Development of a microfluidic system with a bearing micropump for applying fluid shear force in an intervertebral disc degeneration model involving mechanical stimulation
Article information
Abstract
Background
The intervertebral disc (IVD) functions as a shock absorber with viscoelastic tissues, including the gelatinous nucleus pulposus and the collagenous annulus fibrosus (AF). External mechanical loading influences IVD homeostasis but can cause damage and inflammatory reactions, contributing to disc degeneration. There is a lack of in vitro platforms for modeling IVD disease with mechanical stimulation.
Methods
This study aimed to create a mechanical stimulation-based model by subjecting AF cells to shear stress using a micropump system. A micropump platform was used to measure pulsation, flow rate, and shear stress. AF cells were exposed to fluid shear stress of 0.5 and 1 dyne/cm², and morphological changes, cytotoxicity, and cell viability were analyzed through a live/dead assay. Perimeter, area, diameter, and elongation were assessed using live cell imaging and imaging analysis software.
Results
The micropump platform exhibited optimal rotor characteristics for uniform pulsation and shear stress. Fluid shear stress at 0.5 dyne/cm² showed no significant difference from the control group, while 1 dyne/cm² reduced cell adhesion and survival. Comparing the 0.5 dyne/cm² shear stress group and interleukin-1β group to the control, significant decreases in perimeter, area, and diameter were observed.
Conclusion
The study successfully developed a micro-peristaltic pump platform for applying fluid shear stress to cells, identifying an optimal rotor for uniform stress application. The platform effectively modeled IVD disease, revealing reduced cell adhesion and survival under specific shear stress conditions. This platform has potential promise for discovering biomarkers and exploring cellular responsiveness to external stimuli.
Introduction
Human cells and tissues can be mechanically stimulated by the movement of our body. This stimulation manifests as physical properties such as tensile, compressive, shear, and torsional forces [1–3]. These mechanical stimuli are transmitted to the cells within the tissue, prompting them to activate various response mechanisms and signaling pathways [4–6]. The activation of these signaling pathways by mechanical stimulation leads to diverse changes in the expression of genes and proteins within the cells, ultimately affecting proteins including growth factors, signaling molecules, and enzymes that regulate the extracellular matrix. However, when mechanical stimulation exceeds the tissue's tolerance threshold, it can cause damage within the tissue and trigger inflammatory responses. During this process, inflammatory mediators and catabolic enzymes secreted by the cells create an inflammatory microenvironment within the tissue [7,8]. Thus, mechanical stimulation can lead to a variety of changes in both cells and tissues.
Previous studies have shown that various mechanical stimuli can induce morphological, cytoskeletal, and structural changes in cells [9–11]. The spine and intervertebral disc, in particular, are subjected to a high frequency of mechanical stimulation, which plays a crucial role in maintaining spinal homeostasis. The intervertebral disc is composed of 2 distinct tissues: the nucleus pulposus, a gelatinous tissue, and the annulus fibrosus, a surrounding collagenous tissue. Normally, the intervertebral disc is avascular and aneuronal, with blood vessels and nerve endings only present in the outer third of the annulus fibrosus [5,9,12-14]. Excessive mechanical stimulation can damage the intervertebral disc tissue, triggering an inflammatory response. Immune cells within the disc react to this damage, and the release of chemokines draws more immune cells from the blood vessels to the affected area of annulus fibrosus cells. This sequence of events leads to a proliferation of persistent inflammatory responses, primarily driven by interleukin (IL)-1β-mediated inflammation caused by macrophages. This is known to foster an inflammatory microenvironment in the intervertebral disc and cause pain through the expression of inflammatory mediators and neurostimulants [15–18]. In clinical studies, IL-1β expression was found to be significantly increased in the tissues of patients with intervertebral disc degeneration and herniation. Moreover, IL-1β is known to significantly influence the immune response and disease progression in various medical conditions [19–21]. However, the specific mechanisms by which mechanical stimulation affects the reactivity of intervertebral disc cells remain unclear. Overall, mechanical stimulation within the intervertebral disc is a critical trigger for conditions such as intervertebral disc degeneration and prolapse. Therefore, it is essential to develop a platform that can administer mechanical stimulation to cells to further investigate the pathogenic mechanisms involved.
Microfluidic chip technology is being creatively employed in diverse disciplines including machinery, chemistry, healthcare, and more [22–24]. This technology integrates essential processes such as control, mixing, separation, and measurement, leveraging the physical and hydrodynamic properties [25,26]. Physical properties include characteristics like surface tension, density, and viscosity, whereas hydrodynamic properties pertain specifically to Newton’s laws and inertia. The control technology within a microfluidic chip enables precise manipulation of small-volume samples within a sealed microchannel. This control is facilitated by micropump technology, which is categorized into mechanical and non-mechanical types. A mechanical micropump manipulates fluid by mechanically displacing a tube or a polydimethylsiloxane (PDMS) chip using a rotor or an actuator. Conversely, a non-mechanical micropump regulates fluid energy by modulating the volume with electrical and magnetic forces [27–29]. Due to their simplicity and ease of operation, these mechanical micropumps are widely researched and developed. Previous studies have exposed cells to various mechanical stimuli using micropump technology, effectively mimicking the physical conditions present in the human body.
In this study, we constructed a novel platform using a 3-dimensional (3D) printer, designed to apply fluid shear force to cells. This platform was used to deliver mechanical stimulation to the cells of the intervertebral disc, facilitating the development of a model for intervertebral disc degeneration. The effectiveness of the platform was assessed by examining changes in cellular morphology and motility.
Materials and Methods
Ethics statement:
Ethics statement: This study constituted a comprehensive analysis of previously released studies and thus, was not subject to the approval of the institutional review board.
1. Design and fabrication of the micropump
The micropump is constructed using components that are both readily available and mass-produced, as illustrated in Fig. 1A. Each component was designed using a 3D Computer-Aided Design program and subsequently produced with a 3D printer. To accommodate the high humidity conditions within the incubator where the micropump operates, a protective shielding structure was incorporated into its design. The micropump employs a stepper motor to control flow, allowing for the application of various fluid shear stresses essential for constructing the cell culture chamber. Its operating principle involves a peristaltic rotor, connected to the stepper motor, which compresses the silicone tube. This action induces a pressure change within the tube, as depicted in Fig. 1B. Consequently, this pressure alteration causes fluid samples to flow into the tube inlet. The flow rate is adjusted based on the revolutions per minute of the stepper motor and is regulated through the motor drive and the pulse width modulation (PWM) controller, as shown in Fig. 1C.
A schematic diagram of a micropump platform using fluid shear force. (A) Three-dimensional modeling of the micropump platform. (B) Principle of the micropump drive. (C) Micropump control and analysis block diagram. PWM, pulse width modulation.
2. Design and fabrication of fluid shear force chamber chips
The fluid shear force chamber chip is specifically designed to apply a uniform and consistent shear force within the chamber. The design was validated for fluid shear force through 3D simulation. The cell culture chamber dimensions are 10 mm in width, 40 mm in length, and 0.5 mm in height. A fluid shear force model for cells in the channel was developed based on the study by Gaver and Kute [30]. The shear force exerted on cells within the channel can be calculated using the equation provided.
where (τs*)max is the maximum shear stress for a cell in the channel, μ is the viscosity of the fluid (water), Q2-D* is the flow rate of the channel width, and H is the height of the channel. When the maximum shear stress for a cell in the cell culture chamber is designed to be 0.5 dyne/cm2, the flow rate of the micropump is calculated to be about 42.37 μL/min.
The microfluidic shear force chamber chip was constructed using a polyethylene terephthalate film. A mold was created by mixing PDMS (PDMS Sylgard 184A/B; Dow Corning, Midland, MI, USA) with a curing agent and then placing it in a dry oven at 80°C for 2 hours. The resulting PDMS chip was removed from the mold, precisely cut, and punched to achieve the desired shape. The bonding of the PDMS chip was performed using a plasma generator (Femto Science, Hwaseong, Korea). Both the PDMS chip and the slide glass were bonded using plasma within the cell culture chamber. The chip was then sterilized by autoclaving at 121°C for 15 minutes, stored in a drying oven for 2 days, and subjected to hydrophobic surface treatment before use.
3. Functional analysis of the micropump
The performance of the micropump was analyzed in terms of pulsation, flow rate, and fluid shear force using a flow sensor. This sensor was connected to the outlet of the micropump, allowing for the measurement of the instantaneous flow rate over a 5-minute extrusion period. The analysis involved evaluating changes in pulsation, the average flow rate, and the fluid shear force. Instantaneous flow rate values were converted to a specific current level (0–20 mA), and the voltage applied was measured across both ends using an arbitrary resistance. The output current, also measured with an arbitrary resistance, was converted into a digital signal through a 16-bit A/D converter. This signal was then transmitted via I2C communication to the Microcontroller Unit Board (Arduino UNO), where data on pulsation analysis were collected. The average flow rate and fluid shear force were determined by averaging the instantaneous flow rate over 5 minutes for each data point.
4. Live/dead assay
Cytotoxicity analysis was performed using a live/dead assay kit (L3224; Invitrogen, Carlsbad, CA, USA) on cells cultured within a microfluidic chip. The analysis included 2 groups: a control group and an annulus fibrosus cell group. The experimental group was subjected to fluid shear forces of 0.5 and 1 dyne/cm² for 12 hours, followed by a 12-hour rest period. The live/dead assay kit was used according to the manufacturer's instructions. Live cells were detected by green fluorescence (calcein-AM), and dead cells were indicated by red fluorescence (ethidium homodimer-1). Fluorescence images were obtained using the EVOS FL auto-cell imaging system (Thermo Fisher Scientific Inc., Waltham, MA, USA).
5. Morphological analysis of cells
The morphological change analysis involved examining cells from 3 distinct groups: the untreated control group, the annulus fibrosus cell group that underwent a 12-hour rest period following exposure to a fluid shear stress of 0.5 dyne/cm² for 12 hours, and the cell group treated with IL-1β. The samples were fixed with 4% paraformaldehyde and permeabilized in phosphate-buffered saline containing 0.2% Triton X-100 for 10 minutes. They were then blocked with 3% bovine serum albumin for 1 hour at 25ºC (room temperature) and incubated with Alexa 488-conjugated phalloidin (Invitrogen). Subsequently, the cells were stained with 4,6-diamidino-2-phenylindole (Santa Cruz Biotechnology, Dallas, TX, USA). Morphological changes in the cells were assessed using an EVOS FL auto-cell imaging system. Image samples were analyzed with ICY image software (ver. 2.2.0.0) and EIS LSM 9 Zen-blue Edition imaging software ver. 3.2 (Carl Zeiss Microscopy GmbH, Lower Saxony, Germany). The analysis involved defining the outer boundary of the target cell and selecting the region of interest to measure changes in cell length.
6. Statistical analysis
Statistical analysis was presented as mean±standard error of the mean, based on 6 independent experiments using cells cultured in separate technical replications. Differences between experimental groups were analyzed using one-way analysis of variance and Bonferroni’s post-hoc test. All statistical analyses were conducted using GraphPad Prism 9.0 (GraphPad Software, La Jolla, CA, USA). Statistical significance expressed under p value (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).
Results
1. Analysis of micropump pulsation, flow rate and fluid shear stress
We conducted a pulsation analysis of the micropump by examining the instantaneous flow rate using a microflow sensor. This analysis assessed the effects of varying the number of bearings in the rotor while keeping the PWM fixed at 100 Hz, with the goal of optimizing the micropump rotor design. Rotor variants with 2, 4, and 6 bearings were manufactured.
The analysis results indicated average flow rates of 46.38±1.07, 91.94±1.01, and 85.72±0.68 μL/min for rotors with 2, 4, and 6 bearings, respectively, as illustrated in Fig. 2A. Additionally, the pulsation amplitude was assessed, showing values of 294.1, 392.5, and 238.9 for rotors with 2, 4, and 6 bearings, respectively, as depicted in Fig. 2B–D.
Analysis of pulsation by the number of rotor bearings in a micropump. (A) The flow rate according to the number of rotor bearings in a micropump. (B) The pulsation of 2 rotor bearings. (C) The pulsation of 4 rotor bearings. (D) The pulsation of 6 rotor bearings.
Based on these findings, the rotor with 6 bearings emerged as the optimal choice, demonstrating both low amplitude and an appropriate flow rate for the micropump. The performance evaluation of the micropump included an analysis of flow rates, which assessed the values for each PWM according to the motor’s rotation direction, as shown in Fig. 3A. The analysis yielded the following flow rates in the forward direction: 49.62±0.51, 87.95±1.46, 140.11±0.3, 183.68±1.45, 233.19±0.85, and 295.06±0.59 μL/min. In the reverse direction, the flow rates were recorded as 49.29±0.49, 88.37±2.19, 136.16±3.05, 180.65±1.32, 234.3±3.16, and 293.31±1.98 μl/min, as indicated in Fig. 3A.
Micropump performance analysis. (A) Average flow rate analysis according to the micropump rotor rotation direction. (B) Shear force analysis according to the cell culture chamber channel length. PWM, pulse width modulation.
Subsequently, a comparison and analysis were conducted between the theoretical fluid shear stress at 3 outlet points and the actual experimental fluid shear stress, as shown in Fig. 3B. The theoretical values were found to be 0.54, 0.52, and 0.54 dyne/cm², respectively, while the experimental fluid shear stress was measured at 0.53 dyne/cm². These results indicate negligible resistance within the chip, facilitating the consistent application of fluid shear stress under identical conditions in the cell culture section.
2. Toxicity test
The live/dead assay is a fundamental method for assessing cell survival and toxicity. The cell cytotoxicity analysis showed no statistically significant difference in the group exposed to a fluid shear stress of 0.5 dyne/cm2 compared to the control group. However, the group subjected to a fluid shear stress of 1 dyne/cm2 exhibited a decrease in cell viability and adhesion rate, accompanied by noticeable cell contraction.
Subsequently, the control group and the groups exposed to fluid shear stresses of 0.5 dyne/cm² and 1 dyne/cm² underwent live/dead analysis via cell staining, as shown in Fig. 4A. The analysis indicated no significant differences in the viability and toxicity of annulus fibrosus cells between the control group and the group exposed to 0.5 dyne/cm². However, at 1 dyne/cm², there was noticeable cell death among annulus fibrosus cells, as depicted in Fig. 4B.
Analysis of cell viability by microscopy and live/dead staining. (A) Bright-field microscopy analysis. (B) Live/dead staining analysis: Cells were stained with calcein AM (green, live cells) and ethidium homodimer-1 (red, dead cells) to assess cell viability. Scale bar=100 μm.
3. Analysis of morphological changes in annulus fibrosus cells
To examine the morphological changes in intervertebral disc cells due to fluid shear stress, each experimental group was observed using a live cell imaging system, and images were captured. Following this, parameters such as cell perimeter, area, diameter, and elongation for each group were extracted and analyzed using image analysis software.
The control groups exhibited the following measurements: perimeter, 816.45±11.77 μm; area, 123.23±1.88 μm; diameter, 331.28±15.25 μm; and elongation, 3.27±0.22 μm (Fig. 5A). In comparison, the cell populations exposed to fluid shear stress were analyzed with measurements of 384.73±21.58 μm for perimeter, 75.41±3.86 μm for area, 162.56±16.69 μm for diameter, and 2.78±0.18 μm for elongation. The IL-1β cell groups showed measurements of 400.4±15.52 μm for perimeter, 76.02±3.26 μm for area, 185.3±13.79 μm for diameter, and 2.74±0.23 μm for elongation.
Morphological analysis of cells. (A) Morphological analysis of cells using immunocytochemistry (ICC) staining. (B) Quantitative analysis of the perimeter, area, diameter, and elongation for each cell population. Values are presented as mean ± SE of three independent experiments. Statistical significance is indicated as *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001, compared between groups. AF, annulus fibrosus; IL-1β, interleukin 1 beta. Scale bar=100 μm.
The observed data indicated significant reductions in perimeter, area, and diameter in both the fluid shear stress-exposed cell group and the IL-1β cell group when compared to the control group. However, there was no significant difference in elongation, as shown in Fig. 5B.
Discussion
This study aimed to develop a platform for applying mechanical stimulation to annulus fibrosus cells, simulating a degenerative model induced by early-stage mechanical stimulation in the intervertebral disc. The annulus fibrosus tissue of the intervertebral disc features multiple concentric layers that form a lamellar structure. This structure progressively increases in thickness from the outer to the inner layers. Each layer consists of fibers arranged alternately at specific angles, allowing it to withstand loads from tensile strength in various directions [13]. Additionally, annulus fibrosus cells are subjected to diverse mechanical stimuli, including fluid shear stress from interstitial flow within the tissues and tensile compression due to external stimuli [8,13].
Previous research has demonstrated that excessive fluid shear stress increases the production of inflammatory cytokines in annulus fibrosus cells and causes morphological changes in these cells [8,31–33]. However, systems that use constant flow rates and unidirectional fluid flow fall short of replicating the physiological conditions of native IVDs. To address this, our group created a micro-peristaltic pump that can administer repetitive fluid shear stress. Our pump system offers several advantages for related research: it allows precise control over pump speed and direction; it avoids contamination by not coming into direct contact with the fluid; it supports closed-loop circulation of media, which facilitates the analysis of cytokines secreted by the cells; and its compact design makes it suitable for use in a standard incubator. An optimal rotor bearing was selected through experimentation, capable of generating consistent pulsation at 0.25 Hz with a relatively low amplitude of 238.9 μL/min. The selected rotor maintained a linear average flow rate, enabling the uniform application of repetitive fluid shear stress at 0.5 dyne/cm2 without encountering resistance within the cell culture chip. This setup allowed for the application of repeated fluid shear stress to annulus fibrosus cells, inducing an inflammatory response and confirming morphological changes, including alterations in the cell cytoplasm. These observed results are consistent with the morphological changes seen in intervertebral disc cells in actual clinical observations.
In this study, we developed a platform to expose annulus fibrosus cells to cyclic fluid shear stress. Our findings revealed that this type of stress induces changes in annulus fibrosus cells that mirror those seen in the degenerative environment of intervertebral discs, as demonstrated by the analysis of cell morphological changes. However, the scope of this study was limited to examining the morphological alterations in intervertebral disc cells caused by mechanical stimulation. Further research is necessary to conduct molecular biological analyses, which should include examining the genes and proteins of cells affected by external mechanical stimulation and conducting mechanism studies based on these findings. Despite these limitations, the platform developed in this study shows potential for investigating the mechanisms of cell reactivity to external stimuli in various tissues and could ultimately help identify potential biomarkers for disease treatment in future studies.
Notes
Conflict of interest
No potential conflict of interest relevant to this article was reported.
Funding
This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government 2020R1F1A1068910.
Authors’ contributions
Conceptualization: HC, SMB; Formal analysis: TWK; Methodology: JWL; Writing–original draft: SMB, JWL; Writing–review & editing: HC, TWK.
Data availability
Please reach out to the corresponding author to inquire about the availability of data.