Name: a friction testing-bioreactor device for study of synovial joint biomechanics, mechanobiology, and physical regulation
Uploaded: 2022-06-02T13:50:00
Description: Watch this Scientific Journal Video about A Friction Testing-Bioreactor Device for Study of Synovial Joint Biomechanics, Mechanobiology, and Physical Regulation at JoVE.com

This work was supported by the Orthopaedic Scientific Research Foundation, NIH 5R01 AR068133, NIH TERC 5P41EB027062, and NIGMS R01 692 GM083925 (Funder ID: 10.13039/100000057).

Juvenile bovine knee joints, obtained from a local abattoir, were used for the present study. Studies with such bovine specimen samples are exempted from Columbia Institutional Animal Care and Use Committee (IACUC).
1. Designing the friction testing device
NOTE: A schematic representation of the friction testing device is shown in Figure 1. The device is built on a rigid base plate (not shown), which serves as a platform ...

A dynamic mechanical environment exists within the joint as cartilage is subjected to compressive, tensile, and shear forces, and hydrostatic and osmotic pressures44,45. Although cartilage is the main load-bearing tissue of the joint, the synovium also undergoes frictional interactions with the cartilage surface and with itself in regions where the tissue folds. The physical interactions between cartilage and synovium are likely responsible for transferring cells...

Osteoarthritis (OA) is a debilitating, degenerative joint disease that affects more than 32 million American adults, with a healthcare and socio-economic cost of over $16.5 billion1. The disease has classically been characterized by the degradation of articular cartilage and subchondral bone; however, changes to the synovium have recently garnered appreciation as synovitis has been linked to OA symptoms and progression2,3,4. In primary (idiopathic) OA, normal &#39;wear and tear&#39; associated with aging inhibits cartilage&#39;s ability to sustain its load-bearing and lubrication functions. The stresses generated by prolonged sliding contact of articular cartilage layers or sliding contact of cartilage against implant materials have been shown to facilitate delamination wear through subsurface fatigue failure5,6. As a dynamic mechanical environment exists within the joint7,8, 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 has been designed to replicate the motion of the joint with tight control over compressive and frictional loading5,6,9,10,11,12,13.
The present protocol describes a friction testing device that delivers reciprocal, translating motion and compressive load to contacting surfaces of living tissue explants. The computer-controlled device permits user control of the duration of each test, applied load, range of motion of the translation stage, and translation speed. The device is modular, allowing for testing of various counterfaces, such as tissue-on-tissue (cartilage-on-cartilage and synovium-on-cartilage) and tissue-on-glass. In addition to the functional measurements obtained by the tester, tissue and lubricating bath components can be assessed before and after testing to evaluate the biological changes imparted by a given experimental regimen.
Studies of cartilage tribology have been performed for decades, and several techniques have been developed to measure friction coefficients between cartilage and glass and cartilage on cartilage14,15. The different approaches are motivated by the joint and/or the lubrication mechanism of interest. There is often a tradeoff between the control of experimental variables and the recapitulation of physiologic parameters. Pendulum-style devices utilize intact joints as the fulcrum of a simple pendulum where one joint surface translates freely over the second surface14,16,17,18. Instead of using intact joints, friction measurements may be obtained by sliding cartilage explants over desired surfaces14,19,20,21,22,23,24,25. Reported friction coefficients of articular cartilage have varied over a wide range (from 0.002 to 0.5) depending on the operating conditions14,26. Devices have been created to replicate rotary motion23,27,28. Gleghorn et al.26 developed a multi-well custom tribometer to observe cartilage lubrication profiles using Stribeck curve analysis, and a linear oscillatory sliding motion was applied between cartilage against a flat glass counterface.
This device aims to isolate frictional responses and explore the mechanobiology of living tissues under various loading conditions. The device employs a simplified test set-up simulating joint articulation through compressive sliding, which can approximate both rolling and sliding motion with the understanding that the resistance in pure rolling motion is negligible relative to the measured friction coefficient of articular cartilage29. Originally built to study the effects of interstitial fluid pressurization on the frictional response of articular cartilage9, the tester has since been used to explore topics such as frictional effects of removing the superficial zone of cartilage10, lubricating effects of synovial fluid11, cartilage wear hypotheses5,6,30, and synovium-on-tissue friction measurements13. The friction-testing bioreactor can conduct friction experiments under sterile conditions, providing a novel mechanism to explore how frictional forces affect the mechanobiological responses of living cartilage and synovium. This design can be used as a biomimetic bioreactor to study the physical regulation of living joint tissues in response to applied physiologic loading associated with diarthrodial joint articulation.
This study presents a configuration for synovium-on-cartilage friction testing over a range of contact stresses and in different lubricating baths. The articulating surface area of most joints is, to a great extent, synovial tissue31. While synovium-on-cartilage sliding does not occur at primary load-bearing surfaces, the frictional interactions between the two tissues may still have important implications for tissue level repair and cell mechanotransduction. It has previously been shown that fibroblast-like synoviocytes (FLS) residing on the intimal layer of the synovium are mechanosensitive, responding to fluid-induced shear stress32. It has also been demonstrated that stretch33,34 and fluid-induced shear stress35 modulate FLS lubricant production. As such, direct sliding contact between synovium and cartilage may provide another mechanical stimulus to resident cells in the synovium.
Only a few reports on synovium friction coefficients have been published31,36. Estell et al.13 sought to expand on the previous characterization by utilizing biologically relevant counterfaces. With the friction testing device&#39;s ability to test living tissues, it is possible to mimic physiologic tissue interactions during joint articulation to elucidate the role of contact shear stress on synoviocyte function and its contribution to the crosstalk between synovium and cartilage. The latter has been implicated in mediating synovial joint inflammation in arthritis and post-injury. Due to the physical proximity of cartilage to synovium and synovial fluid, which contain synoviocytes that exhibit multipotent capacity, including chondrogenesis, it is postulated that synoviocytes play a role in cartilage homeostasis and repair by engrafting to the articular surface. In this context, physical contact and reciprocal shearing of cartilage-synovium and synovium-synovium may increase the accessibility of synoviocytes to regions of cartilage damage37,38,39,40. Studies utilizing synovium-on-cartilage configurations will not only provide insights into joint gross tissue mechanics and tribology, but they may also lead to new strategies for maintaining joint health.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Aluminum foil</td>
			<td>Reynolds Group Holdings</td>
			<td>Reynolds Wrap</td>
			<td>Sterile tissue harvest</td>
		</tr>
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			<td>Aluminum-framed acrylic enclosure</td>
			<td>Custom made</td>
			<td></td>
			<td>Friction tester component</td>
		</tr>
		<tr>
			<td>Autoclavable instant sealing sterilization pouches</td>
			<td>Fisherbrand</td>
			<td>01-812-54</td>
			<td>Sterilization of tools</td>
		</tr>
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			<td>Autoclave</td>
			<td>Buxton</td>
			<td></td>
			<td>Sterilization of tools</td>
		</tr>
		<tr>
			<td>Beaker (250 mL)</td>
			<td>Pyrex Vista</td>
			<td>70000</td>
			<td>Tissue harvest</td>
		</tr>
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			<td>Betadine (Povidone Iodine Prep Solution)</td>
			<td>Medline Industries, LP</td>
			<td>MDS093906</td>
			<td>Sterile tissue harvest</td>
		</tr>
		<tr>
			<td>Biological safety cabinet</td>
			<td>Labconco</td>
			<td>Purifier Logic+ Class II, Type A2 BSC</td>
			<td>Sterile tissue harvest</td>
		</tr>
		<tr>
			<td>Biospy punch</td>
			<td>Steritool Inc.</td>
			<td>50162</td>
			<td>Tissue harvest</td>
		</tr>
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			<td>Box cutter</td>
			<td>American Safety Razor Company</td>
			<td>94-120-71</td>
			<td>Tissue harvest</td>
		</tr>
		<tr>
			<td>Circular acrylic-sillicone post (synovium)</td>
			<td>Custom made</td>
			<td></td>
			<td>Tissue mounting</td>
		</tr>
		<tr>
			<td>Culture media</td>
			<td>Custom made</td>
			<td></td>
			<td>DMEM (Cat No. 11-965-118; Gibco) supplemented with 50 &#956;g/mL L-proline (Cat. No. P5607; Sigma), 100 &#956;g/mL sodium pyruvate (Cat. No. S8636; Sigma), 1% ITS (Cat. No. 354350; Corning), and 1% antibiotic&#8211;antimycotic (Cat. No. 15-240-062, Gibco)</td>
		</tr>
		<tr>
			<td>Cyanoacrylate (Loctite 420 Clear)</td>
			<td>Henkel</td>
			<td>135455</td>
			<td>Tissue mounting</td>
		</tr>
		<tr>
			<td>Dead weights</td>
			<td>OHAUS</td>
			<td></td>
			<td>Normal load</td>
		</tr>
		<tr>
			<td>Ethanol 200 proof</td>
			<td>Decon Labs, Inc.</td>
			<td>2701</td>
			<td>Dilute to 70 %</td>
		</tr>
		<tr>
			<td>Fixed base</td>
			<td>ThorLabs, Inc.</td>
			<td>SB1T</td>
			<td>Friction tester component</td>
		</tr>
		<tr>
			<td>Forceps (synovium harvest)</td>
			<td>Fine Science Tools</td>
			<td>11019-12</td>
			<td>Tissue harvest</td>
		</tr>
		<tr>
			<td>Forceps (synovium mounting)</td>
			<td>Excelta</td>
			<td>3C-S-PI</td>
			<td>Tissue mounting</td>
		</tr>
		<tr>
			<td>Horizontal linear encoder (for translating stage)</td>
			<td>RSF Electronics, Inc.</td>
			<td>MSA 670.63</td>
			<td>Friction tester component; system resolution of 1 &#181;m</td>
		</tr>
		<tr>
			<td>Hot glue gun and glue</td>
			<td>FPC Corporation</td>
			<td>Surebonder Pro 4000A</td>
			<td>Tissue mounting</td>
		</tr>
		<tr>
			<td>LabVIEW</td>
			<td>National Instruments Corporation</td>
			<td>LabVIEW&#160; 2010</td>
			<td>Friction testing program</td>
		</tr>
		<tr>
			<td>Load cell</td>
			<td>JR3 Inc.</td>
			<td>20E12A-M25B</td>
			<td>Friction tester component; 0.0019 lbs resolution in x&#38;y, 0.0038 lbs resolution in z</td>
		</tr>
		<tr>
			<td>Loading platen</td>
			<td>Custom made</td>
			<td></td>
			<td>Tissue mounting</td>
		</tr>
		<tr>
			<td>O-ring</td>
			<td>Parker</td>
			<td>S1138AS568-009</td>
			<td>Tissue mounting</td>
		</tr>
		<tr>
			<td>Petri dish (60 mm)</td>
			<td>Falcon</td>
			<td>351007</td>
			<td>Tissue mounting</td>
		</tr>
		<tr>
			<td>PivotLok Work Positioner (tibia holder)</td>
			<td>Industry Depot, Pivot Lok</td>
			<td>PL325</td>
			<td>Tissue harvest</td>
		</tr>
		<tr>
			<td>Removable base</td>
			<td>ThorLabs, Inc.</td>
			<td>SB1B</td>
			<td>Friction tester component</td>
		</tr>
		<tr>
			<td>Ring stand</td>
			<td></td>
			<td></td>
			<td>Tissue harvest</td>
		</tr>
		<tr>
			<td>Scalpel blades</td>
			<td>Havel&#39;s Inc.</td>
			<td>FSC22</td>
			<td>Tissue harvest</td>
		</tr>
		<tr>
			<td>Scalpel handle</td>
			<td>FEATHER Safety Razor Co., Ltd.</td>
			<td>No. 4</td>
			<td>Tissue harvest</td>
		</tr>
		<tr>
			<td>Screwdriver</td>
			<td>Wera</td>
			<td>3334</td>
			<td>Tissue harvest</td>
		</tr>
		<tr>
			<td>Stage</td>
			<td>JMAR</td>
			<td></td>
			<td>Friction tester component</td>
		</tr>
		<tr>
			<td>Stepper motor</td>
			<td>Oriental Motor Co., Ltd.</td>
			<td>PK266-03B</td>
			<td>Friction tester component</td>
		</tr>
		<tr>
			<td>Suction tool</td>
			<td>Virtual Industries, Inc.</td>
			<td>PEN-VAC Vacuum Pen</td>
			<td>Tissue mounting</td>
		</tr>
		<tr>
			<td>Support rod</td>
			<td>Custom made</td>
			<td></td>
			<td>Tissue mounting</td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td>Fine Science Tools</td>
			<td>14061-09</td>
			<td>Tissue mounting</td>
		</tr>
		<tr>
			<td>Synovial fluid (bovine)</td>
			<td>Animal Technologies, Inc.</td>
			<td></td>
			<td>Friction testing bath</td>
		</tr>
		<tr>
			<td>Testing bath</td>
			<td>Custom made</td>
			<td></td>
			<td>Phosphate-Buffered Saline (PBS) with protease inhibitors: 0.04% isothiazolone-base biocide (Proclin 950 Cat. No. 46878-U; Sigma) and 0.1% protease inhibitor - 0.05 M ethylenediaminetetraacetic acid, EDTA (Cat. No. 0369; Sigma)</td>
		</tr>
		<tr>
			<td>Tissue culture incubator</td>
			<td>Fisher Scientific</td>
			<td>Isotemp</td>
			<td>Sterile culture</td>
		</tr>
		<tr>
			<td>Vertical linear encoder (for loading stage)</td>
			<td>Renishaw</td>
			<td>T1031-30A</td>
			<td>Friction tester component; 20 nm resolution</td>
		</tr>
		<tr>
			<td>Voice coil actuator</td>
			<td>H2W Technologies</td>
			<td>NCC20-15-027-1RC</td>
			<td>Friction tester component</td>
		</tr>
	</tbody>
</table>

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.

A synovium-on-cartilage configuration was used to friction test juvenile bovine explants. The synovium was mounted on a 10 mm diameter acrylic loading platen such that the intimal layer would be in contact with the underlying cartilage. A tibial strip was used as the cartilage counterface (Figure 6A). Tibial strips were cut with a depth of approximately 1.4 mm and a size of 10 mm x 30 mm. The samples were tested for 1 h at 37 &#176;C in a phosphate-buffered saline (PBS) bath or a bovine syno...

Watch this Scientific Journal Video about A Friction Testing-Bioreactor Device for Study of Synovial Joint Biomechanics, Mechanobiology, and Physical Regulation at JoVE.com

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

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 ...

Traditionally we've used the tester to study non-living tissues to calculate friction coefficients but we are significantly expanding the tester's capabilities to test living tissues and to evaluate biological interactions. The friction testing device delivers reciprocal translating motion and compressive load to contacting surfaces of living tissue explants. The device is modular, allowing for the testing of various biological counterfaces.The method could provide insight into how frictional forces affect the mechanical and mechanicobiological responses of living cartilage and synovium, which may lead to new strategies for maintaining joint health. Begin by harvesting the juvenile bovine synovium. Using a scalpel blade, trace the outline of the synovium region of interest.Using forceps, grasp one end of the synovium and gently lift to stretch the synovium distal to the underlying bone. Use a scalpel blade to remove the synovium from the bone and place the tissue in appropriate culture media or testing bath solution. Then harvest the juvenile bovine cartilage by securing the tibia in an adjustable holder.Remove the meniscus carefully while avoiding contact with the cartilage surface. On the outer edges of the tibial plateau, use a box cutter to cut perpendicular to the cartilage toward the bone. Cut completely through the cartilage to make straight edges or sides.Remove excess tissue. On the outside edges, use the box cutter to make a clean cut at the interface between the bone and the cartilage. To remove the tibial strip from the plateau surface, gently insert a flathead screwdriver below the cut and gently rotate to loosen the articular cartilage from the subchondral bone.As the sample loosens, slowly push the screwdriver forward until the cartilage strip detaches from the bone. Ensure that the screwdriver is pushed towards the bone, not the cartilage. Using a box cutter, cut the tibial plateau surface to produce rectangular samples of desired size and thickness.Place tissue in appropriate culture media or testing bath solution. If the tibial strip is used as the bottom counterface, remove the detachable magnetic base and glue a 60-millimeter diameter Petri dish to the top surface of the detachable base. With the Petri dish glued in place, attach the detached base to the fixed base and mark the Petri dish to indicate a sliding direction.Apply a small amount of cyanoacrylate to the center of the dish. Align the tibial strip with the sliding direction of the stage. Gently press the cartilage strip onto the dish.Restore the removable magnetic base to its paired fixed base in the friction tester. Fill the Petri dish with the desired testing bath solution. If the synovium is used as the top counterface, remove the loading platen and support rod from the friction tester.Place the synovium on top of the circular platen. To secure the synovium, spread an O-ring over its circumference. Using forceps, gently pull at the synovium to stretch tissue taut and flat beneath the O-ring.Trim excess tissue with surgical scissors. Restore the loading platen and support rod to the friction tester. Adjust the vertical height of the loading platen such that the synovium hovers over the bottom counterface and is submerged in the testing bath.Insert the mounted specimens into the friction tester device. Open the analog data build MF DAQ. Initialize Load PID and Trigger Dynamic Caller Windows in the program.Run the analog data build MF DAQ and initialize load PID window by pressing the Run button. Navigate to the Stepper tab in the Trigger Dynamic Caller window. Specify the acceleration, speed, and distance of the translation stage in the user input boxes.To input the test duration, click on the Open Folder button at the bottom right of the Time State table and select the Stepper Time Index file path. Then specify the test duration again in the Voice Coil tab. Select the Voice Coil Index file path by clicking on the Open Folder button at the bottom right of the Time State table and select the file.This must be performed whether the voice coil is used or not. Apply the normal load. If using deadweights, place desired weights on the linear bearings above the loading platen.Ensure that the load applied plus the weight of the loading platen and support rod do not surpass the load cell-rated capacity. Select the path and file name for data storage using the Open Folder button to the right of the Right to File box. Save the file with a txt extension.Center the bottom counterface underneath the top counterface and set this as the zero X position. To do this, run the Trigger Dynamic Caller window by pressing the Run button. In the Stepper tab click on the Home button to move the stage to the last saved zero X position.If counterfaces are not aligned, move the stage by clicking the green left and right arrow buttons. When the desired location is reached, click on the zero button to save the current stage location as the new zero X position. Stop the Trigger Dynamic Caller window by clicking on the Stop button.Once the top and bottom counterfaces are centered, initiate friction testing of the samples by starting the cyclic movement of the stage. Once the stage moves, slowly bring the top counterface into contact with the bottom. Let the test run, collecting the friction testing data.After the desired testing duration, stop the test by pressing the Stop button and by unloading the specimens by raising the top counterface and moving it out of contact with the bottom counterface. Use the custom code to calculate the friction coefficient and the hysteresis per cycle. Ensure a single folder contains all relevant files.Open the Friction Cycle run. m file. Click on the Run button in the script.Select the raw data file to analyze and the desired save location. The friction coefficient and hysteresis plots will be output by MATLAB. A synovium-on-cartilage configuration was used to friction test juvenile bovine explants where the synovium was mounted on a 10-millimeter diameter acrylic loading platen such that the intimal layer would be in contact with the underlying cartilage.A tibial strip was used as the cartilage counterface. An effective friction coefficient was calculated from the average of FT divided by FN over each reciprocating cycle and then plotted against test duration to yield a friction coefficient-versus-time plot. For each test, friction coefficient values were averaged over the entire test.In a PBS testing bath, the average friction coefficient values increased as the contact stress increased. Conversely, the average friction coefficient values remained similar as the contact stress increased in a bovine synovial fluid bath. Remember to start collecting load data before bringing the surfaces into contact.This ensures a proper tare load can be calculated. Tissue and lubricating bath components can be assessed before and after testing to evaluate the biological changes imparted by a given experimental regimen.

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