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).

The authors have nothing to disclose.

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 and releasing mesenchymal stem cells into the joint environment, offering a potential cell source to contribute to (limited) articular cartilage repair mechanisms37,38,39,40. The frictional properties of both cartilage and synovium have important implications for joint maintenance and degeneration through tissue wear13. A device capable of delivering reciprocal translating motion and compressive loading is presented to study the mechanical and mechanobiological processes responsible for joint homeostasis and disease progression.
The selection of testing parameters and specimen mounting are two critical steps of the protocol. The device applies a compressive load with either dead weights or a voice coil actuator. The custom software program allows for control over various parameters such as test duration, stage speed, and travel path. An issue may arise if the test duration is too short; when this is the case, the short duration does not allow the friction coefficient&#160;&#956; to reach equilibrium (&#956;eq). If the &#956;eq output is desired, the user must select an appropriate test duration that will be able to capture the tissue behavior until it becomes constant. Samples can reach equilibrium within a few hours of testing, depending on the size of the contact area on the tissue46. The type of test must also be considered. The device has been used in the stationary contact area and migrating contact area configurations to study cartilage friction properties5,6,9,11,12,47. The travel path, stage speed, and congruence of the two counterfaces can be manipulated to produce the desired testing mode. It is recommended to create real-time plots in the LabVIEW program user interface to assist in monitoring a test. Helpful plots include horizontal stage position vs. time, normal force vs. time, and tangential force vs. horizontal stage position (hysteresis, Figure 5C). For example, the top counterface must only rest on the bottom counterface to ensure the full prescribed load is applied. The applied load value can be confirmed by viewing the normal load real-time plot (Figure 5A). The mounting of specimens must be secure to prevent tissue slipping or tearing that will provide erroneous measurements. Synovium tearing due to improper mounting will result in an incorrect friction coefficient, as the mounting surface underneath the synovium will be exposed. This error may be detected by monitoring real-time hysteresis curves. The device&#39;s real-time assessment of functional properties is distinct from other friction testing systems.
All raw data needs to be written to a file that can be imported and processed by the desired data processing software. It is recommended to collect data at a frequency of at least 10 data points/second and to save raw data to a .csv or .txt file. The friction coefficient can be calculated for each position in each cycle by using the equation <img alt="Equation 1" src="/files/ftp_upload/63880/63880eq01.jpg" style="margin: auto;" /> where t and n refer to the tangential and normal forces, respectively, and where + and - refer to the forward and backward strokes, respectively, per cycle5. This formula recognizes that the sign of F-t is opposite to that of F+t. Normal force (Fn) is defined as the force in line with the applied load (z-direction, Figure 1), while tangential force (Ft) is the force parallel with sliding (x-direction, Figure 1). The cycle-average friction coefficient can be calculated by taking the mean of &#956; for all positions in a given cycle. The creep displacement is calculated by normalizing the vertical displacement of the top counterface such that the initial displacement is zero and the subsequent displacements are relative to the initial displacement. If desired, standard tissue assessments and media analyses can be performed on the tested explants and aliquots of the testing bath solution. Prior to analysis, it is recommended to record the testing bath volume to be used in data processing or normalization.
The modular counterfaces have enabled the adaptation of multiple testing configurations. Early studies used glass-on-cartilage testing to elucidate the role of interstitial fluid load support in cartilage tribology9,10. The importance of interstitial fluid pressurization was further validated by comparing stationary and migrating contact area tests for cartilage-on-cartilage and cartilage against glass11. Oungoulian et al.6 evaluated the wear mechanism of articular cartilage against metal alloys used in hemiarthroplasties and showed that the stresses generated by sliding contact for 4 h facilitated delamination wear through subsurface fatigue failure. This work was followed by Durney et al.5, who demonstrated that delamination wear can still occur when friction remains low under a migrating contact area configuration. Most recently, Estell et al.13 reported for the first time the friction properties of the synovium in testing conditions that mimicked native interactions with underlying tissues (cartilage and synovium) and in conditions that mimicked an osteoarthritic state (diluted synovial fluid bath with cartilage wear particles). Ultimately, the design flexibility of the friction testing device has permitted a wide range of experiments to be conducted, contributing to the greater understanding of cartilage and synovium tribology.
One limitation of the current system is that it can only maintain aseptic testing conditions for a few hours. This is achieved through the acrylic enclosure, sterilizing media-contacting components via autoclave, and spraying the testing device with 70% ethanol. The acrylic enclosure also includes a heating element and constant temperature monitoring capabilities. The heating element heats the air within the box, controlling the temperature of the inside environment, and can be controlled externally to avoid exposing the samples to the outside environment. Aseptic conditions can be further achieved by harvesting the specimens in a sterile biological safety cabinet (BSC) and assembling the specimens inside the BSC within a sterile container that can interface with the support rod and fixed base. For long-term studies, the acrylic enclosure can be outfitted with the necessary materials to provide a more sterile environment (ultraviolet light, proper airflow and filtration, and self-regulating temperature control). Another limitation is that the current friction testing device is configured to test a single top and bottom counterface. A multi-specimen counterface approach can be attained by altering the loading platen and removable base design, converting the current friction testing device to a bioreactor with a multi-well capacity to apply physiologic loading of cartilage-on-cartilage and synovium-on-cartilage. A working prototype using a 6-well plate has been created (Figure 7). The design reserves the ability to modulate top and bottom counterfaces as desired. The top of the plate is stationary and secured to a tissue culture incubator rack, while the bottom of the plate is attached to a translating stage. Similar to the current friction testing device, dead weight can be added to prescribe a normal load. With the bioreactor in a sterile environment, media can be sampled over time to evaluate biological responses to loading regimens. The next design iteration will look to create a stand-alone bioreactor that incorporates computer-controlled translation. If the complexity of the friction testing device were to be maintained in the bioreactor, changes to tissue mechanical and mechanobiological properties could be measured longitudinally.
A friction testing device that permits control over the delivery of reciprocal translating motion and normal load to two contacting biological counterfaces is described. In this study, a synovium-on-cartilage configuration was utilized to demonstrate the modularity of the device and the ability to study the frictional responses of living tissues. The representative results reaffirmed the role of synovial fluid in providing boundary lubrication to reduce wear and friction of the diarthrodial joint. The device permits the execution of multi-scale experiments ranging from bulk friction to mechanotransduction. The design can operate under sterile conditions for a few hours and can be converted to a long-term bioreactor to recapitulate the compressive sliding of the joint, thereby facilitating the study of biomechanics, mechanobiology, and physical regulation of living joint tissues. Future studies will contribute to understanding how healthy and diseased physical environments influence joint maintenance.

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><tbody><tr><td>Aluminum foil</td><td>Reynolds Group Holdings</td><td>Reynolds Wrap</td><td>Sterile tissue harvest</td></tr><tr><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><tr><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><tr><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><tr><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 &amp;mu;g/mL L-proline (Cat. No. P5607; Sigma), 100 &amp;mu;g/mL sodium pyruvate (Cat. No. S8636; Sigma), 1% ITS (Cat. No. 354350; Corning), and 1% antibiotic&amp;ndash;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 &amp;micro;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&amp;nbsp; 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&amp;amp;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&#039;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 synovial fluid (SF) bath. The SF bath consisted of a 50/50 mixture of PBS and bovine SF. The stage acceleration was 100 mm/s2, the stage speed was 1 mm/s, and the stage path distance was 2.5 mm6,9,42. Dead weights were used to apply various normal loads resulting in contact stresses of 180, 230, and 300 kPa11,43.
After one hour, the tissues were unloaded, and the friction coefficients were assessed. An effective friction coefficient&#160;&#956; was calculated from the average of Ft/Fn over each reciprocating cycle and then plotted against test duration to yield a friction coefficient vs. time plot (Figure 6B). For each test, values of&#160;&#956; were averaged over the entire test (all cycles) to produce &#956;avg. In a PBS testing bath, the &#956;avg values increased as the contact stress increased. The &#956;avg,PBS increased from 0.015 &#177; 0.005 at 180 kPa, to 0.019 &#177; 0.005 at 230 kPa, to 0.022 &#177; 0.010 at 300 kPa. Conversely, the &#8203;&#956;avg values remained similar as the contact stress increased in a SF bath (Figure 6C). The &#956;avg,SF was 0.013 &#177; 0.002 at 180 kPa, 0.011 &#177; 0.001 at 230 kPa, and 0.011 &#177; 0.001 at 300 kPa.
Overall, the results demonstrate the ability of the friction tester device to concomitantly apply reciprocal sliding and normal load to two biological counterfaces. In this study, synovium-on-cartilage samples tested in an SF bath did not display an increase in friction coefficient when the contact stress was increased, thereby supporting the notion that SF contributes to the low wear and low friction properties of the joint through a boundary lubrication mechanism.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63880/63880fig01.jpg" /> 
Figure 1: Schematic of two-axis custom friction testing device (left) and cross-section of loaded sample in Petri dish (right). The stage is attached to a motor which induces sliding motion and causes the bottom contacting surface to articulate against the top contacting surface. The load cell collects real-time load measurements, while the loading stage linear encoder collects real-time creep displacement measurements. The figure has been modified with permission from Reference10. <a href="https://www.jove.com/files/ftp_upload/63880/63880fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63880/63880fig02.jpg" /> 
Figure 2: Bovine synovium harvest. (A) The patellar tendon is severed using a horizontal incision superior to the tibia. (B,C) The patella is removed by making two anterior-to-posterior cuts in the shape of a V (dotted lines). (D) The outline of the synovium is traced with a scalpel blade. (E) The synovium is then stretched distal to the underlying bone and removed. Scale bar = 5 cm. <a href="https://www.jove.com/files/ftp_upload/63880/63880fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63880/63880fig03.jpg" /> 
Figure 3: Bovine femoral cartilage plug harvest. (A) A 15.9 mm diameter biopsy punch is inserted normal to the femoral condyle articular cartilage surface until the bone is reached. (B) The punch and plug are removed. Scale bar = 16 mm. <a href="https://www.jove.com/files/ftp_upload/63880/63880fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63880/63880fig04.jpg" /> 
Figure 4: Bovine cartilage tibial strip harvest. (A) The meniscus is removed from the tibial plateau. (B) The plateau edges are cut to make straight sides (inset). (C) The inside of the plateau is scored to create a strip. (D) A cut is made at the cartilage-bone interface. (E) A screwdriver is inserted below the cut. (F) The strip is removed. Scale bar = 10 mm. <a href="https://www.jove.com/files/ftp_upload/63880/63880fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63880/63880fig05.jpg" /> 
Figure 5: LabVIEW user interface. The custom program allows for controlling various test parameters such as stage acceleration, stage speed, travel path, and test duration. (A) Real-time applied load plot (Fz&#160;vs. t where Fz is the normal load Fn), (B) stepper position (ux&#160;vs. t), and (C) hysteresis plot (Fx&#160;vs. ux, where Fx is the tangential force Ft) are shown. <a href="https://www.jove.com/files/ftp_upload/63880/63880fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/63880/63880fig06.jpg" /> 
Figure 6: Synovium-on-cartilage friction measurements. (A) The friction testing device configured for juvenile bovine synovium (inset) on a tibial cartilage strip. (B) Representative friction coefficient (&#956;) as a function of time plot. (C) The friction coefficient for various contact stresses (180 kPa, blue; 230 kPa, red; 300 kPa, green) in a phosphate-buffered saline (PBS, closed circle) or bovine synovial fluid (SF, open circle) bath. Error bars are mean with standard deviation. <a href="https://www.jove.com/files/ftp_upload/63880/63880fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/63880/63880fig07.jpg" /> 
Figure 7: Friction bioreactor. (A) Schematic of friction bioreactor with stationary top counterfaces and moving bottom counterfaces. (B) A side-view and (C) bottom view of the bioreactor applying physiologic shear in a synovium-on-cartilage configuration. (D) The bioreactor is housed inside of a tissue culture incubator. <a href="https://www.jove.com/files/ftp_upload/63880/63880fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental Coding Files.&#160;<a href="https://www.jove.com/files/ftp_upload/63880/63880_Hung_Supplementary_Coding_Files.zip" target="_blank">Please click here to download this file.</a>

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 ...

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Unit 1

Friction

The Joints

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2.8K Views.  Columbia University. The present protocol describes a friction testing device that applies simultaneous reciprocal sliding and normal load to two contacting biological counterfaces. 

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

Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix (ECM) components within the tumor microenvironment. Hyaluronic acid (HA) is one stromal ECM component that is known to facilitate tumor progression by enhancing invasion, growth, and angiogenesis1. Interaction of HA with its cell surface receptor CD44 induces signaling events that promote tumor cell growth, survival, and migration, thereby increasing metastatic spread2-3. HA is an anionic, nonsulfated glycosaminoglycan composed of repeating units of D-glucuronic acid and D-N-acetylglucosamine. Due to the presence of carboxyl and hydroxyl groups on repeating disaccharide units, native HA is largely hydrophilic and amenable to chemical modifications that introduce sulfate groups for photoreative immobilization 4-5. Previous studies involving the immobilizations of HA onto surfaces utilize the bioresistant behavior of HA and its sulfated derivative to control cell adhesion onto surfaces6-7. In these studies cell adhesion preferentially occurs on non-HA patterned regions.
To analyze cellular interactions with exogenous HA, we have developed patterned functionalized surfaces that enable a controllable study and high-resolution visualization of cancer cell interactions with HA. We utilized microcontact printing (uCP) to define discrete patterned regions of HA on glass surfaces. A "tethering" approach that applies carbodiimide linking chemistry to immobilize HA was used 8. Glass surfaces were microcontact printed with an aminosilane and reacted with a HA solution of optimized ratios of EDC and NHS to enable HA immobilization in patterned arrays. Incorporating carbodiimide chemistry with mCP enabled the immobilization of HA to defined regions, creating surfaces suitable for in vitro applications. Both colon cancer cells and breast cancer cells implicitly interacted with the HA micropatterned surfaces. Cancer cell adhesion occurred within 24 hours with proliferation by 48 hours. Using HA micropatterned surfaces, we demonstrated that cancer cell adhesion occurs through the HA receptor CD44. Furthermore, HA patterned surfaces were compatible with scanning electron microscopy (SEM) and allowed high resolution imaging of cancer cell adhesive protrusions and spreading on HA patterns to analyze cancer cell motility on exogenous HA.

A novel approach that allows the high-resolution analysis of cancer cell interactions with exogenous hyaluronic acid (HA) is described. Patterned surfaces are fabricated by combining carbodiimide chemistry and microcontact printing.

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix ...

Experiments on Ultrasonic Lubrication Using a Piezoelectrically-assisted Tribometer and Optical Profilometer

Friction and wear are detrimental to engineered systems. Ultrasonic lubrication is achieved when the interface between two sliding surfaces is vibrated at a frequency above the acoustic range (20 kHz). As a solid-state technology, ultrasonic lubrication can be used where conventional lubricants are unfeasible or undesirable. Further, ultrasonic lubrication allows for electrical modulation of the effective friction coefficient between two sliding surfaces. This property enables adaptive systems that modify their frictional state and associated dynamic response as the operating conditions change. Surface wear can also be reduced through ultrasonic lubrication. We developed a protocol to investigate the dependence of friction force reduction and wear reduction on the linear sliding velocity between ultrasonically lubricated surfaces. A pin-on-disc tribometer was built which differs from commercial units in that a piezoelectric stack is used to vibrate the pin at 22 kHz normal to the rotating disc surface. Friction and wear metrics including effective friction force, volume loss, and surface roughness are measured without and with ultrasonic vibrations at a constant pressure of 1 to 4 MPa and three different sliding velocities: 20.3, 40.6, and 87 mm/sec. An optical profilometer is utilized to characterize the wear surfaces. The effective friction force is reduced by 62% at 20.3 mm/sec. Consistently with existing theories for ultrasonic lubrication, the percent reduction in friction force diminishes with increasing speed, down to 29% friction force reduction at 87 mm/sec. Wear reduction remains essentially constant (49%) at the three speeds considered.

We present a protocol for using a piezoelectrically-assisted tribometer and optical profilometer to investigate the dependence of ultrasonic wear and friction reduction on linear velocity, contact pressure, and surface properties.

Friction and wear are detrimental to engineered systems. Ultrasonic lubrication is achieved when the interface between two sliding surfaces is vibrated at ...

Engineering

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

A Novel Stretching Platform for Applications in Cell and Tissue Mechanobiology

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have contributed dramatically to the understanding of basic mechanobiology. These techniques have been extensively used to demonstrate how the onset and progression of various diseases are heavily influenced by mechanical cues. This article presents a multi-functional biaxial stretching (BAXS) platform that can either mechanically stimulate single cells or quantify the mechanical stiffness of tissues. The BAXS platform consists of four voice coil motors that can be controlled independently. Single cells can be cultured on a flexible substrate that can be attached to the motors allowing one to expose the cells to complex, dynamic, and spatially varying strain fields. Conversely, by incorporating a force load cell, one can also quantify the mechanical properties of primary tissues as they are exposed to deformation cycles. In both cases, a proper set of clamps must be designed and mounted to the BAXS platform motors in order to firmly hold the flexible substrate or the tissue of interest. The BAXS platform can be mounted on an inverted microscope to perform simultaneous transmitted light and/or fluorescence imaging to examine the structural or biochemical response of the sample during stretching experiments. This article provides experimental details of the design and usage of the BAXS platform and presents results for single cell and whole tissue studies. The BAXS platform was used to measure the deformation of nuclei in single mouse myoblast cells in response to substrate strain and to measure the stiffness of isolated mouse aortas. The BAXS platform is a versatile tool that can be combined with various optical microscopies in order to provide novel mechanobiological insights at the sub-cellular, cellular and whole tissue levels.

We present in this article a novel stretching platform that can be used to investigate single cell responses to complex anisotropic biaxial mechanical deformation and quantify the mechanical properties of biological tissues.

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have ...

A Multi-Cue Bioreactor to Evaluate the Inflammatory and Regenerative Capacity of Biomaterials under Flow and Stretch

The use of resorbable biomaterials to induce regeneration directly in the body is an attractive strategy from a translational perspective. Such materials induce an inflammatory response upon implantation, which is the driver of subsequent resorption of the material and the regeneration of new tissue. This strategy, also known as in situ tissue engineering, is pursued to obtain cardiovascular replacements such as tissue-engineered vascular grafts. Both the inflammatory and the regenerative processes are determined by the local biomechanical cues on the scaffold (i.e., stretch and shear stress). Here, we describe in detail the use of a custom-developed bioreactor that uniquely enables the decoupling of stretch and shear stress on a tubular scaffold. This allows for the systematic and standardized evaluation of the inflammatory and regenerative capacity of tubular scaffolds under the influence of well-controlled mechanical loads, which we demonstrate on the basis of a dynamic co-culture experiment using human macrophages and myofibroblasts. The key practical steps in this approach&#8212;the construction and setting up of the bioreactor, preparation of the scaffolds and cell seeding, application and maintenance of stretch and shear flow, and sample harvesting for analysis&#8212;are discussed in detail.

The goal of this protocol is to execute a dynamic co-culture of human macrophages and myofibroblasts in tubular electrospun scaffolds to investigate material-driven tissue regeneration, using a bioreactor which enables the decoupling of shear stress and cyclic stretch.

The use of resorbable biomaterials to induce regeneration directly in the body is an attractive strategy from a translational perspective. Such materials ...

Stiffness Measurement of Soft Silicone Substrates for Mechanobiology Studies Using a Widefield Fluorescence Microscope

Soft tissues in the human body typically have stiffness in the kilopascal (kPa) range. Accordingly, silicone and hydrogel flexible substrates have been proven to be useful substrates for culturing cells in a physical microenvironment that partially mimics in vivo conditions. Here, we present a simple protocol for characterizing the Young's moduli of isotropic linear elastic substrates typically used for mechanobiology studies. The protocol consists of preparing a soft silicone substrate on a Petri dish or stiff silicone, coating the top surface of the silicone substrate with fluorescent beads, using a millimeter-scale sphere to indent the top surface (by gravity), imaging the fluorescent beads on the indented silicone surface using a fluorescence microscope, and analyzing the resultant images to calculate the Young's modulus of the silicone substrate. Coupling the substrate's top surface with a moduli extracellular matrix protein (in addition to the fluorescent beads) allows the silicone substrate to be readily used for cell plating and subsequent studies using traction force microscopy experiments. The use of stiff silicone, instead of a Petri dish, as the base of the soft silicone, enables the use of mechanobiology studies involving external stretch. A specific advantage of this protocol is that a widefield fluorescence microscope, which is commonly available in many labs, is the major equipment necessary for this procedure. We demonstrate this protocol by measuring the Young's modulus of soft silicone substrates of different elastic moduli.

Substrates with stiffness in the kilopascal-range are useful to study the response of cells to physiologically relevant micro-environment stiffness. Using just a widefield fluorescence microscope, the Young's modulus of soft silicone gels can be determined using an indentation with a suitable sphere.

Soft tissues in the human body typically have stiffness in the kilopascal (kPa) range. Accordingly, silicone and hydrogel flexible substrates have been ...

Biomechanical Testing of Murine Tendons

Tendon disorders are common, affect people of all ages, and are often debilitating. Standard treatments, such as anti-inflammatory drugs, rehabilitation, and surgical repair, often fail. In order to define tendon function and demonstrate efficacy of new treatments, the mechanical properties of tendons from animal models must be accurately determined. Murine animal models are now widely used to study tendon disorders and evaluate novel treatments for tendinopathies; however, determining the mechanical properties of mouse tendons has been challenging. In this study, a new system was developed for tendon mechanical testing that includes 3D-printed fixtures that exactly match the anatomies of the humerus and calcaneus to mechanically test supraspinatus tendons and Achilles tendons, respectively. These fixtures were developed using 3D reconstructions of native bone anatomy, solid modeling, and additive manufacturing. The new approach eliminated artifactual gripping failures (e.g., failure at the growth plate failure rather than in the tendon), decreased overall testing time, and increased reproducibility. Furthermore, this new method is readily adaptable for testing other murine tendons and tendons from other animals.

The protocol describes efficient and reproducible tensile biomechanical testing methods for murine tendons through the use of custom-fit 3D printed fixtures.

Tendon disorders are common, affect people of all ages, and are often debilitating. Standard treatments, such as anti-inflammatory drugs, rehabilitation, ...

Creation of a Knee Joint-on-a-Chip for Modeling Joint Diseases and Testing Drugs

The high prevalence of debilitating joint diseases like osteoarthritis (OA) poses a high socioeconomic burden. Currently, the available drugs that target joint disorders are mostly palliative. The unmet need for effective disease-modifying OA drugs (DMOADs) has been primarily caused by the absence of appropriate models for studying the disease mechanisms and testing potential DMOADs. Herein, we describe the establishment of a miniature synovial joint-mimicking microphysiological system (miniJoint) comprising adipose, fibrous, and osteochondral tissue components derived from human mesenchymal stem cells (MSCs). To obtain the three-dimensional (3D) microtissues, MSCs were encapsulated in photocrosslinkable methacrylated gelatin before or following differentiation. The cell-laden tissue constructs were then integrated into a 3D-printed bioreactor, forming the miniJoint. Separate flows of osteogenic, fibrogenic, and adipogenic media were introduced to maintain the respective tissue phenotypes. A commonly shared stream was perfused through the cartilage, synovial, and adipose tissues to enable tissue crosstalk. This flow pattern allows the induction of perturbations in one or more of the tissue components for mechanistic studies. Furthermore, potential DMOADs can be tested via either &#34;systemic administration&#34; through all the medium streams or &#34;intraarticular administration&#34; by adding the drugs to only the shared &#34;synovial fluid&#34;-simulating flow. Thus, the miniJoint can serve as a versatile in vitro platform for efficiently studying disease mechanisms and testing drugs in personalized medicine.

We provide detailed methods for generating four types of tissues from human mesenchymal stem cells, which are used to recapitulate the cartilage, bone, fat pad, and synovium in the human knee joint. These four tissues are integrated into a customized bioreactor and connected through microfluidics, thus generating a knee joint-on-a-chip.

The high prevalence of debilitating joint diseases like osteoarthritis (OA) poses a high socioeconomic burden. Currently, the available drugs that target ...

A Probing Device for Quantitatively Measuring the Mechanical Properties of Soft Tissues during Arthroscopy

Probing in arthroscopic surgery is performed by pulling or pushing the soft tissue, which provides feedback for understanding the condition of the soft tissue. However, the output is only qualitative based on the &#8220;surgeon&#8217;s feeling&#8221;. Herein is described a probing device developed to address this issue by measuring the resistance of soft tissues quantitatively with a tri-axial force sensor. Under both conditions (i.e., pull- and push-probing certain tissues mimicking the acetabular labrum and cartilage), this probing device is found to be useful for measuring some mechanical properties in joints during arthroscopy.

Probing during arthroscopy surgery is normally done to assess the condition of the soft tissue, but this approach has always been subjective and qualitative. This report describes a probing device that can measure the resistance of the soft tissue quantitatively with a tri-axial force sensor during arthroscopy.

Probing in arthroscopic surgery is performed by pulling or pushing the soft tissue, which provides feedback for understanding the condition of the soft ...

Medicine

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.

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 ...