osteochondral pins offers a simple geometry model to investigate the tribology of articular cartilage sliding against native cartilage or any implant material14 and might further be used in whole joint simulation models15. Metal-on-cartilage pairings show accelerated cartilage wear, extracellular matrix disruption, and decreased cell viability in the superficial zone compared with a cartilage-on-cartilage pairing16. Damage to the cartilage occurred mainly in the form of delamination between the superficial and middle zones17. However, the mechanisms leading to cartilage degeneration are not fully understood. This protocol provides a comprehensive analysis of the biosynthetic activity of articular cartilage. By the determination of metabolic activity and gene expression levels of catabolic genes, early indications for cartilage breakdown might be identified. The advantage of in vitro tribological experiments is that loading parameters can be adjusted to imitate various loading conditions.
Hence, the following protocol is suitable to simulate a metal-on-cartilage pairing, representing an experimental hemiarthroplasty model.

<table border="0" cellpadding="0" cellspacing="0" style="width:773px;" width="772">
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	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:297px;">Amphotericin B</td>
			<td style="width:191px;">Sigma&#8208;Aldrich Chemie GmbH</td>
			<td style="width:119px;">A-2942-100ML</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">buffered formaldehyde solution 4%</td>
			<td style="width:191px;">VWR</td>
			<td style="width:119px;">97131000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell Proliferation Kit II (XTT)</td>
			<td>Roche Diagnostics</td>
			<td style="width:119px;">11465015001</td>
			<td>XTT-based ex vivo toxicology assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">CoCrMo raw material</td>
			<td style="width:191px;">Acnis International</td>
			<td style="width:119px;"></td>
			<td>CoCrMo rods 6mm in diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CryoStar NX70 Cryostat</td>
			<td>Thermo Fischer Scientific</td>
			<td style="width:119px;"></td>
			<td>cryosectioning device</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">dimethyl sulfoxide (DMSO)</td>
			<td style="width:191px;">Sidma-Aldrich Chemie</td>
			<td style="width:119px;">D 2438-10ML</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Dulbecco&#8217;s modified Eagle&#8217;s medium</td>
			<td style="width:191px;">Sigma&#8208;Aldrich Chemie GmbH</td>
			<td style="width:119px;"></td>
			<td>medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">fetal bovine serum</td>
			<td style="width:191px;">Gibco</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Hyaluronic acid</td>
			<td style="width:191px;">Anika Therapeutics Inc.</td>
			<td style="width:119px;"></td>
			<td>component of lubricating solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">iCycler</td>
			<td>BioRad</td>
			<td style="width:119px;"></td>
			<td>thermal cycler</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leica microscope DM&#8208;1000</td>
			<td>Leica</td>
			<td style="width:119px;"></td>
			<td>microscope for histology</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LightCycler 480 Sealing Foil</td>
			<td style="width:191px;">Roche Diagnostics</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LightCycler 96</td>
			<td>Roche Diagnostics</td>
			<td style="width:119px;"></td>
			<td>thermal cycler for PCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MagNA Lyser Green Beads</td>
			<td>Roche Diagnostics</td>
			<td style="width:119px;">3358941001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Osteochondral Autograft Transfer System (OATS)</td>
			<td style="width:191px;">Arthrex Inc.</td>
			<td style="width:119px;"></td>
			<td>cutting tube for harvesting osteochondral cylinders</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">osteosoft</td>
			<td style="width:191px;">Merck</td>
			<td style="width:119px;">1017279010</td>
			<td>decalcifier-solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Penicillin /Streptomycin</td>
			<td style="width:191px;">Sigma&#8208;Aldrich Chemie GmbH</td>
			<td style="width:119px;">P4333-100ML</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:297px;">phosphate&#8208;buffered saline</td>
			<td style="width:191px;">Sigma&#8208;Aldrich Chemie GmbH</td>
			<td style="width:119px;"></td>
			<td>PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Prescale Low Pressure</td>
			<td style="width:191px;">Fujifilm</td>
			<td style="width:119px;"></td>
			<td>pressure indicating film</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNeasy Fibrous Tissue Kit</td>
			<td style="width:191px;">QIAGEN</td>
			<td style="width:119px;">74404</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Synergy 2</td>
			<td>BioTek Instruments</td>
			<td style="width:119px;"></td>
			<td>plate reader</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Tetra&#8208;Falex MUST</td>
			<td style="width:191px;">Falex Tribology</td>
			<td style="width:119px;"></td>
			<td>Tribometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue&#8208; Tek O.C.T.</td>
			<td style="width:191px;">SAKURA</td>
			<td style="width:119px;">4583</td>
			<td>embedding formulation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Transcriptor First Strand cDNA Synthesis Kit</td>
			<td>Roche Diagnostics</td>
			<td style="width:119px;">40897030001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#946;-mercaptoethanol</td>
			<td style="width:191px;">Sidma-Aldrich Chemie</td>
			<td style="width:119px;">M3148</td>
			<td></td>
		</tr>
	</tbody>
</table>

biotribological testing and analysis of articular cartilage sliding against metal for implants

Osteochondral defects in middle-aged patients might be treated with focal metallic implants. First developed for defects in the knee joint, implants are now available for the shoulder, hip, ankle and the first metatarsalphalangeal joint. While providing pain reduction and clinical improvement, progressive degenerative changes of the opposing cartilage are observed in many patients. The mechanisms leading to this damage are not fully understood. This protocol describes a tribological experiment to simulate a metal-on-cartilage pairing and comprehensive analysis of the articular cartilage. Metal implant material is tested against bovine osteochondral cylinders as a model for human articular cartilage. By applying different loads and sliding speeds, physiological loading conditions can be imitated. To provide a comprehensive analysis of the effects on the articular cartilage, histology, metabolic activity and gene expression analysis are described in this protocol. The main advantage of tribological testing is that loading parameters can be adjusted freely to simulate in vivo conditions. Furthermore, different testing solutions might be used to investigate the influence of lubrication or pro-inflammatory agents. By using gene expression analysis for cartilage-specific genes and catabolic genes, early changes in the metabolism of articular chondrocytes in response to mechanical loading might be detected.

The contact area and contact pressure must be confirmed using a pressure measurement film (Figure 1). Physiological loading condition can be confirmed by comparing with reference imprints for defined contact pressures. During testing, the coefficient of friction is monitored constantly. With a migrating contact area, a low friction coefficient can be maintained for at least 1 h (Figure 2). Using Safranin O staining the extracellular matrix composition and struct...

Watch this Scientific Journal Video about Biotribological Testing and Analysis of Articular Cartilage Sliding against Metal for Implants at JoVE.com

Biotribological Testing and Analysis of Articular Cartilage Sliding against Metal for Implants

This protocol describes the preparation, biotribological testing, and analysis of osteochondral cylinders sliding against metal implant material. Outcome measures included in this protocol are metabolic activity, gene expression and histology.

Osteochondral defects in middle-aged patients might be treated with focal metallic implants. First developed for defects in the knee joint, implants are ...

Our protocol makes it possible to test the sliding of metal for orthopedic implants against articular cartilage and thereby investigating the effects of focal implants or hemiarthroplasty on the biosynthetic activity of particular chondracytes the main advantage of this technique is that it provides a comprehensive insight into both the tribological properties and biologic effects of mechanical loading in the cartilage pairing. This technique might compliment the clinical findings after surgical procedures like implantation of a focal metallic implants to treat an osteochondral defect of the knee or hemiarthroplasty of the hip. Demonstrating the procedure will be Christopher Bauer a postdoc from my laboratory here at the Danube University.Use a commercially available reciprocating tribometer with a cylinder on plate configuration, vertical loading capabilities and adjustable load and sliding speed. Additionally, a liquid cell is required to perform the tests in a lubricating solution. Fix the osteochondral cylinders on the bottom sample holder with the marking aligned with the sliding direction.Begin by determining the contact pressure in the cobalt chromium molybdenum on cartilage system using a pressure measurement film. Place the pressure measurement film at the interface, and apply a static load for 30 seconds to determine initial contact pressure, contact size and shape. Mount the cobalt chromium molybdenum cylinders onto the upper load cell.Add the testing solution into the liquid cell to submerge the osteochondral cylinder and cover the metal cartilage sliding interface. Set testing parameters such as described in normal force stroke and sliding speed which will be maintained throughout the test. Start reciprocal sliding of the metal cylinder against the articular cartilage immersed in the lubricating solution.Monitoring the coefficient of friction throughout the experiment, terminate the experiment. After the desire testing period remove the osteochondral plug from the sample holder rinse it with PBS and store it in medium until further biological analysis. Keep control samples and the testing solution at room temperature for the duration of the test and analyze them together with the samples that have been exposed to mechanical loading.Place, a 24 well plate on a scale and zero it. Rinse the osteochondral plug with PBS and place it in a Petri dish. Then use a scalpel to cut the cartilage from the graft in one piece bisect the cartilage in two equal pieces so that the contact area is equally distributed onto both cartilage pieces and mince one half into approximately one millimeter cubed pieces.Use the second half for gene expression analysis transfer the minced cartilage into one well of the prepared at 24 well plate and determine the tissue weight repeat this process for each sample and add one milliliter of growth medium to each well of the plate. Add 500 microliters of XTT solution to each well and mix then incubate the plate at 37 degrees Celsius and 5%carbon dioxide for four hours after incubation remove the supernatant and transfer it to a five milliliter tube. Extract the tetrazolium product by adding 500 microliters of DMSO to the cartilage tissue in each well and apply continuous agitation for one hour at room temperature, remove the DMSO solution and pull it with the previously collected XTT solution.Transfer 100 microliters of each sample in triplicates to a 96 well plate. Use a plate reader to measure the absorbance at a wavelength of 492 nanometers, and a reference wavelength of 690 nanometers. To isolate the RNA mince the second half of the cartilage tissue obtained from the osteochondral plug into small pieces transfer the tissue to a tube containing ceramic beads and 300 microliters of licensed buffer.With 1%beta mercaptoethanol use a commercial lysate to homogenize the tissue applying 6, 500 RPM for 20 seconds four times with a 20 minute cooling phase after each run. Add 20 microliters of proteinase K and 580 microliters of RNAs free water to each sample, and incubate them at 55 degrees Celsius for 30 minutes. Centrifuge the samples for three minutes at 10, 000 times G and transfer the supernatant to 1.5 milliliter tubes.Add 0.5 volumes of 90%ethanol to each tube and mix then transfer 700 microliters of the sample to an RNA binding column in a two milliliter collection tube and centrifuge it at 8, 000 times G for 15 seconds. Discard the flow-through and repeat the centrifigation step for the rest of the lysate. Add a 350 microliters of buffer RW one to the column.Centrifuge it at 8, 000 times G for 15 seconds and discard the flow-through. Mix 10 microliters of DNA stock solution and 70 microliters of buffer RDD. Add the solution to the RNA purification membrane and incubate it at room temperature for 15 minutes then add 350 microliters of buffer RW one to the column.Centrifuge it at 8, 000 times G for 15 seconds and discard the flow-through add 500 microliters of buffer RPE to the RNA purification column and Centrifuge it at 8, 000 times G for 15 seconds discard the flow through and to add another 500 microliters of buffer RPE to the RNA purification column then centrifuge it at 8, 000 times G for two minutes place the column in a new 1.5 milliliter collection tube and add 30 microliters of RNAs free water centrifuge the tube at 8, 000 times G for one minute, to allude the RNA synthesized cDNA using a commercial kit thaw and mix all regions, add the RNA sample and perform the reaction and the thermal cycler as described in the text manuscript. Add nine microliters of RTQ PCR master mix, and one microliter of cDNA to each well of a 96 well plate with each sample in triplicates glows the PCR plate with ceiling oil and centrifuge it at 877 times G for 10 minutes at four degrees Celsius perform RTQ PCR and a precision and thermal cycler. According to manuscript directions.Prior to testing the contact area and contact pressure at the metal cartilage interface must be confirmed using a pressure measurement film. Physiological loading condition can then be confirmed by comparing the obtained imprint with reference inference for defined contact A low friction coefficient can be maintained for at least one hour with a migrating contact area. The extracellular matrix composition and structure can be determined with saffron and O staining.The intensity of saffron and O staining is proportional to the proteoglycan content. The proteoglycan content varies over the articular surface but should be uniform throughout the tissue section in baseline samples, show extraction of glycosaminoglycans which can be counteracted by mechanical loading. Metabolic activity of the bovine articular chondracytes is independent of the harvesting site but shows an increase with mechanical loading.The gene expression levels of cartilage specific genes increased with physiological loading conditions. Whereas catabolic genes are up-regulated with stationary contact area. Following this procedure, additional analysis can be performed including determination of cartilage web products and analysis of articular surface using scanning electron microscopy and furthermore we try to optimize lubrication of articular cartilage in various tribological pairings.

Research

JoVE Journal

Medicine

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