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Stiffness Mediated-Mechanosensations of Airway Smooth Muscle Cells on Linear Stiffness Gradient Hydrogels

Airflow limitation in obstructive airway disease is characterized by narrowing of the airway lumen from excessive contraction of airway smooth muscle (ASM) and remodeling of the airway wall which includes changes in the extracellular matrix (ECM) of the ASM layer.*

Previous studies on human airway smooth muscle cells ( hASMC ) have independently assessed the influence of extracellular matrix (ECM) proteins on substrates of supra-physiological stiffnesses, such as tissue culture plastic or glass.*

While the influence of discrete substrate stiffness on hASMC behavior has been examined, manipulation of both substrate stiffness and ECM proteins simultaneously (as expected in disease) has not been extensively modeled in vitro.*

In the article “Stiffness Mediated-Mechanosensation of Airway Smooth Muscle Cells on Linear Stiffness Gradient Hydrogels” Yong Hwee Tan, Kimberley C. W. Wang, Ian L. Chin, Rowan W. Sanderson, Jiayue Li, Brendan F. Kennedy, Peter B. Noble and Yu Suk Choi highlight the interplay and complexities between stiffness and ECM protein type on hASMC mechanosensation, relevant to airway remodelling in obstructive airway diseases.*

The authors first determined a physiological range of ASM layer stiffness using a porcine airway and used these empirical recordings to inform the fabrication of a linear stiffness gradient platform coated with different ECM proteins.*

Using this linear stiffness gradient platform, Yong Hwee Tan et al. profiled hASMC morphology, contractile function with alpha-smooth muscle actin (αSMA) and mechanisms of mechanosensation, specifically with nuclear translocalization of Yes-associated protein (YAP) and lamin-A expression.*

Yong Hwee Tan et al.’s assessment of hASMC mechanosensation utilized an innovative hydrogel platform delivering a linear stiffness gradient to understand stiffness-mediated cell behavior with an ECM substrate for cellular adhesion. *

The employment of a stiffness gradient that was designed after empirical measurements performed on ex vivo ASM tissue, enabled the presentation of physiologically relevant stiffnesses to study hASMC behavior.*

Using this platform, the authors of the article found that hASMC mechanosense underlying mechanical cues more than the types of proteins they are anchored to by screening hASMC morphology, contractile phenotype, and mechanomarker expression, with a few exceptions.*

While the authors acknowledge that the findings from their study were done using cells from only one donor they still think that their study provides a proof of concept for the relevance of hASMC mechanosensation to ECM stiffness, and is another step in the right direction for understanding the pathophysiological impact of airway remodeling in obstructive diseases and exploring potential avenues for improving therapy through greater fidelity of in vitro platforms that include key concepts of mechanosensation. *

Yong Hwee Tan et al. wanted to use the same method which is used to assess hydrogel stiffness, namely atomic force microscopy (AFM), to measure ASM stiffness.*

However, nanoscale measurements of ASM strips by AFM proved to be difficult due to an uneven tissue surface after de-epithelialization (Figure S1C, Supporting Information of the cited article), resulting in false force triggering.

To validate the translation of stiffness values measured from macroscale compression (ASM strips) to nanoscale indentation (AFM on hydrogels), Yong Hwee Tan et al. fabricated additional hydrogels of four different stiffnesses using well-characterized polyacrylamide and compared the stiffness of hydrogels measured by uniaxial compression tester and atomic force microscopy (Figure S2A, Supporting Information of the cited article).

The nanoscale stiffness of hydrogels was assessed using an atomic force microscope (AFM) with NanoWorld triangular Pyrex-Nitride PNP-TR AFM probes (the longer AFM cantilever beam – CB 2 – with 200 µm length was used).

These AFM tips probed hydrogels immersed in 1 × PBS with 2 nN, an approach velocity of 2 µm s−1 and a retraction velocity of 10 µm s−1.

Young’s modulus was determined from linear portions of contact-generated force curves using a custom-written code in Igor Pro.

All probe indentations were made in triplicate and averaged for a stiffness measurement in kilopascals (kPa).

An example force curve is shown in Figure S2B, Supporting Information of the cited article. Validation of a linear stiffness gradient was achieved with eight indentations on the hydrogel, 2 mm away from both edges of the hydrogel and at 1 mm intervals along the stiffness gradient axis. Measurements were plotted against displacement from the hydrogel edge (soft to stiff) (Figure 2B of the cited article).

Figure 2 from Yong Hwee Tan et al. 2024 “Stiffness Mediated-Mechanosensation of Airway Smooth Muscle Cells on Linear Stiffness Gradient Hydrogels”:Linear stiffness gradient hydrogel fabrication. A) A schematic of a two-step polymerization process. i) 120 µL of mixed polyacrylamide (PA) solution (% acrylamide + % bis-acrylamide) was added to the primary mold and left to polymerize under ii) a methacrylated coverslip for 20 min. iii) Wedge-shaped 1° gel was removed and flipped for placement of a iv) secondary mold before v) addition of a second 120 µL PA solution and polymerized under a vi) dichlorodimethylsilane-coated coverslip for 20 min. vii) Removal of coverslip and mold completes the fabrication of bi-layered stiffness gradient hydrogel. viii) the dotted arrow indicating the direction of gradient and atomic force microscopy (AFM) measurement. B) Young's moduli gradient measured by AFM. Twelve hydrogels were selected (one gel per batch) and assessed for stiffness, yielding a gradient of 4.0 kPa mm−1, with a range of 1.7 ± 1.2 to 29.6 ± 4.3 kPa (R2 = 0.998, n = 8). Data are presented as mean ± SD. NanoWorld triangular Pyrex-Nitride PNP-TR AFM probes were used to assess the stiffness of the hydrogels with atomic force microscopy. — Figure 2 from Yong Hwee Tan et al. 2024 “Stiffness Mediated-Mechanosensation of Airway Smooth Muscle Cells on Linear Stiffness Gradient Hydrogels”:
Linear stiffness gradient hydrogel fabrication. A) A schematic of a two-step polymerization process. i) 120 µL of mixed polyacrylamide (PA) solution (% acrylamide + % bis-acrylamide) was added to the primary mold and left to polymerize under ii) a methacrylated coverslip for 20 min. iii) Wedge-shaped 1° gel was removed and flipped for placement of a iv) secondary mold before v) addition of a second 120 µL PA solution and polymerized under a vi) dichlorodimethylsilane-coated coverslip for 20 min. vii) Removal of coverslip and mold completes the fabrication of bi-layered stiffness gradient hydrogel. viii) the dotted arrow indicating the direction of gradient and atomic force microscopy (AFM) measurement. B) Young’s moduli gradient measured by AFM. Twelve hydrogels were selected (one gel per batch) and assessed for stiffness, yielding a gradient of 4.0 kPa mm−1, with a range of 1.7 ± 1.2 to 29.6 ± 4.3 kPa (R2 = 0.998, n = 8). Data are presented as mean ± SD.

Figure S2 from Yong Hwee Tan et al. 2024 “Stiffness Mediated-Mechanosensation of Airway Smooth Muscle Cells on Linear Stiffness Gradient Hydrogels”:(A) Correlation of Young’s modulus from macroscale stiffness (UCT) assessment with nanoindentation (AFM), was conducted using cylindrical PA hydrogels of different Acrylamide %/Bis-acrylamide % derived from Tse and Engler [47] 10 %/0.06 %, 10 %/0.1 %, 10 %/0.15 % and 10 %/0.3 % (Linear regression, P < 0.0001, R2 = 0.9288, n = 4). Data are presented as mean  SEM. (B) An example force curve from atomic force microscopy with an approach velocity of 2 μm/s, until a 2 nN trigger force was registered, and retraction of indenter at 10 μm/s. NanoWorld triangular Pyrex-Nitride PNP-TR AFM probes were used to assess the stiffness of the hydrogels with atomic force microscopy. — Figure S2 from Yong Hwee Tan et al. 2024 “Stiffness Mediated-Mechanosensation of Airway Smooth Muscle Cells on Linear Stiffness Gradient Hydrogels”:
(A) Correlation of Young’s modulus from macroscale stiffness (UCT) assessment with nanoindentation (AFM), was conducted using cylindrical PA hydrogels of different Acrylamide %/Bis-acrylamide % derived from Tse and Engler [47] 10 %/0.06 %, 10 %/0.1 %, 10 %/0.15 % and 10 %/0.3 % (Linear regression, P < 0.0001, R2 = 0.9288, n = 4). Data are presented as mean  SEM. (B) An example force curve from atomic force microscopy with an approach velocity of 2 μm/s, until a 2 nN trigger force was registered, and retraction of indenter at 10 μm/s.

*Yong Hwee Tan, Kimberley C. W. Wang, Ian L. Chin, Rowan W. Sanderson, Jiayue Li, Brendan F. Kennedy, Peter B. Noble and Yu Suk Choi
Stiffness Mediated-Mechanosensation of Airway Smooth Muscle Cells on Linear Stiffness Gradient Hydrogels
Advanced Healthcare Materials 2024, 2304254
DOI: https://doi.org/10.1002/adhm.202304254

The article “Stiffness Mediated-Mechanosensation of Airway Smooth Muscle Cells on Linear Stiffness Gradient Hydrogels” by Yong Hwee Tan, Kimberley C. W. Wang, Ian L. Chin, Rowan W. Sanderson, Jiayue Li, Brendan F. Kennedy, Peter B. Noble and Yu Suk Choi is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third-party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit https://creativecommons.org/licenses/by/4.0/.

A beginner’s guide to the Characterization of Hydrogel Microarchitecture for Cellular Applications

Hydrogel materials show a number of properties which make them interesting candidates to be utilized to mimic the extracellular matrix (ECM). Therefore, these materials are attractive for use in biological applications such as tissue engineering, cell culture 3D bioprinting and more.

Are you planning to use hydrogels for the first time in your research?

Then have a look at the insightful article “A beginner’s guide to the Characterization of Hydrogel Microarchitecture for Cellular Applications” by Francisco Drusso Martinez-Garcia, Tony Fischer, Alexander Hayn, Claudia Tanja Mierke, Janette Kay Burgess and Martin Conrad Harmsen.

In their article the authors describe and evaluate the different technologies that are most commonly used to assess hydrogel microarchitecture.

Francisco Drusso Martinez-Garcia et al. explain the working principle of the various methods and also discuss the merits and limitations of each of them in view of their usefulness for the characterization of hydrogels.

They introduce and explore the pros and cons of the following methods: Scanning Electron Microscopy (SEM), Cryogenic Scanning Electron Microscopy (Cryo-SEM), Environmental Scanning Electron Microscopy (ESEM), Micro-Computed Tomography (µ-CT), Confocal Laser Scanning Microscopy (CLSM), Second Harmonic Generation and Atomic Force Microscopy (AFM).*

Atomic force microscopy (AFM) can be used to investigate the hydrogel surface topology as well as a hydrogel’s mechanical properties. The latter can be achieved through mathematical modelling of force-distance curves.

When using the AFM to characterize the elasticity of a hydrogel sample it is essential to take the stiffness of the investigated material into account when choosing what kind of AFM probe to use for these experiments.

If an AFM cantilever used for probing a soft sample is too stiff (if the force constant/spring constant is too high) this might result in a poor signal-to-noise ratio.

If a soft AFM probe (an AFM probe with an AFM cantilever with a low force constant) is chosen to investigate a soft material this should lead to a better signal-to-noise ratio. On the other hand, if an AFM cantilever is too soft (if the force constant is too low) then it might not be stiff enough to indent the investigated material.

Another critical factor is the shape and the size of the AFM tip.

Spheroidal AFM probes might stick to the material, resulting in artefacts, disrupted force–distance curves, or even damaged AFM cantilevers. If the AFM tip is much smaller than the pore size of the hydrogel, it might get stuck in the fibrous network microarchitecture.

On the other hand, if the spherical AFM tip, e.g. as in colloidal AFM probes (a sphere glued to end of a tipless AFM cantilever), is too large, the weight of the sphere can have a negative influence on the spring characteristics of the AFM cantilever.

All these factors and more as described in the cited article have to be carefully weighed before deciding on the settings of the atomic force microscope and choosing an AFM probe for the investigation of a specific hydrogel.

NanoWorld tipless ArrowTL2 cantilever arrays with polystyrene beads glued to them were used by the authors of this beginner’s guide to achieve the AFM data presented in the article.*

Figure 6. from Francisco Drusso Martinez-Garcia et al. 2022: Atomic force microscopy. (A) Equipment. (B) Schematic of an AFM setup with a four-quadrant photodiode (1), in which the four-quadrant photodiode (1) receives a laser (2) reflected from a cantilever (3), in this case positioned over a hydrogel (4) mounted in a piezo stage (5). For example, the height differences in a sample (4) are measured by adjusting the stage using piezo elements (5) to counter the cantilever bending on a nanometer scale. (C) The AFM can then generate a surface heightmap of the hydrogels such as a GelMA hydrogel (shown). AFM can also be used to determine the mechanical properties of hydrogels. (D) Schematic of the AFM technique to determine the elastic moduli of hydrogels with a tipless cantilever (1), spheroidal probe (2, red), hydrogel (3), and stiff substrate (4). As the cantilever represents a spring with a known spring constant, the cantilever bending due to elastic counterforces exerted by the soft material is correlated with the piezo stage height (4). (E) The so-called force–distance curves are recorded. Data from a collagen type-I hydrogel (3.0 g/L) are shown. (F) Young’s moduli of a 1.5 g/L and 3.0 g/L collagen type-I hydrogel. Outliers indicated by ◆. AFM equipment detailed in Appendix A of the cited article. NanoWorld tipless ArrowTL2 cantilever arrays with polystyrene beads glued to them were used by the authors of this beginner’s guide to achieve the AFM data presented in the article. — Figure 6. from Francisco Drusso Martinez-Garcia et al. 2022:
Atomic force microscopy. (A) Equipment. (B) Schematic of an AFM setup with a four-quadrant photodiode (1), in which the four-quadrant photodiode (1) receives a laser (2) reflected from a cantilever (3), in this case positioned over a hydrogel (4) mounted in a piezo stage (5). For example, the height differences in a sample (4) are measured by adjusting the stage using piezo elements (5) to counter the cantilever bending on a nanometer scale. (C) The AFM can then generate a surface heightmap of the hydrogels such as a GelMA hydrogel (shown). AFM can also be used to determine the mechanical properties of hydrogels. (D) Schematic of the AFM technique to determine the elastic moduli of hydrogels with a tipless cantilever (1), spheroidal probe (2, red), hydrogel (3), and stiff substrate (4). As the cantilever represents a spring with a known spring constant, the cantilever bending due to elastic counterforces exerted by the soft material is correlated with the piezo stage height (4). (E) The so-called force–distance curves are recorded. Data from a collagen type-I hydrogel (3.0 g/L) are shown. (F) Young’s moduli of a 1.5 g/L and 3.0 g/L collagen type-I hydrogel. Outliers indicated by ◆. AFM equipment detailed in Appendix A of the cited article.

*Francisco Drusso Martinez-Garcia, Tony Fischer, Alexander Hayn, Claudia Tanja Mierke, Janette Kay Burgess and Martin Conrad Harmsen
A Beginner’s Guide to the Characterization of Hydrogel Microarchitecture for Cellular Applications
Gels 2022, 8(9), 535
DOI: https://doi.org/10.3390/gels8090535

The article “A Beginner’s Guide to the Characterization of Hydrogel Microarchitecture for Cellular Applications” by Francisco Drusso Martinez-Garcia, Tony Fischer, Alexander Hayn, Claudia Tanja Mierke, Janette Kay Burgess and Martin Conrad Harmsen is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third-party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit https://creativecommons.org/licenses/by/4.0/.

MACC1-Induced Collective Migration Is Promoted by Proliferation Rather Than Single Cell Biomechanics

High metastasis-associated in colon cancer 1 (MACC1) expression is associated with metastasis, tumor cell migration, and increased proliferation in colorectal cancer. Tumors with high MACC1 expression show a worse prognosis and higher invasion into neighboring structures. However, the mediation of the pro-migratory effects is still a matter of investigation.*

In their study “MACC1-Induced Collective Migration Is Promoted by Proliferation Rather Than Single Cell Biomechanics” Tim Hohmann, Urszula Hohmann, Mathias Dahlmann, Dennis Kobelt, Ulrike Stein and Faramarz Dehghani aim to elucidate the impact of single cell biomechanics and proliferation on MACC1-dependent migration.*

The authors found that MACC1 expression associated with increased collective migration, caused by increased proliferation, and no changes in single cell biomechanics. Thus, targeting proliferation in high-MACC1-expressing tumors may offer additional effects on cell migration.*

The mechanical properties of single cells were assessed in the form of the Young’s modulus and cortex tension; both were measured using atomic force microscopy. Briefly, cells were seeded on a petri dish and measured 15 min after seeding to avoid slippage of individual cells. Measurements were conducted using a tipless NanoWorld Arrow-TL2 AFM cantilever array to apply a force of 1 nN that led to deformations of 1–2 µm. The Young’s modulus was calculated using the Hertz model.*

Figure 1 from “MACC1-Induced Collective Migration Is Promoted by Proliferation Rather Than Single Cell Biomechanics” by Tim Hohmann et al. Single cell properties of high- and low-MACC1-expressing colon carcinoma cells. (A,B) depict the results of the biomechanical measurements for the Young´s modulus and the cortex tension. (C,D) show the results of live cell imaging of single cells for the mean speed and the contact area with the substrate. Sample sizes: (A) nSW480/EV = 35; nSW480/MACC1 = 33; nSW620/shMACC1 = 40; nSW620/shCTL = 40. (B) nSW480/EV = 33; nSW480/MACC1 = 31; nSW620/shMACC1 = 25; nSW620/shCTL = 26. (C,D) nSW480/EV = 66; nSW480/MACC1 = 98; nSW620/shMACC1 = 102; nSW620/shCTL = 111. Asterisk depicts statistically significant results with p < 0.05. Box plots show the median (red line), 25 and 75 percentile (box), non-outlier range (whiskers), and outliers (red dots). The mechanical properties of single cells were assessed in the form of the Young’s modulus and cortex tension; both were measured using atomic force microscopy. Briefly, cells were seeded on a petri dish and measured 15 min after seeding to avoid slippage of individual cells. Measurements were conducted using a NanoWorld Arrow-TL2 AFM cantilever to apply a force of 1 nN that led to deformations of 1–2 µm. The Young’s modulus was calculated using the Hertz model. — Figure 1 from “MACC1-Induced Collective Migration Is Promoted by Proliferation Rather Than Single Cell Biomechanics” by Tim Hohmann et al.
Single cell properties of high- and low-MACC1-expressing colon carcinoma cells. (A,B) depict the results of the biomechanical measurements for the Young´s modulus and the cortex tension. (C,D) show the results of live cell imaging of single cells for the mean speed and the contact area with the substrate. Sample sizes: (A) nSW480/EV = 35; nSW480/MACC1 = 33; nSW620/shMACC1 = 40; nSW620/shCTL = 40. (B) nSW480/EV = 33; nSW480/MACC1 = 31; nSW620/shMACC1 = 25; nSW620/shCTL = 26. (C,D) nSW480/EV = 66; nSW480/MACC1 = 98; nSW620/shMACC1 = 102; nSW620/shCTL = 111. Asterisk depicts statistically significant results with p < 0.05. Box plots show the median (red line), 25 and 75 percentile (box), non-outlier range (whiskers), and outliers (red dots).

*Tim Hohmann, Urszula Hohmann, Mathias Dahlmann, Dennis Kobelt, Ulrike Stein and Faramarz Dehghani
MACC1-Induced Collective Migration Is Promoted by Proliferation Rather Than Single Cell Biomechanics
Cancers 2022, 14(12), 2857
DOI: https://doi.org/10.3390/cancers14122857

Open Access

The article “MACC1-Induced Collective Migration Is Promoted by Proliferation Rather Than Single Cell Biomechanics” by Tim Hohmann, Urszula Hohmann, Mathias Dahlmann, Dennis Kobelt, Ulrike Stein and Faramarz Dehghani is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit https://creativecommons.org/licenses/by/4.0/.