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 grinding paper starting with a grain size of 500. Use grinding paper in increasing order up to a grain size of 4000.</li>
	<li>Polish the cylinder with 3 &#181;m and 1 &#181;m paste to achieve a surface roughness that is within the tolerance level of surface finish requirements for metallic surgical implants (ISO 5832-12:2019) and total and partial joint replacement implants (ISO 21534:2007). 
	NOTE: The average surface roughness is determined using a confocal microscope.</li>
	<li>Cut CoCrMo rods (&#216; of 6 mm) to cylinders with a length of 10 mm.</li>
</ol>
2. Harvesting of osteochondral cylinders
<ol>
	<li>Use bovine stifle joints from skeletally mature animals (aged 18-24 months at the time of sacrifice) and keep them contained and cooled until dissection within 24 h after sacrifice. 
	NOTE: Joints are purchased from the local butcher. The joint remains closed until dissection.</li>
	<li>To harvest cylindrical osteochondral plugs under aseptic conditions, disinfect the knee and perform an arthrotomy and expose the medial femoral condyle. 
	NOTE: The dissection has to be performed with caution not to damage the articular surface.</li>
	<li>Inspect the articular surface for macroscopic damages. 
	NOTE: Discard the sample if the cartilage lacks its whitish, smooth and glossy appearance or if there is blistering, fissures or larger defects.</li>
	<li>Align the cutting tube perpendicular to the articular surface of the weight-bearing area and drive the device into the cartilage and subchondral bone by firm strokes with a hammer. At 15 mm penetration depth, twist the device clockwise with a sudden motion.</li>
	<li>Remove the device, insert the white knob and screw it in until the bottom end of the osteochondral plug is visible.</li>
	<li>Mark the anteroposterior orientation of the samples with a sterile marker in order to arrange the osteochondral cylinder accordingly during testing. 
	NOTE: The three-dimensional collagen network and its complex architecture facilitate the unique mechanical properties of articular cartilage and should be considered in the orientation of the samples.</li>
	<li>Rinse the sample with phosphate-buffered saline (PBS) to wash off blood and fat tissue.</li>
	<li>Repeat the steps mentioned above to harvest the desired number of osteochondral plugs (8 mm diameter, 15 mm length). 
	NOTE: Typically, 9 to 12 osteochondral cylinders can be harvested from the weight bearing area on the medial femoral condyle.</li>
	<li>Place the samples in Dulbecco&#8217;s modified Eagle&#8217;s medium containing 10% fetal bovine serum, supplemented with antibiotics (penicillin 200 U/mL; streptomycin 0.2 mg/mL) and amphotericin B 2.5 &#181;g/mL and store them at 4 &#176;C until testing to maintain viability.</li>
	<li>Analyze control osteochondral plugs immediately after harvesting to establish baseline values (see analysis section).</li>
</ol>
3. Tribological testing
<ol>
	<li>Perform the experiments using a commercially available reciprocating tribometer with a cylinder-on-plate configuration. Requirements for the device are vertical loading and adjustable load and sliding speed. Furthermore, a liquid cell enables to perform the tests in a lubricating solution.</li>
	<li>Determine the contact pressure in the CoCrMo-on-cartilage system using a pressure measurement film. Place the pressure measurement film at the interface and apply static load for 30 s to determine initial contact pressure, contact size and shape. Owing to the convexity of the metal cylinder and the articular cartilage, the initial contact area has an elliptical shape in this configuration. 
	NOTE: The pressure measurement film reacts to the applied pressure showing red discoloration of zones where the threshold pressure is reached or exceeded. For 1 N of load, the contact pressure was determined around 2 MPa by visual comparison with defined contact pressures.</li>
	<li>Fix the osteochondral cylinders on the bottom sample holder with the marking aligned with the sliding direction, and mount the CoCrMo cylinders onto the upper load cell.</li>
	<li>Add the testing solution (PBS with 3 g/L hyaluronic acid) into the liquid cell that results in submerging the osteochondral cylinder and covering the metal-cartilage sliding interface.</li>
	<li>Set the testing parameters (prescribed normal force, stroke and sliding speed), which are then applied and maintained throughout the test. 
	NOTE: The stroke length of the reciprocating motion must be set according to the contact area to create a migrating contact area (MCA). For plugs that are 8 mm in diameter, a 2 mm stroke allows adequate rehydration of the cartilage.</li>
	<li>Start reciprocal sliding of the CoCrMo cylinder against the articular cartilage immersed in the lubricating solution with the set loading parameters.</li>
	<li>Monitor the coefficient of friction (COF) during the experiments. 
	NOTE: The COF is assessed automatically but can be calculated using the equation &#956;=F/W (&#956; - coefficient of friction; F - frictional force; W - normal load applied by the system).</li>
	<li>Terminate the experiment after the desired testing period.</li>
	<li>Remove the osteochondral plug from the sample holder, rinse it with PBS and store it in medium until further biological analysis (see below).</li>
	<li>Submerge control samples in the testing solution at room temperature for the duration of the test and analyze together with samples that have been exposed to mechanical loading.</li>
</ol>
4. Analysis
NOTE: Osteochondral cylinder are analyzed for metabolic activity and gene expression to investigate biological activity; histology is performed to study cartilage surface integrity and the underlying matrix.
<ol>
	<li>Histology
	<ol>
		<li>For histological analysis, immerse the osteochondral plugs in 4% buffered formaldehyde solution at room temperature until further processing.</li>
		<li>Rinse the samples with PBS and place them into a plastic vessel.</li>
		<li>Add an excess of the ready-to-use decalcifier-solution so that all samples are covered.</li>
		<li>Apply constant agitation for 4 weeks for complete decalcification.</li>
		<li>After decalcification, embed the samples in water soluble glycols and resins and store them at &#8722;80 &#176;C.</li>
		<li>Obtain 6 &#181;m sections by cryosectioning transversal to the contact area.</li>
		<li>Subsequently, prepare the samples for Safranin O staining and Fastgreen counterstaining using a manufacturer&#8217;s protocol.</li>
		<li>Capture histological images using a microscope and process using imaging processing software.</li>
	</ol>
	</li>
	<li>Metabolic activity 
	NOTE: The metabolic activity of chondrocytes in the articular cartilage are investigated with an XTT-based ex vivo toxicology assay.
	<ol>
		<li>Rinse the osteochondral plug using PBS and place the sample in a Petri dish.</li>
		<li>Place a 24-well plate on a scale and zero the scale.</li>
		<li>Cut off the cartilage from the osteochondral graft with a scalpel in one piece.</li>
		<li>Bisect the cartilage in two equal pieces so that the contact area is equally distributed onto both cartilage pieces and mince one half to 1 mm&#179; pieces. The second half is used for gene expression analysis.</li>
		<li>Transfer the minced cartilage into one well of the prepared 24-well plate and determine the tissue weight.</li>
		<li>Repeat the steps mentioned above for each sample and add 1 mL of growth medium to each well of the plate.</li>
		<li>Add the XTT solution (490 &#181;L of XTT labelling reagent and 10 &#181;L of activation reagent) according to the manufacturer&#8217;s instruction and mix.</li>
		<li>Incubate the plate at 37 &#176;C and 5% CO2 for 4 h.</li>
		<li>After incubation, remove the supernatant and transfer it to a 5 mL tube.</li>
		<li>Extract the tetrazolium product by adding 0.5 mL of dimethyl sulfoxide (DMSO) to the cartilage tissue in the 24-well plate and apply continuous agitation for 1 h at room temperature.</li>
		<li>Remove the DMSO solution and pool it with the previously collected XTT solution.</li>
		<li>Transfer 100 &#181;L of the sample in triplicates in a 96-well plate on a plate reader and measure the absorbance at a wavelength of 492 nm and a reference wavelength at 690 nm.</li>
		<li>Normalize the resulting absorbance values to the wet weight of each sample and perform analysis using software.</li>
	</ol>
	</li>
	<li>Gene expression analysis
	<ol>
		<li>RNA isolation 
		NOTE: RNA isolation is carried out using a commercial kit (Table of Materials) according to the instructions provided by the manufacturer with small amendments.
		<ol>
			<li>Mince the second half of the cartilage tissue obtained from the osteochondral plug into small pieces.</li>
			<li>Transfer them to a tube containing ceramic beads and 300 &#181;L of Lysis Buffer (containing 1% &#946;-mercaptoethanol). 
			NOTE: The samples can be frozen in liquid nitrogen until further processing.</li>
			<li>Thaw the samples for 2 min and use the commercial lyser for homogenization of the tissue. Apply 6500 rpm for 20 s (homogenization step) four times with a 2 min cooling phase after each run (at 4 &#176;C using the commercial lyser cooling device) to fully disrupt the tissue.</li>
			<li>Add 20 &#181;L of proteinase K and 580 &#181;L of RNase-free water to each tube and incubate them at 55 &#176;C for 30 min.</li>
			<li>Centrifuge the samples for 3 min at 10,000 x g and transfer the supernatant to a 1.5 mL tube.</li>
			<li>Add 0.5 volumes of 90% ethanol to each tube and mix.</li>
			<li>Transfer 700 &#181;L of the sample to an RNA binding column placed in a 2 mL collection tube and centrifuge at 8,000 x g for 15 s.</li>
			<li>Discard the flow-through and repeat the centrifugation step for the complete lysate.</li>
			<li>Add 350 &#181;L of Buffer RW1 to the column, centrifuge at 8,000 x g for 15 s, and discard the flow-through.</li>
			<li>Mix 10 &#181;L of DNase stock solution and 70 &#181;L of Buffer RDD. Add the solution to the RNA purification membrane and incubate it at room temperature for 15 min.</li>
			<li>Add 350 &#181;L of Buffer RW1 to the column and centrifuge at 8,000 x g for 15 s. Discard the flow-through.</li>
			<li>Add 500 &#181;L of Buffer RPE and centrifuge at 8,000 x g for 15 s. Discard the flow-through.</li>
			<li>Add 500 &#181;L of Buffer RPE to the RNA purification column and centrifuge at 8,000 x g for 2 min.</li>
			<li>Place the column in a 1.5 mL collection tube and add 30 &#181;L of RNase-free water. Centrifuge at 8,000 x g for 1 min.</li>
			<li>Store the isolated RNA at -80 &#176;C until cDNA synthesis.</li>
		</ol>
		</li>
		<li>cDNA synthesis 
		NOTE: To synthesize complementary DNA (cDNA) from messenger RNA (mRNA) a commercial kit (Table of Materials) was used. RNA from bacteriophage MS2 was added to stabilize isolated RNA during cDNA synthesis.
		<ol>
			<li>Thaw and mix the reagents. The composition for a single reaction is shown in Table 1.</li>
			<li>Add 16 &#181;L of RNA sample to the volume for a single reaction (14 &#181;L).</li>
			<li>Perform cDNA synthesis in a thermal cycler using the following parameters: 10 min at 25 &#176;C (primer annealing), 60 min at 50 &#176;C (DNA synthesis), 5 min at 85 &#176;C (denaturation) and 5 min at 20 &#176;C (cooling phase).</li>
			<li>Store cDNA at -20 &#176;C until real-time quantitative polymerase chain reaction (RT-qPCR).</li>
		</ol>
		</li>
		<li>RT-qPCR 
		NOTE: For RT-qPCR of bovine samples, primers and probes were designed by using commercial Real-Time qPCR software (e.g., IDT) for the genes GAPDH (Glyceraldehyde 3-phosphate dehydrogenase), COL2A1 (Collagen type 2), ACAN (Aggrecan), COL1A1 (Collagen type 1), MMP-1 (Matrix Metalloproteinase-1), and MMP-13 (Matrix Metalloproteinase-13). Bovine primers and double quenched probes were provided by IDT. The reagents used for a single reaction to evaluate the efficiency and gene expression are displayed in Table 2.
		<ol>
			<li>Dispense the master mix of a single reaction (9 &#181;L) to each well of a 96-well PCR plate and add 1 &#181;L of cDNA to each reaction. Perform tests for each sample in triplicates.</li>
			<li>Close the PCR plate using sealing oil and centrifuge at 877 x g for 10 min at 4 &#176;C.</li>
			<li>Perform RT-qPCR using a precision thermal cycler with the following protocol: 95 &#176;C for 10 min, 45 cycles of amplification (95 &#176;C for 10 s, annealing for 30 s, cDNA synthesis), and 37 &#176;C for 30 s. 
			NOTE: Specific annealing temperatures are required for each primer.</li>
			<li>Use GAPDH along with the target genes to confirm efficiency.</li>
			<li>Use the provided software to calculate the efficiency of each gene.</li>
			<li>Normalize the cycle threshold (CT) values to the expression of the reference gene GAPDH and use the &#916;&#916;CT method for quantification.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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The authors declare that they have no competing interests.

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

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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 ...

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

Research

JoVE Journal

Medicine

3.8K Views.  Danube University Krems. Ons protocol maakt het mogelijk om het schuiven van metaal voor orthopedische implantaten te testen tegen gewrichtskraakbeen en daarmee de effecten van focale implantaten of hemiarthroplastie op de biosynthetische activiteit van bepaalde chondracyten te onderzoeken het belangrijkste voordeel van deze techniek is dat het een uitgebreid inzicht biedt in zowel de tribologische eigenschappen als de biologische effecten van mechanische belasting in het kraakbeenkoppeling. Deze techniek kan complim...

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

Potentiodynamic Corrosion Testing

Different metallic materials have different polarization characteristics as dictated by the open circuit potential, breakdown potential, and passivation potential of the material. The detection of these electrochemical parameters identifies the corrosion factors of a material. A reliable and well-functioning corrosion system is required to achieve this.
Corrosion of the samples was achieved via a potentiodynamic polarization technique employing a three-electrode configuration, consisting of reference, counter, and working electrodes. Prior to commencement a baseline potential is obtained. Following the stabilization of the corrosion potential (Ecorr), the applied potential is ramped at a slow rate in the positive direction relative to the reference electrode. The working electrode was a stainless steel screw. The reference electrode was a standard Ag/AgCl. The counter electrode used was a platinum mesh. Having a reliable and well-functioning in vitro corrosion system to test biomaterials provides an in-expensive technique that allows for the systematic characterization of the material by determining the breakdown potential, to further understand the material's response to corrosion. The goal of the protocol is to set up and run an in vitro potentiodynamic corrosion system to analyze pitting corrosion for small metallic medical devices.

Here, we present a protocol to set up and run an in vitro potentiodynamic corrosion system to analyze pitting corrosion for small metallic medical devices.

Different metallic materials have different polarization characteristics as dictated by the open circuit potential, breakdown potential, and passivation ...

Environment

Multi-Scale Modification of Metallic Implants With Pore Gradients, Polyelectrolytes and Their Indirect Monitoring In vivo

Metallic implants, especially titanium implants, are widely used in clinical applications. Tissue in-growth and integration to these implants in the tissues are important parameters for successful clinical outcomes. In order to improve tissue integration, porous metallic implants have being developed. Open porosity of metallic foams is very advantageous, since the pore areas can be functionalized without compromising the mechanical properties of the whole structure. Here we describe such modifications using porous titanium implants based on titanium microbeads. By using inherent physical properties such as hydrophobicity of titanium, it is possible to obtain hydrophobic pore gradients within microbead based metallic implants and at the same time to have a basement membrane mimic based on hydrophilic, natural polymers. 3D pore gradients are formed by synthetic polymers such as Poly-L-lactic acid (PLLA) by freeze-extraction method. 2D nanofibrillar surfaces are formed by using collagen/alginate followed by a crosslinking step with a natural crosslinker (genipin). This nanofibrillar film was built up by layer by layer (LbL) deposition method of the two oppositely charged molecules, collagen and alginate. Finally, an implant where different areas can accommodate different cell types, as this is necessary for many multicellular tissues, can be obtained. By, this way cellular movement in different directions by different cell types can be controlled. Such a system is described for the specific case of trachea regeneration, but it can be modified for other target organs. Analysis of cell migration and the possible methods for creating different pore gradients are elaborated. The next step in the analysis of such implants is their characterization after implantation. However, histological analysis of metallic implants is a long and cumbersome process, thus for monitoring host reaction to metallic implants in vivo an alternative method based on monitoring CGA and different blood proteins is also described. These methods can be used for developing in vitro custom-made migration and colonization tests and also be used for analysis of functionalized metallic implants in vivo without histology.

Multi-Scale Modification of Metallic Implants With Pore Gradients, Polyelectrolytes and Their Indirect Monitoring In vivo

In this video, we will demonstrate modification techniques for porous metallic implants to improve their functionality and to control cell migration. Techniques include development of pore gradients to control cell movement in 3D and production of basement membrane mimics to control cell movement in 2-D. Also, a HPLC-based method for monitoring implant integration in-vivo via analysis of blood proteins is described.

Metallic implants, especially titanium implants, are widely used in  clinical applications. Tissue in-growth and integration to these implants in  the ...

Bioengineering

In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint

This study demonstrates a novel biomechanics testing protocol. The advantage of this protocol includes the use of an in situ loading device coupled to a high resolution X-ray microscope, thus enabling visualization of internal structural elements under simulated physiological loads and wet conditions. Experimental specimens will include intact bone-periodontal ligament (PDL)-tooth fibrous joints. Results will illustrate three important features of the protocol as they can be applied to organ level biomechanics: 1) reactionary force vs. displacement: tooth displacement within the alveolar socket and its reactionary response to loading, 2) three-dimensional (3D) spatial configuration and morphometrics: geometric relationship of the tooth with the alveolar socket, and 3) changes in readouts 1 and 2 due to a change in loading axis, i.e. from concentric to eccentric loads. Efficacy of the proposed protocol will be evaluated by coupling mechanical testing readouts to 3D morphometrics and overall biomechanics of the joint. In addition, this technique will emphasize on the need to equilibrate experimental conditions, specifically reactionary loads prior to acquiring tomograms of fibrous joints. It should be noted that the proposed protocol is limited to testing specimens under ex vivo conditions, and that use of contrast agents to visualize soft tissue mechanical response could lead to erroneous conclusions about tissue and organ-level biomechanics.

In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint

In this study, the use of an in situ loading device coupled with micro-X-ray computed tomography for fibrous joint biomechanics will be discussed. Experimental readouts identifiable with an overall change in joint biomechanics will include: 1) reactionary force vs. displacement, i.e. tooth displacement within the alveolar socket and its reactionary response to loading, 2) three-dimensional (3D) spatial configuration and morphometrics, i.e.&#160;geometric relationship of the tooth with the alveolar socket, and 3) changes in readouts 1 and 2 due to a change in loading axis, i.e. concentric or eccentric loads.

This study demonstrates a novel biomechanics testing protocol. The advantage of this protocol includes the use of an in situ loading device coupled to a ...

An Improved Mechanical Testing Method to Assess Bone-implant Anchorage

Recent advances in material science have led to a substantial increase in the topographical complexity of implant surfaces, both on a micro- and a nano-scale. As such, traditional methods of describing implant surfaces -&#160;namely numerical determinants of surface roughness -&#160;are inadequate for predicting in vivo performance. Biomechanical testing provides an accurate and comparative platform to analyze the performance of biomaterial surfaces. An improved mechanical testing method to test the anchorage of bone to candidate implant surfaces is presented. The method is applicable to both early and later stages of healing and can be employed for any range of chemically or mechanically modified surfaces -&#160;but not smooth surfaces. Custom rectangular implants are placed bilaterally in the distal femora of male Wistar rats and collected with the surrounding bone. Test specimens are prepared and potted using a novel breakaway mold and the disruption test is conducted using a mechanical testing machine. This method allows for alignment of the disruption force exactly perpendicular, or parallel, to the plane of the implant surface, and provides an accurate and reproducible means for isolating an exact peri-implant region for testing.

An improved method to mechanically test bone anchorage to candidate implant surfaces is presented. This method allows for alignment of the disruption force exactly perpendicular, or parallel, to the plane of the implant surface, and provides an accurate means to direct the disruption forces to an exact peri-implant region.

Recent advances in material science have led to a substantial increase in the topographical complexity of implant surfaces, both on a micro- and a ...

Determining Tribocorrosion Rate and Wear-Corrosion Synergy of Bulk and Thin Film Aluminum Alloys

The increasing complexity and severity of service conditions in areas, such as aerospace and marine industries, nuclear systems, microelectronics, batteries, and biomedical devices, etc., impose great challenges on the reliable performance of alloys exposed to extreme conditions where mechanical and electrochemical attack co-exist. Finding ways for alloys to mitigate the combined attack of wear and corrosion (i.e., tribocorrosion) under such extreme conditions is thus highly critical for improving their reliability and service lifetime when used in such conditions. The challenge lies in the fact that wear and corrosion are not independent of each other, but rather work synergistically to accelerate the total material loss. Thus, a reliable method to evaluate the tribocorrosion resistance of metals and alloys is needed. Here, a protocol for measuring the tribocorrosion rate and wear-corrosion synergy of Al-based bulk and thin film samples in a corrosive environment under room temperature is presented.

Here, we present a protocol to measure the tribocorrosion rate and wear-corrosion synergy of thin film and bulk Al alloys in simulated sea water at room temperature.

The increasing complexity and severity of service conditions in areas, such as aerospace and marine industries, nuclear systems, microelectronics, ...

Engineering

Biomechanical Characterization of Human Soft Tissues Using Indentation and Tensile Testing

Regenerative medicine aims to engineer materials to replace or restore damaged or diseased organs. The mechanical properties of such materials should mimic the human tissues they are aiming to replace; to provide the required anatomical shape, the materials must be able to sustain the mechanical forces they will experience when implanted at the defect site. Although the mechanical properties of tissue-engineered scaffolds are of great importance, many human tissues that undergo restoration with engineered materials have not been fully biomechanically characterized. Several compressive and tensile protocols are reported for evaluating materials, but with large variability it is difficult to compare results between studies. Further complicating the studies is the often destructive nature of mechanical testing. Whilst an understanding of tissue failure is important, it is also important to have knowledge of the elastic and viscoelastic properties under more physiological loading conditions.
This report aims to provide a minimally destructive protocol to evaluate the compressive and tensile properties of human soft tissues. As examples of this technique, the tensile testing of skin and the compressive testing of cartilage are described. These protocols can also be directly applied to synthetic materials to ensure that the mechanical properties are similar to the native tissue. Protocols to assess the mechanical properties of human native tissue will allow a benchmark by which to create suitable tissue-engineered substitutes.

Tissue biomechanics is important for maintaining cell shape and function and for determining phenotype. This report demonstrates non-destructive mechanical protocols for characterizing elastic and viscoelastic properties of human soft tissues, which can be directly applied to tissue-engineered substrates to allow a close matching of engineered materials to native tissue.

Regenerative medicine aims to engineer materials to replace or restore damaged or diseased organs. The mechanical properties of such materials should ...

Novel Diagnostics in Revision Arthroplasty: Implant Sonication and Multiplex Polymerase Chain Reaction

In orthopedic patients, foreign body-associated infections, especially periprosthetic joint infections (PJIs), are a devastating complication of arthroplasty. Infection requires complex treatment, may result in long hospitalization and causes considerable costs. Multiple surgical revisions can be necessary in these patients, with a loss in function as well as in quality of life.
The routine preoperative diagnostics include blood examination for C-reactive protein (CRP) and other biomarkers, as well as joint aspirate analysis for cell count, differentiation, and culture. Intraoperative specimens for histology and microbiology are also standard procedure. The microbiological examination of removed implants with sonication, in combination with the implementation of molecular biology techniques in microbiology, represent two novel techniques currently employed to enhance the differential diagnostics of PJI.
We present here the step-wise procedure of analyzing joint aspirate and sonication fluid, using a cartridge-based multiplex polymerase chain reaction (PCR) system. Results were matched against conventional cultures and consensus criteria for PJI. Conventional microbiological cultures from tissue biopsies, joint aspirate and sonication fluid showed a sensitivity of 66.7%, 66.7%, and 88.9%, respectively, and a specificity of 82.3%, 54.6%, and 61.5%, respectively. The PCR diagnostic of the sonication fluid and the joint fluid showed a sensitivity of 50.0% and 55.6%, respectively, and both a specificity of 100.0%. Both PCR diagnostics combined had a sensitivity of 66.7% and a specificity of 100.0%. The multiplex PCR therefore presents a rapid diagnostic tool with moderate sensitivity but high specificity in diagnosing PJI.

Differential diagnostics in painful arthroplasty is crucial for treatment success. Joint aspiration is routinely performed preoperatively. Multiplex polymerase chain reaction of the joint aspirate and the sonication fluid, a feasible tool for quick pathogen detection from these samples is described.

In orthopedic patients, foreign body-associated infections, especially periprosthetic joint infections (PJIs), are a devastating complication of ...

A Friction Testing-Bioreactor Device for Study of Synovial Joint Biomechanics, Mechanobiology, and Physical Regulation

In primary osteoarthritis (OA), normal 'wear and tear' associated with aging inhibits the ability of cartilage to sustain its load-bearing and lubrication functions, fostering a deleterious physical environment. The frictional interactions of articular cartilage and synovium may influence joint homeostasis through tissue level wear and cellular mechanotransduction. To study these mechanical and mechanobiological processes, a device capable of replicating the motion of the joint is described. The friction testing device controls the delivery of reciprocal translating motion and normal load to two contacting biological counterfaces. This study adopts a synovium-on-cartilage configuration, and friction coefficient measurements are presented for tests performed in a phosphate-buffered saline (PBS) or synovial fluid (SF) bath. The testing was performed for a range of contact stresses, highlighting the lubricating properties of SF under high loads. This friction testing device can be used as a biomimetic bioreactor for studying the physical regulation of living joint tissues in response to applied physiologic loading associated with diarthrodial joint articulation.

The present protocol describes a friction testing device that applies simultaneous reciprocal sliding and normal load to two contacting biological counterfaces.

In primary osteoarthritis (OA), normal 'wear and tear' associated with aging inhibits the ability of cartilage to sustain its load-bearing and lubrication ...

Addressing Practical Issues in Atomic Force Microscopy-Based Micro-Indentation on Human Articular Cartilage Explants

Without a doubt, atomic force microscopy (AFM) is currently one of the most powerful and useful techniques to assess micro and even nano-cues in the biological field. However, as with any other microscopic approach, methodological challenges can arise. In particular, the characteristics of the sample, sample preparation, type of instrument, and indentation probe can lead to unwanted artifacts. In this protocol, we exemplify these emerging issues on healthy as well as osteoarthritic articular cartilage explants. To this end, we first show via a step-by-step approach how to generate, grade, and visually classify ex vivo articular cartilage discs according to different stages of degeneration by means of large 2D mosaic fluorescence imaging of the whole tissue explants. The major strength of the ex vivo model is that it comprises aged, native, human cartilage that allows the investigation of osteoarthritis-related changes from early onset to progression. In addition, common pitfalls in tissue preparation, as well as the actual AFM procedure together with the subsequent data analysis, are also presented. We show how basic but crucial steps such as sample preparation and processing, topographic sample characteristics caused by advanced degeneration, and sample-tip interaction can impact data acquisition. We also subject to scrutiny the most common problems in AFM and describe, where possible, how to overcome them. Knowledge of these limitations is of the utmost importance for correct data acquisition, interpretation, and, ultimately, the embedding of findings into a broad scientific context.

We present a step-by-step approach to identify and address the most common problems associated with atomic force microscopy micro-indentations. We exemplify the emerging problems on native human articular cartilage explants characterized by various degrees of osteoarthritis-driven degeneration.

Without a doubt, atomic force microscopy (AFM) is currently one of the most powerful and useful techniques to assess micro and even nano-cues in the ...

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.

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

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