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.

biotribological-testing-analysis-articular-cartilage-sliding-against

Research

JoVE Journal

Medicine

A Novel Application of Musculoskeletal Ultrasound Imaging

Ultrasound is an attractive modality for imaging muscle and tendon motion during dynamic tasks and can provide a complementary methodological approach for biomechanical studies in a clinical or laboratory setting. Towards this goal, methods for quantification of muscle kinematics from ultrasound imagery are being developed based on image processing. The temporal resolution of these methods is typically not sufficient for highly dynamic tasks, such as drop-landing. We propose a new approach that utilizes a Doppler method for quantifying muscle kinematics. We have developed a novel vector tissue Doppler imaging (vTDI) technique that can be used to measure musculoskeletal contraction velocity, strain and strain rate with sub-millisecond temporal resolution during dynamic activities using ultrasound. The goal of this preliminary study was to investigate the repeatability and potential applicability of the vTDI technique in measuring musculoskeletal velocities during a drop-landing task, in healthy subjects. The vTDI measurements can be performed concurrently with other biomechanical techniques, such as 3D motion capture for joint kinematics and kinetics, electromyography for timing of muscle activation and force plates for ground reaction force. Integration of these complementary techniques could lead to a better understanding of dynamic muscle function and dysfunction underlying the pathogenesis and pathophysiology of musculoskeletal disorders.

We describe a new ultrasound-based vector tissue Doppler imaging technique to measure muscle contraction velocity, strain and strain rate with sub-millisecond temporal resolution during dynamic activities. This approach provides complementary measurements of dynamic muscle function and could lead to a better understanding of mechanisms underlying musculoskeletal disorders.

Ultrasound is an attractive modality for imaging muscle and tendon  motion during dynamic tasks and can provide a complementary methodological  approach ...

Techniques for Processing Eyes Implanted with a Retinal Prosthesis for Localized Histopathological Analysis: Part 2 Epiretinal Implants with Retinal Tacks

Retinal prostheses for the treatment of certain forms of blindness are gaining traction in clinical trials around the world with commercial devices currently entering the market. In order to evaluate the safety of these devices, in preclinical studies, reliable techniques are needed. However, the hard metal components utilised in some retinal implants are not compatible with traditional histological processes, particularly in consideration for the delicate nature of the surrounding tissue. Here we describe techniques for assessing the health of the eye directly adjacent to a retinal implant secured epiretinally with a metal tack.
Retinal prostheses feature electrode arrays in contact with eye tissue. The most commonly used location for implantation is the epiretinal location (posterior chamber of the eye), where the implant is secured to the retina with a metal tack that penetrates all the layers of the eye. Previous methods have not been able to assess the proximal ocular tissue with the tack in situ, due to the inability of traditional histological techniques to cut metal objects. Consequently, it has been difficult to assess localized damage, if present, caused by tack insertion.
Therefore, we developed a technique for visualizing the tissue around a retinal tack and implant. We have modified an established technique, used for processing and visualizing hard bony tissue around a cochlear implant, for the soft delicate tissues of the eye. We orientated and embedded the fixed eye tissue, including the implant and retinal tack, in epoxy resin, to stabilise and protect the structure of the sample. Embedded samples were then ground, polished, stained, and imaged under various magnifications at incremental depths through the sample. This technique allowed the reliable assessment of eye tissue integrity and cytoarchitecture adjacent to the metal tack.

Here we describe histological techniques for visualising ocular tissue directly adjacent to a metal epiretinal tack and retinal prosthesis.

Retinal prostheses for the treatment of certain forms of blindness are gaining traction in clinical trials around the world with commercial devices ...

Proximal Cadaveric Femur Preparation for Fracture Strength Testing and Quantitative CT-based Finite Element Analysis

Cadaveric fracture testing is routinely used to understand factors that affect proximal femur strength. Because ex vivo biological tissues are prone to lose their mechanical properties over time, specimen preparation for experimental testing must be performed carefully to obtain reliable results that represent in vivo conditions. For that reason, we designed a protocol and a set of fixtures to prepare the femoral specimens such that their mechanical properties experienced minimal changes. The femora were kept in a frozen state except during preparation steps and mechanical testing. The relevant clinical measures of total hip and femoral neck bone mineral density (BMD) were obtained with a clinical dual X-ray absorptiometry (DXA) bone densitometer, and the 3D geometry and distribution of bone mineral were obtained using CT with a calibration phantom for quantitative estimations based on the greyscale values. Any possible bone disease, fracture, or the presence of implants or artifacts affecting the bone structure, was ruled out with X-ray scans. For preparation, all bones were carefully cleaned of excess soft tissue, and were cut and potted at the internal rotation angle of interest. A cutting fixture allowed the distal end of the bone to be cut off leaving the proximal femur at a desired length. To allow positioning of the femoral neck at prescribed angles during later CT scanning and mechanical testing, the proximal femoral shafts were potted in polymethylmethacrylate (PMMA) using a fixture designed specifically for desired orientations. The data collected from our experiments were then used for validation of quantitative computed tomography (QCT)-based finite element analysis (FEA), as described in a different protocol. In this manuscript, we present the protocol for the precise bone preparation for mechanical testing and subsequent QCT/FEA modeling. The current protocol was successfully applied to prepare about 200 cadaveric femora over a 6-year time period.

We present a robust protocol on how to carefully preserve and prepare cadaveric femora for fracture testing and quantitative computed tomography imaging. The method provides precise control over input conditions for the purpose of determining relationships between bone mineral density, fracture strength, and defining finite element model geometry and properties.

Cadaveric fracture testing is routinely used to understand factors that affect proximal femur strength.  Because ex vivo biological tissues are prone to ...

Oral Biofilm Formation on Different Materials for Dental Implants

Dental implants and their prosthetic components are prone to bacterial colonization and biofilm formation. The use of materials that provides low microbial adhesion may reduce the prevalence and progression of peri-implant diseases. In view of the oral environment complexity and oral biofilm heterogeneity, microscopy techniques are needed that can enable a biofilm analysis of the surfaces of teeth and dental materials. This article describes a series of protocols implemented for comparing oral biofilm formation on titanium and ceramic materials for prosthetic abutments, as well as the methods involved in oral biofilms analyses at the morphological and cellular levels. The in situ model to evaluate oral biofilm formation on titanium and zirconia materials for dental prosthesis abutments as described in this study provides a satisfactory preservation of the 48 h biofilm, thereby demonstrating methodological adequacy. Multiphoton microscopy allows the analysis of an area representative of the biofilm formed on the test materials. In addition, the use of fluorophores and the processing of the images using multiphoton microscopy allows the analysis of the bacterial viability in a very heterogeneous population of microorganisms. The preparation of biological specimens for electron microscopy promotes the structural preservation of biofilm, images with good resolution, and no artifacts.

Here, we present a protocol to evaluate oral biofilm formation on titanium and zirconia materials for dental prosthesis abutments, including the analysis of bacterial cells viability and morphological characteristics. An in situ model associated with powerful microscopy techniques is used for the oral biofilm analysis.

Dental implants and their prosthetic components are prone to bacterial colonization and biofilm formation. The use of materials that provides low ...

Handheld Metal Detector Screening for Metallic Foreign Body Ingestion in Children

Coins are the most common ingested metallic foreign bodies among children. The goal of this protocol is to assess the accuracy and feasibility of using a handheld metal detector to detect ingested metallic foreign bodies in children. We propose that by introducing handheld metal detector screening early in the triage process of children with high suspicion of metallic foreign body ingestion, the number of radiographs being ordered to localize the metallic foreign body can be reduced in this radio-sensitive population. The study protocol requires the screening of the participants for history of foreign body ingestion and exclusion of patients with respiratory distress or metallic implants. The patient changes to hospital gown and items that could contain metal like eyeglasses, earrings, pendants, and ornaments are removed. The patient is positioned in the center of the room away from other metallic interferences. The working status of the handheld metal detector is first confirmed by eliciting a positive audio-visual signal. Then the screening is done in an erect position with head in extension to expose the neck, from the level of the chin to the level of the hip joint, to cover the anatomical areas from neck to pelvis in a zig-zag manner both anteriorly and posteriorly. A positive audio-visual signal is carefully noted during the scanning for the presence of metallic foreign body. Relevant radiographs are ordered as per the area detected on the metal detector screening. The handheld metal detector was able to precisely identify all the coins among the ingested metallic foreign bodies in our study. The handheld metal detector could not consistently detect non-coin metallic foreign bodies. This protocol demonstrates the accuracy of handheld metal detector in the identification and localization of coins and coin like metallic foreign bodies.

Here, we present a protocol to use the handheld metal detector, to screen for the presence of ingested metallic foreign bodies in children who present to the pediatric emergency medicine department with a history of foreign body ingestion.

Coins are the most common ingested metallic foreign bodies among children. The goal of this protocol is to assess the accuracy and feasibility of using a ...

Hemocompatibility Testing of Blood-Contacting Implants in a Flow Loop Model Mimicking Human Blood Flow

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the body&#39;s circulatory system demands a reliable and multiparametric approach that evaluates the possible hematologic complications caused by these devices (i.e., activation and destruction of blood components). Comprehensive in vitro hemocompatibility testing of blood-contacting implants is the first step towards successful in vivo implementation. Therefore, extensive analysis according to the International Organization for Standardization 10993-4 (ISO 10993-4) is mandatory prior to clinical application. The presented flow loop describes a sensitive model to analyze the hemostatic performance of stents (in this case, neurovascular) and reveal adverse effects. The use of fresh human whole blood and gentle blood sampling are essential to avoid the preactivation of blood. The blood is perfused through a heparinized tubing containing the test specimen by using a peristaltic pump at a rate of 150 mL/min at 37 &#176;C for 60 min. Before and after perfusion, hematologic markers (i.e., blood cell count, hemoglobin, hematocrit, and plasmatic markers) indicating the activation of leukocytes (polymorphonuclear [PMN]-elastase), platelets (&#946;-thromboglobulin [&#946;-TG]), the coagulation system (thombin-antithrombin III [TAT]), and the complement cascade (SC5b-9) are analyzed. In conclusion, we present an essential and reliable model for extensive hemocompatibility testing of stents and other blood-contacting devices prior to clinical application.

This protocol describes a comprehensive hemocompatibility evaluation of blood-contacting devices using laser-cut neurovascular implants. A flow loop model with fresh, heparinized human blood is applied to mimic blood flow. After perfusion, various hematologic markers are analyzed and compared to the values gained directly after blood collection for hemocompatibility evaluation of the tested devices.

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the ...

Novel Percutaneous Approach for Deployment of 3D Printed Coronary Stenosis Implants in Swine Models of Ischemic Heart Disease

Minimally invasive methods for creating models of focal coronary narrowing in large animals are challenging. Rapid prototyping using three-dimensionally (3D) printed coronary implants can be employed to percutaneously create a focal coronary stenosis. However, reliable delivery of the implants can be difficult without the use of ancillary equipment. We describe the use of a mother-and-child coronary guide catheter for stabilization of the implant and for effective delivery of the 3D printed implant to any desired location along the length of the coronary vessel. The focal coronary narrowing was confirmed under coronary cineangiography and the functional significance of the coronary stenosis was assessed using gadolinium-enhanced first-pass cardiac perfusion MRI. We showed that reliable delivery of 3D printed coronary implants in swine models (n = 11) of ischemic heart disease can be achieved through repurposing mother-and-child coronary guide catheters. Our technique simplifies the percutaneous delivery of coronary implants to create closed-chest swine models of focal coronary artery stenosis and can be performed expeditiously, with a low procedural failure rate.

We describe a novel, cost-effective, and efficient technique for percutaneous delivery of three-dimensionally printed coronary implants to create closed-chest swine models of ischemic heart disease. The implants were fixed in place using a mother-and-child extension catheter with high success rate.

Minimally invasive methods for creating models of focal coronary narrowing in large animals are challenging. Rapid prototyping using three-dimensionally ...

3D Planning and Printing of Patient Specific Implants for Reconstruction of Bony Defects

We are in the midst of the 3D era in most aspects of life, and especially in medicine. The surgical discipline is one of the major players in the medical field using the constantly developing 3D planning and printing capabilities. Computer-assisted design (CAD) and computer assisted manufacturing (CAM) are used to describe the 3D planning and manufacturing of the product. The planning and manufacturing of 3D surgical guides and reconstruction implants is performed almost exclusively by engineers. As technology advances and software interfaces become more user-friendly, it raises a question regarding the possibility of transferring the planning and manufacturing to the clinician. The reasons for such a shift are clear: the surgeon has the idea of what he wants to design, and he also knows what is feasible and could be used in the operating room. It allows him to be prepared for any scenario/unexpected results during the operation and allows the surgeon to be creative and express his new ideas using the CAD software. The purpose of this method is to provide clinicians with the ability to create their own surgical guides and reconstruction implants. In this manuscript, a detailed protocol will provide a simple method for segmentation using segmentation software and implant planning using a 3D design software. Following the segmentation and stl file production using segmentation software, the clinician could create a simple patient specific reconstruction plate or a more complex plate with a cradle for bone graft positioning. Surgical guides can be created for accurate resection, hole preparation for proper reconstruction plate positioning or for bone graft harvesting and re-contouring. A case of lower jaw reconstruction following plate fracture and nonunion healing of a trauma sustained injury is detailed.

This protocol describes the use of 3D planning and printing for reconstruction of bony defects. We use segmentation tools to create 3D models followed by 3D design software to create patient specific implants for reconstruction purposes concomitant to ablative surgery or as a second stage.

We are in the midst of the 3D era in most aspects of life, and especially in medicine. The surgical discipline is one of the major players in the medical ...

Treatment of Facial Deformities using 3D Planning and Printing of Patient-Specific Implants

Technological advancements in surgical planning and patient-specific implants are constantly evolving. One can either adopt the technology to achieve better results, even in the less experienced hand, or continue without it. As technology develops and becomes more user-friendly, we believe it is time to allow the surgeon the option to plan his/her operations and create his/her own patient-specific surgical guides and fixation plates allowing him full control over the process. We present here a protocol for 3D planning of the operation followed by 3D planning and printing of surgical guides and patient-specific fixation implants. During this process we use two commercial computer-assisted design (CAD) software. We also use a fused deposition&#160;modeling printer for the surgical guides and a selective laser sintering printer for the titanium patient-specific fixation implants. The process includes computed tomography (CT) imaging acquisition, 3D segmentation of the skull and facial bones from the CT, 3D planning of the operations, 3D planning of patient-specific fixation implant according to the final position of the bones, 3D planning of surgical guides for performing an accurate osteotomy and preparing the bone for the fixation plates, and 3D printing of the surgical guides and the patient-specific fixation plates. The advantages of the method include full control over the surgery, planned osteotomies and fixation plates, significant reduction in price, reduction in operation duration, superior performance and highly accurate results. Limitations include the need to master the CAD programs.

As technology develops and becomes more user-friendly, planning of operations and patient-specific surgical guides and fixation plates should be performed by the surgeon. We present a protocol for 3D planning of orthognathic skeletal movements and 3D planning and printing of patient-specific fixation plates and surgical guides.

Technological advancements in surgical planning and patient-specific implants are constantly evolving. One can either adopt the technology to achieve ...

Development and Evaluation of 3D-Printed Cardiovascular Phantoms for Interventional Planning and Training

Catheter-based interventions are standard treatment options for cardiovascular pathologies. Therefore, patient-specific models could help training physicians&#39; wire-skills as well as improving planning of interventional procedures. The aim of this study was to develop a manufacturing process of patient-specific 3D-printed models for cardiovascular interventions.
To create a 3D-printed elastic phantom, different 3D-printing materials were compared to porcine biological tissues (i.e., aortic tissue) in terms of mechanical characteristics. A fitting material was selected based on comparative tensile tests and specific material thicknesses were defined. Anonymized contrast-enhanced CT-datasets were collected retrospectively. Patient-specific volumetric models were extracted from these datasets and subsequently 3D-printed. A pulsatile flow loop was constructed to simulate the intraluminal blood flow during interventions. Models&#39; suitability for clinical imaging was assessed by x-ray imaging, CT, 4D-MRI and (Doppler) ultrasonography. Contrast medium was used to enhance visibility in x-ray-based imaging. Different catheterization techniques were applied to evaluate the 3D-printed phantoms in physicians&#39; training as well as for pre-interventional therapy planning.
Printed models showed a high printing resolution (~30 &#181;m) and mechanical properties of the chosen material were comparable to physiological biomechanics. Physical and digital models showed high anatomical accuracy when compared to the underlying radiological dataset. Printed models were suitable for ultrasonic imaging as well as standard x-rays. Doppler ultrasonography and 4D-MRI displayed flow patterns and landmark characteristics (i.e., turbulence, wall shear stress) matching native data. In a catheter-based laboratory setting, patient-specific phantoms were easy to catheterize. Therapy planning and training of interventional procedures on challenging anatomies (e.g., congenital heart disease (CHD)) was possible.
Flexible patient-specific cardiovascular phantoms were 3D-printed, and the application of common clinical imaging techniques was possible. This new process is ideal as a training tool for catheter-based (electrophysiological) interventions and can be used in patient-specific therapy planning.

Here we present development of a mock circulation setup for multimodal therapy evaluation, pre-interventional planning, and physician-training on cardiovascular anatomies. With the application of patient-specific tomographic scans, this setup is ideal for therapeutic approaches, training, and education in individualized medicine.