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.
