Design of a flexing organ-chip to model in situ loading of the intervertebral disc

A. AF physiological strain analysis to design the annulus-on-a-chip

Strains applied to the intervertebral disc are translated to the AF and NP.77. A. White and M. Panjabi, Clinical Biomechanics of the Spine (Lippincott, 1978). Degeneration reduces internal pressure from the NP, increasing the magnitude of AF strains and the prevalence of tears.3535. H. A. L. Guerin and D. M. Elliott, “Degeneration affects the fiber reorientation of human annulus fibrosus under tensile load,” J. Biomech. 39, 1410–1418 (2006). https://doi.org/10.1016/j.jbiomech.2005.04.007,3636. G. D. O’Connell, E. J. Vresilovic, and D. M. Elliott, “Human intervertebral disc internal strain in compression: The effect of disc region, loading position, and degeneration,” J. Orthop. Res. 29, 547–555 (2011). https://doi.org/10.1002/jor.21232 When designing the AoC, we chose to replicate strains in a degenerated posterior AF1212. N. M. Shahraki, A. Fatemi, A. Agarwal, and V. K. Goel, “Prediction of clinically relevant initiation and progression of tears within annulus fibrosus,” J. Orthop. Res. 35, 113–122 (2017). https://doi.org/10.1002/jor.23346 because of its susceptibility to tearing and initiating IDD.66. A. C. Schwarzer, C. N. Aprill, R. Derby, J. Fortin, G. Kine, and N. Bogduk, “The prevalence and clinical features of internal disc disruption in patients with chronic low back pain,” Spine 20, 1878–1883 (1995). https://doi.org/10.1097/00007632-199509000-00007Much of the literature on AF mechanics has focused on uniaxial tissue stretching,35–3735. H. A. L. Guerin and D. M. Elliott, “Degeneration affects the fiber reorientation of human annulus fibrosus under tensile load,” J. Biomech. 39, 1410–1418 (2006). https://doi.org/10.1016/j.jbiomech.2005.04.00737. G. D. O’Connell, H. L. Guerin, and D. M. Elliott, “Theoretical and uniaxial experimental evaluation of human annulus fibrosus degeneration,” J. Biomech. Eng. 131, 111007 (2009). https://doi.org/10.1115/1.3212104 which applies large tensile strains in one direction and unconstrained contractile strains in the transverse directions due to the cell’s Poisson’s ratio. In contrast, in situ loading with additional boundary conditions results in more complex constraints that may increase the risk of AF tissue failure.3838. N. Berger-Roscher, G. Casaroli, V. Rasche, T. Villa, F. Galbusera, and H.-J. Wilke, “Influence of complex loading conditions on intervertebral disc failure,” Spine 42, E78–E85 (2017). https://doi.org/10.1097/BRS.0000000000001699,3939. M. Zhou, R. D. Huff, Y. Abubakr, and G. D. O’Connell, “Torque- and muscle-driven flexion induce disparate risks of in vitro herniation: A multiscale and multiphasic structure-based finite element study,” J. Biomech. Eng. 144, 061005 (2022). https://doi.org/10.1115/1.4053402 For example, under combined flexion, compression, and axial rotation, the posterior AF experiences inversely proportional tensile and contractile strains with low and variable circumferential strains.3232. D. Amin, C. Moawad, and J. Costi, “New findings confirm regional internal disc strain changes during simulation of repetitive lifting motions,” Ann. Biomed. Eng. 47, 1378–1390 (2019). https://doi.org/10.1007/s10439-019-02250-z,3636. G. D. O’Connell, E. J. Vresilovic, and D. M. Elliott, “Human intervertebral disc internal strain in compression: The effect of disc region, loading position, and degeneration,” J. Orthop. Res. 29, 547–555 (2011). https://doi.org/10.1002/jor.21232,3838. N. Berger-Roscher, G. Casaroli, V. Rasche, T. Villa, F. Galbusera, and H.-J. Wilke, “Influence of complex loading conditions on intervertebral disc failure,” Spine 42, E78–E85 (2017). https://doi.org/10.1097/BRS.0000000000001699 By considering the strains on an “engineering element” within the posterior AF we can consider different strain ratios with respect to the orientation commonly used for the disc [e.g., axial:radial or circumferential:radial strain ratios; Fig. 1(e)]. These strain ratios make it possible to translate from whole disc loading of all types on one scale to strain on the smaller scale in the AF.To create a target for strain orientation and proportionality for the AoC, we analyzed results from Amin et al.,3232. D. Amin, C. Moawad, and J. Costi, “New findings confirm regional internal disc strain changes during simulation of repetitive lifting motions,” Ann. Biomed. Eng. 47, 1378–1390 (2019). https://doi.org/10.1007/s10439-019-02250-z which evaluated axial, radial, and circumferential strains during the cyclic application of combined flexion, compression, and axial rotation on 12 degenerated discs (Pfirrmann grades II-III). We integrated the contributions from each strain direction by calculating strain ratios (axial:radial and circumferential:radial) across these discs at 20 000 cycles. The means and standard deviations were calculated from ratios of averaged strains to provide a physiological relevant strain window for the posterior AF. Circumferential:radial and axial:radial ratios were 0.05±0.35 and −0.95±1.17, respectively. The large standard deviations compared to the mean are due to the variation in circumferential strain directionality and not from variation in axial or radial strains. Ultimately, the AoC strain ratios were designed to fit within this physiologically relevant window by leveraging bending mechanics.

B. Device design and development

Several microfluidic devices have been developed which apply complex strains to cells that are viewable under brightfield microscopes.3434. A. Mainardi, E. Cambria, P. Occhetta, I. Martin, A. Barbero, S. Schären, A. Mehrkens, and O. Krupkova, “Intervertebral disc-on-a-chip as advanced in vitro model for mechanobiology research and drug testing: A review and perspective,” Front. Bioeng. Biotechnol. 9, 1–18 (2021). https://doi.org/10.3389/fbioe.2021.826867,4040. K. Kaarj and J. Y. Yoon, “Methods of delivering mechanical stimuli to organ-on-a-chip,” Micromachines 10, 700 (2019). https://doi.org/10.3390/mi10100700 While these devices do not replicate the complex strains needed for the AF, their designs were referenced in the development of the AoC.A device created by Hsieh et al. in 2014, for example, applies complex strains to annular hydrogel structures using unconfined compression.3030. H.-Y. Hsieh, G. Camci-Unal, T.-W. Huang, R. Liao, T.-J. Chen, A. Paul, F.-G. Tseng, and A. Khademhosseini, “Gradient static-strain stimulation in a microfluidic chip for 3d cellular alignment,” Lab. Chip. 14, 482–493 (2014). https://doi.org/10.1039/C3LC50884F Concentric rings of hydrogel structures enable a gradient of complex strains. Compression is applied statically for simplicity. In 2019, Lee et al. also used unconfined compression on hydrogel structures but added dynamic loading to the device.4141. D. Lee, A. Erickson, T. You, A. T. Dudley, and S. Ryu, “Pneumatic microfluidic cell compression device for high-throughput study of chondrocyte mechanobiology,” Lab. Chip. 18, 2077–2086 (2018). https://doi.org/10.1039/C8LC00320C Arranged in an array, these hydrogel structures are cylindrical and can receive various magnitudes of compressive strains. The same year, another form of dynamic complex strain was developed on a cartilage-on-a-chip device by Occhetta et al.4242. P. Occhetta, A. Mainardi, E. Votta, Q. Vallmajo-Martin, M. Ehrbar, I. Martin, A. Barbero, and M. Rasponi, “Hyperphysiological compression of articular cartilage induces an osteoarthritic phenotype in a cartilage-on-a-chip model,” Nat. Biomed. Eng. 3, 545–557 (2019). https://doi.org/10.1038/s41551-019-0406-3 This example uses rectangular posts along a 300 μm wide culture chamber to apply confined hyperphysiological compression. The culture chamber enables uniformity of applied strain by minimizing gradients as compared to the unconfined cylindrical structures.

Upon analysis of these existing microfluidic devices which apply complex strain, overarching design requirements for the AoC were established. The AoC needs a confined chamber like the cartilage-on-a-chip but one subject to bending to accomplish uniform physiological relevant strain. Like previous devices, the AoC also needs to be observable at all times during loading under a brightfield microscope. Unlike previous devices, the AoC needs to be operational with or without a hydrogel to enable various types of modeling. Additionally, the AoC needs to be simple in both design and in operation to allow for the easy adoption of existing protocols performed on other microfluidic organ-chip platforms with small amounts of tissue, such as running polymerase chain reaction-enzyme-linked immunosorbent assays (PCR-ELISA), microscale Western blotting, or immunofluorescence imaging necessary for future biological work.

To satisfy these design requirements, the AoC became a deformable, optically clear, mechanically actuated organ-chip device that applies strains to cells through bending [Figs. 1(a)1(c)]. The device was made of elastomeric polydimethylsiloxane (PDMS) with a 300×50μm2 channel created using standard soft lithography methods (further described in Sec. ).The AF consists of alternating layers of fiber-reinforced tissue called lamellae. The 300 μm channel width was chosen to approximate the width of one lamellae (average lamellae thickness=420μm±60μm in adults 53–76 years old).4343. F. Marchand and A. M. Ahmed, “Investigation of the laminate structure of lumbar disc anulus fibrosus,” Spine 15, 402–410 (1990). https://doi.org/10.1097/00007632-199005000-00011 The channel height (50 μm) was selected to be several times greater than the diameter of an AF cell (∼20μm). As a result, cells have access to media and can be distributed along the channel length as they flow unobstructed. Meanwhile, the ratio of channel width to height was kept small (6:1) to limit the possibility of channel collapse during manufacturing and testing. FE simulations later showed a 6% decrease in mean channel height at the chip’s maximum flexion state (Fig. S1A in the supplementary material), indicative of no channel collapse (Fig. S1B in the supplementary material).Lastly, the channel length was chosen to be the longest possible to accommodate the greatest number of cells, minimize differences in strain along its length and fall within known lengths for nutrient diffusion in the AF. The AF relies on diffusion of nutrients and waste primarily through the disc’s cartilaginous endplates given limited peripheral vasculature reaching only the outermost AF lamellae. Diffusion across the endplate alone results in a distance of at least 7–8  mm4444. C. M. De Geer, “Intervertebral disk nutrients and transport mechanisms in relation to disk degeneration: A narrative literature review,” J. Chiropr. Med. 17, 97–105 (2018). https://doi.org/10.1016/j.jcm.2017.11.006 with greater diffusion lengths by the time AF cells at the midheight of the AF are reached.1515. G. D. O’Connell, W. Johannessen, E. J. Vresilovic, and D. M. Elliott, “Human internal disc strains in axial compression measured noninvasively using magnetic resonance imaging,” Spine 32, 2860–2868 (2007). https://doi.org/10.1097/BRS.0b013e31815b75fb A half channel length of 8.5 mm, or 8.5 mm from the nearest port, falls within this physiological length for diffusion.

In total, the channel recapitulates a subunit of the AF at a much smaller scale than the whole disc. To account for this smaller scale, in situ whole disc loading, which includes a combination of axial and rotational loads, was converted to the multiaxial strains of different ratios (circumferential:radial and axial:radial) to be applied on the channel.

The device applied scalable and uniform strains on the channel when it was flexed over a rigid cylinder or barrel structure. The design of the cylinder and device thickness were made such that the axial, radial, and circumferential (or hoop) strains [Fig. 1(d)] were scaled to match physiological levels for the outer AF by repositioning the location of channel through the thickness of the PDMS device. In this design, radial and axial strains correspond to strains in the x- and y-directions, respectively [Fig. 1(e)]. For proof-of-concept testing, we kept the radius of curvature for the barrel constant and adjusted the location of the channel in the device to match strains observed in the posterior AF of degenerated discs. In the future, manufacturing could be simplified such that the channel location remains fixed while the extent of device bending dictates the applied strain.Device flexing was achieved by powering servo motors that lifted the device’s edges while keeping the channel in the focal plane of the microscope [Figs. 2(a) and 2(b)]. During device development, the applied strain due to flexion was approximated using beam theory. Tensile radial strain (ϵx) was approximated using the device thickness (t), barrel radius of curvature (r), and the distance between the channel and the neutral axis (yc), which was assumed to be equivalent to the centroidal axis [Fig. 2(c)]. Thus, the applied radial strain increased linearly by moving the channel further from the neutral axis (along the negative y-direction) or by decreasing the radius of curvature, ρ,Lastly, long-term device durability against cyclic loading was assessed. We applied 10% strain at 0.5 Hz for 66 K cycles to represent a week-long cell based study. Cycle count was selected based on daily activity. Rohlmann et al. measured a daily median of 4400 spine movements (most often in flexion) or ∼30 K movements each week.4545. A. Rohlmann, T. Consmüller, M. Dreischarf, M. Bashkuev, A. Disch, E. Pries, G. N. Duda, and H. Schmidt, “Measurement of the number of lumbar spinal movements in the sagittal plane in a 24-hour period,” Eur. Spine J. 23, 2375–2384 (2014). https://doi.org/10.1007/s00586-014-3588-0 To ensure that the AoC could withstand this week’s worth of loading cycles, an accelerated life test was performed for more than 2× the expected weekly loading cycles (66 K). Visual inspection of the AoC following accelerated life testing showed that the device was durable enough to withstand long-term cell-stretching studies and showed no signs of wear or fracture. The packing tape connecting the device to plastic cylinders connected to the servo motors exhibited subtle wear, but no signs of fracture. Servo motors and the apparatus to anchor each component remained intact.

C. Comparing measured device strains to in situ physiological strains in the AF

To confirm that the AoC could recapitulate the complex strains within the posterior AF, we compared measured device strain ratios to those found in Amin et al.3232. D. Amin, C. Moawad, and J. Costi, “New findings confirm regional internal disc strain changes during simulation of repetitive lifting motions,” Ann. Biomed. Eng. 47, 1378–1390 (2019). https://doi.org/10.1007/s10439-019-02250-z To this end, two sets of five devices were fabricated using the first-order beam theory approximation (see Sec. ). While the device could be designed for higher peak strains (15%–20%) as seen in other cell studies,26–2826. S. Molladavoodi, J. McMorran, and D. Gregory, “Mechanobiology of annulus fibrosus and nucleus pulposus cells in intervertebral discs,” Cell Tissue Res. 379, 429–444 (2020). https://doi.org/10.1007/s00441-019-03136-127. L. Ning, L. Gao, F. Zhang, X. Li, and T. Wang, “Mechanical stretch induces annulus fibrosus cell senescence through activation of the rhoa/rock pathway,” BioMed. Res. Int. 2021, 2021 (2021). https://doi.org/10.1155/2021/532112128. E. Cambria, M. J. Arlt, S. Wandel, O. Krupkova, W. Hitzl, F. S. Passini, O. N. Hausmann, J. G. Snedeker, S. J. Ferguson, and K. Wuertz-Kozak, “Trpv4 inhibition and crispr-cas9 knockout reduce inflammation induced by hyperphysiological stretching in human annulus fibrosus cells,” Cells 9, 1736 (2020). https://doi.org/10.3390/cells9071736,4646. M. Likhitpanichkul, O. M. Torre, J. Gruen, B. A. Walter, A. C. Hecht, and J. C. Iatridis, “Do mechanical strain and TNF-α interact to amplify pro-inflammatory cytokine production in human annulus fibrosus cells?,” J. Biomech. 49, 1214–1220 (2016). https://doi.org/10.1016/j.jbiomech.2016.02.029 initial device characterization and stretching process development was limited to 5%–10% strain based on the moderate to high physiological loading within the AF.2626. S. Molladavoodi, J. McMorran, and D. Gregory, “Mechanobiology of annulus fibrosus and nucleus pulposus cells in intervertebral discs,” Cell Tissue Res. 379, 429–444 (2020). https://doi.org/10.1007/s00441-019-03136-1 Measured applied strains in the x-direction were 7.4±0.8% and 11.9±1.5%, respectively. The relatively low standard deviation in the strains indicated that the device fabrication and loading was repeatable and robust.As expected, measured strains were consistently higher than the first-order approximation. This suggested that a more comprehensive 3D computational model was needed to describe strain fields within the device. Additionally, this model could be used to determine axial and circumferential strains (see Sec. ), which are difficult to measure experimentally.After modeling the 3D strain magnitude and orientation in the AoC, we calculated strain ratios within the channel to compare to the ratios in posterior AF tissue from degenerated human lumbar bone-disc-bone motion segments under flexion in situ (Table I). The standard deviations within the AoC’s ratios are due to the variation along the length of the device channel. We considered the AoC as physiologically relevant since the circumferential:radial and the axial:radial ratios fit within the window of posterior AF ratios. Strain ratios for a conventional PDMS uniaxial cell stretcher were included to illustrate how physiological relevant loads cannot be achieved with a traditional cell stretching method that relies on the Poisson’s effect of PDMS.Table icon

TABLE I. Comparison of strain ratios in the posterior AF within bone-disc-bone segments under flexion, the AoC, and commercial uniaxial cell stretchers.

Circumferential:radialAxial:radialPosterior AF0.05 ± 0.353232. D. Amin, C. Moawad, and J. Costi, “New findings confirm regional internal disc strain changes during simulation of repetitive lifting motions,” Ann. Biomed. Eng. 47, 1378–1390 (2019). https://doi.org/10.1007/s10439-019-02250-z−0.95 ± 1.173232. D. Amin, C. Moawad, and J. Costi, “New findings confirm regional internal disc strain changes during simulation of repetitive lifting motions,” Ann. Biomed. Eng. 47, 1378–1390 (2019). https://doi.org/10.1007/s10439-019-02250-zAnnulus-on-a-chip−0.0025 ± 0.07−0.77 ± 0.06Uniaxial cell stretcheran/a−0.55 to −0.44747. S. Dogru, B. Aksoy, H. Bayraktar, and B. E. Alaca, “Poisson’s ratio of pdms thin films,” Polym. Test. 69, 375–384 (2018). https://doi.org/10.1016/j.polymertesting.2018.05.044

D. Modeling device strains with finite element simulation

While strains in the channel were measured optically in one direction (radial), a finite element (FE) analysis was used to verify strain uniformity along the channel and assess strains in the other two directions. FE models were also used to assess strain heterogeneity, which can be used to alter the channel design for maximizing channel size and cell count. We developed models of three device configurations, with target strains of 0%, 5%, and 10% to validate our uniaxial strain measurements and establish the relationship between target strains throughout the bending cycle. We visualized the models’ strain in the x-direction [Fig. 3(a)], which was considered to be the “radial strain” with respect to orientation in the AF [Fig. 1(e)]. Strains in the y- and z-directions correspond to axial and circumferential strains, respectively, and are labeled accordingly.The FE results aligned well with our one-dimensional strain measurements taken under brightfield microscopy [Fig. 3(b)]. We then selected the “high strain” model (10% target) and varied the bending angle to achieve specific strain magnitudes within the channel. We defined the bending angle as being the angle from beneath the device to its outer edge with respect to the horizontal plane [Fig. 3(c), inset]. For 5% and 10% target strain, the model informed us to apply a bending angle of 9.8° and 19.2°, respectively [Fig. 3(c)]. Moreover, due to the linear relationship between strain and bending angle below 20°, we can apply controlled loading sequences of varying strains without varying the channel position. For example, we can alternate between 5% and 10% strain for each consecutive cycle or provide a 5% strain for 100 cycles before transitioning to applying a 10% strain for 100 cycles, and so on. The applied strain began to taper at 20°, which was due to the radius of curvature for the 3D printed barrel. For subsequent loading sequences, we utilized results from the model to modify the servo motor control sequence to ensure that the device applied 10% strain.The model provided insights into strain uniformity along the channel length. We observed a change in strain and strain ratios toward the ends of the channel [Figs. 3(d) and 3(e)]. These changes are likely due to the tendency of the device to bend into a hyperbolic paraboloid shape, imposing a unique strain distribution that cannot be estimated with the pure beam bending assumption. The curvature becomes more pronounced toward edges of the device, resulting in strain deviations at ∼2 mm away from the center of the device. A more uniform strain distribution may be obtained by redesigning the device with a shorter channel to standardize applied strain along the full length of the channel. Alternatively, the device may be widened and the channel length maintained to create a uniform strain distribution while avoiding a reduction in cell population within the channel. Using the model, we can iterate on the AoC design and achieve target cell populations and applied strain distributions without the need for extensive lab time or fabrication.

E. AF cell selection and culturing

Bovine AF cells were chosen for the AoC proof-of-concept testing due to accessibility and similarities with the human AF cells.1515. G. D. O’Connell, W. Johannessen, E. J. Vresilovic, and D. M. Elliott, “Human internal disc strains in axial compression measured noninvasively using magnetic resonance imaging,” Spine 32, 2860–2868 (2007). https://doi.org/10.1097/BRS.0b013e31815b75fb,48–5248. Z. Li, Y. Gehlen, F. Heizmann, S. Grad, M. Alini, R. G. Richards, D. Kubosch, N. Südkamp, K. Izadpanah, E. J. Kubosch et al., “Preclinical ex-vivo testing of anti-inflammatory drugs in a bovine intervertebral degenerative disc model,” Front. Bioeng. Biotechnol. 8, 583 (2020). https://doi.org/10.3389/fbioe.2020.0058349. M. Calió, B. Gantenbein, M. Egli, L. Poveda, and F. Ille, “The cellular composition of bovine coccygeal intervertebral discs: A comprehensive single-cell rnaseq analysis,” Int. J. Mol. Sci. 22, 4917 (2021). https://doi.org/10.3390/ijms2209491750. J. Desrochers and N. A. Duncan, “Strain transfer in the annulus fibrosus under applied flexion,” J. Biomech. 43, 2141–2148 (2010). https://doi.org/10.1016/j.jbiomech.2010.03.04551. G. D. O’Connell, E. J. Vresilovic, and D. M. Elliott, “Comparison of animals used in disc research to human lumbar disc geometry,” Spine 32, 328–333 (2007). https://doi.org/10.1097/01.brs.0000253961.40910.c152. C. Daly, P. Ghosh, G. Jenkin, D. Oehme, and T. Goldschlager, “A review of animal models of intervertebral disc degeneration: Pathophysiology, regeneration, and translation to the clinic,” BioMed. Res. Int. 2016, 5952165 (2016). https://doi.org/10.1155/2016/5952165 Static cell culture media was chosen instead of continuous flow to mimic AF tissue, which relies more on diffusion of nutrients than convection given its lack of vasculature.4444. C. M. De Geer, “Intervertebral disk nutrients and transport mechanisms in relation to disk degeneration: A narrative literature review,” J. Chiropr. Med. 17, 97–105 (2018). https://doi.org/10.1016/j.jcm.2017.11.006 To increase the reserve of nutrients without relying on convection, two pipet tips filled with 150 μL of media were placed in each port hole and replaced every third day inside a cell culture hood like other microphysiological system designs.5353. B. Charrez, V. Charwat, B. A. Siemons, I. Goswami, C. Sakolish, Y.-S. Luo, H. Finsberg, A. G. Edwards, E. W. Miller, I. Rusyn et al., “Heart muscle microphysiological system for cardiac liability prediction of repurposed covid-19 therapeutics,” Front. Pharmacol. 12, 4 (2021). https://doi.org/10.3389/fphar.2021.684252 Prior to strain experiments, we confirmed using live/dead imaging that AF cells could sustain long-duration static culture [3-weeks; Fig. 4(a)] three times longer than required for a typical study. The highest resolution live/dead fluorescent image was captured only on the last day of culture to limit the stress on the cells while the sample was transferred to a microscope in a separate facility. Cells within the channel were found to be clustered near the ports, as expected, but also covered the remainder of the channel floor in a sparse monolayer. The number of living cells neared 2000 without any dead cells. However, it is possible that dead cells were washed from the channel when the live/dead solution was added. Only a few cells were positioned just above the sparse monolayer by adhering to corner surfaces between the channel floor and sidewalls. Seventy percent of manufactured devices were used in the study; the other 30% of manufactured devices were removed from testing primarily because of manufacturing defects, which included delamination of PDMS layers. Bubbles in the channel, a low cell population (∼300 cells), and channel occlusion due to cell clumping were other, less frequent reasons to remove devices from testing. Future work will focus on reducing these losses.The effects of a protein coating on cell adhesion in the channel were considered during chip development. In the channel, fibronectin coated PDMS at various concentrations (0, 0.03, 0.06, 0.125, 0.25, and 0.5 mg/ml) was compared to the plasma treated PDMS. Cell adhesion for each treatment was determined by observing the number of cells that adhered in the channel before spreading one hour, one day, three days, and seven days after. As far as cell adhesion and spreading, fibronectin performed similarly to plasma treated PDMS in the channel (Fig. S2B in the supplementary material). While fibronectin may play an important role in cell adhesion in the presence of excessive loading, it was eliminated from the protocol for simplicity at this stage.We did not design the surface of the PDMS channel to include select areas for cell attachment; thus, cells were able to multiply during the culture period. The cell population was observed multiplying over multiple days using brightfield images until a high confluency was reached and maintained (Fig. S2A in the supplementary material).

While the cell population of the current design is too low for sufficient RNA yield for gene expression analysis, the device dimensions can be scaled to increase cell population while maintaining similar strain profiles. The devices can also be pooled to accumulate the required number of cells for the analysis.

F. Applying cyclic load to AF cells in the device

To establish the feasibility of the AoC as a platform for studying cell mechanobiology within load-bearing tissues, we seeded the device’s microchannel with a sparse population of bovine AF cells (hundreds of cells) to limit any effects of population size on cellular strain. While a greater cell density could be used, such that more cell–cell interactions occur, a consistent cell density should be used between treatment groups. We allowed the cells to proliferate for 3 days before applying cyclic loading. A strain of 3.5% at 0.5 Hz for 75 cycles was chosen to represent low physiological loading.5454. G. Sowa, P. Coelho, N. Vo, R. Bedison, A. Chiao, C. Davies, R. Studer, and J. Kang, “Determination of annulus fibrosus cell response to tensile strain as a function of duration, magnitude, and frequency,” J. Orthop. Res. 29, 1275–1283 (2011). https://doi.org/10.1002/jor.21388 We took brightfield images of the channel before loading, with applied strain, and after the cyclic loading. By processing the brightfield images with ImageJ, we observed cell deformity and strains in parallel (radial) and perpendicular (circumferential) directions relative to the channel. Despite the lower resolution (10× objective lens), consistent differences can be seen in annotated brightfield images of the cells between the strained and unstrained channel [Fig. 4(b)]. However, with our initial study looking at cells during chip deformation, we did not observe significant cell migration and the image resolution was not sufficient to track cell deformations. However, we think that such a system can be used with better imaging facilities to study cell behaviors with loading. An additional brightfield image was taken after strain was applied to confirm that cells remained adhered to the channel for at least an hour after loading [Fig. 4(c)]. Meanwhile, static devices were used as controls to identify changes to cell viability, migration, and morphology due to the cell microenvironment within the chip. Cell migration was observed without mechanical loading (Fig. S3 in the supplementary material), which will need to be considered when measuring cell changes under mechanical load.

G. Limitations

The AoC has been proven to be durable and effective at applying physiological strains within 0%–10%. However, in situ strains in the AF can be hyperphysiological (15%–20% and greater).26–2826. S. Molladavoodi, J. McMorran, and D. Gregory, “Mechanobiology of annulus fibrosus and nucleus pulposus cells in intervertebral discs,” Cell Tissue Res. 379, 429–444 (2020). https://doi.org/10.1007/s00441-019-03136-127. L. Ning, L. Gao, F. Zhang, X. Li, and T. Wang, “Mechanical stretch induces annulus fibrosus cell senescence through activation of the rhoa/rock pathway,” BioMed. Res. Int. 2021, 2021 (2021). https://doi.org/10.1155/2021/532112128. E. Cambria, M. J. Arlt, S. Wandel, O. Krupkova, W. Hitzl, F. S. Passini, O. N. Hausmann, J. G. Snedeker, S. J. Ferguson, and K. Wuertz-Kozak, “Trpv4 inhibition and crispr-cas9 knockout reduce inflammation induced by hyperphysiological stretching in human annulus fibrosus cells,” Cells 9, 1736 (2020). https://doi.org/10.3390/cells9071736,3232. D. Amin, C. Moawad, and J. Costi, “New findings confirm regional internal disc strain changes during simulation of repetitive lifting motions,” Ann. Biomed. Eng. 47, 1378–1390 (2019). https://doi.org/10.1007/s10439-019-02250-z,4646. M. Likhitpanichkul, O. M. Torre, J. Gruen, B. A. Walter, A. C. Hecht, and J. C. Iatridis, “Do mechanical strain and TNF-α interact to amplify pro-inflammatory cytokine production in human annulus fibrosus cells?,” J. Biomech. 49, 1214–1220 (2016). https://doi.org/10.1016/j.jbiomech.2016.02.029 Strains in the 15%–20% range are achievable on the AoC by either decreasing the radius of curvature, ρ or the distance, yc [Fig. 2(c)]. However, additional long-term cyclic testing is needed to evaluate device durability given these higher strains.

Furthermore, to measure strains more accurately than with brightfield images, a reference and a deformed image can be acquired using fluorescent protein tags or live cell dyes (e.g., live/dead or f-actin). Digital image correlation can then convert these images to strain maps. While we detected morphological differences in strained vs unstrained bovine AF cells, measuring these differences and converting them to strains maps using digital image correlation remains challenging due to low-contrast images. Ongoing work is focused on establishing high-contrast images, which would enable digital image correlation as well as automatic, live-cell segmentation.

It should also be noted that cell access to nutrients depends on position along the length of the channel, which may play a role in viability as well as response to loads. For this reason, cell position will be taken into account as a possible variable in the future.

Additionally, the throughput in terms of cells and test replicates are limited. For a higher throughput of cells per device, all dimensions can be scaled to increase the volume and surface area of the channel while still creating a similar strain environment. For a higher throughput of tests, multiple devices with the existing dimensions could be placed in an array and actuated simultaneously.

Also, while culturing cells in the current design, pipet tips acting as media reservoirs are open to the air, which increases the possibility of contamination. To mitigate the risk of contamination, filtered or barrier pipet tips can be used instead of traditional pipet tips so as to close off the external environment while still allowing for any changes in pressure.

Lastly, while bovine AF cells were cultured in the AoC channel in a monolayer for proof of concept, 3D cultures and therefore 3D loading conditions have yet to be explored with bovine cells or human cells. Upcoming challenges include introducing and adhering cell–gel constructs within the channel to the PDMS surface, and visualizing 3D strains under microscopy. A gel adds complexity as it limits the diffusion of nutrients, waste, and gas; but this effect can be mitigated by perfusion channels. Tools and techniques exist for addressing each of these concerns as the AoC matures in complexity and capability. Functionalization of the PDMS surface5555. Y. J. Chuah, Y. T. Koh, K. Lim, N. V. Menon, Y. Wu, and Y. Kang, “Simple surface engineering of polydimethylsiloxane with polydopamine for stabilized mesenchymal stem cell adhesion and multipotency,” Sci. Rep. 5, 1–12 (2015). https://doi.org/10.1038/srep18162,5656. Y. J. Chuah, S. Kuddannaya, M. H. A. Lee, Y. Zhang, and Y. Kang, “The effects of poly (dimethylsiloxane) surface silanization on the mesenchymal stem cell fate,” Biomater. Sci. 3, 383–390 (2015). https://doi.org/10.1039/C4BM00268G can facilitate the cell–substrate adhesion and ensure proper force transfer from the surface to the construct. Visualizing fluorescent-tagged actin within the cells using confocal microscopy can allow for 3D strain measurements on the cells. Computational modeling can predict the effects of perfusion channel dimensions and spacing on diffusion and loading before upgrades are made to the existing proof of concept. This ongoing work is founded upon the present proof-of-concept study to validate the physiological relevance of the AoC.

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