Airflow limitation in obstructive airway illness is characterised by narrowing of the airway lumen from extreme contraction of airway {smooth} muscle (ASM) and transforming of the airway wall which incorporates adjustments within the extracellular matrix (ECM) of the ASM layer.*
Earlier research on human airway {smooth} muscle cells ( hASMC ) have independently assessed the affect of extracellular matrix (ECM) proteins on substrates of supra-physiological stiffnesses, resembling tissue tradition plastic or glass.*
Whereas the affect of discrete substrate stiffness on hASMC conduct has been examined, manipulation of each substrate stiffness and ECM proteins concurrently (as anticipated in illness) has not been extensively modeled in vitro.*
Within the article “Stiffness Mediated-Mechanosensation of Airway Clean 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 spotlight the interaction and complexities between stiffness and ECM protein kind on hASMC mechanosensation, related to airway remodelling in obstructive airway illnesses.*
The authors first decided a physiological vary of ASM layer stiffness utilizing a porcine airway and used these empirical recordings to tell the fabrication of a linear stiffness gradient platform coated with completely different ECM proteins.*
Utilizing this linear stiffness gradient platform, Yong Hwee Tan et al. profiled hASMC morphology, contractile perform with alpha-smooth muscle actin (αSMA) and mechanisms of mechanosensation, particularly with nuclear translocalization of Sure-associated protein (YAP) and lamin-A expression.*
Yong Hwee Tan et al.’s evaluation of hASMC mechanosensation utilized an revolutionary hydrogel platform delivering a linear stiffness gradient to grasp stiffness-mediated cell conduct with an ECM substrate for mobile adhesion. *
The employment of a stiffness gradient that was designed after empirical measurements carried out on ex vivo ASM tissue, enabled the presentation of physiologically related stiffnesses to check hASMC conduct.*
Utilizing this platform, the authors of the article discovered that hASMC mechanosense underlying mechanical cues greater than the varieties of proteins they’re anchored to by screening hASMC morphology, contractile phenotype, and mechanomarker expression, with a number of exceptions.*
Whereas the authors acknowledge that the findings from their examine had been executed utilizing cells from just one donor they nonetheless assume that their examine gives a proof of idea for the relevance of hASMC mechanosensation to ECM stiffness, and is one other step in the appropriate course for understanding the pathophysiological influence of airway transforming in obstructive illnesses and exploring potential avenues for bettering remedy by better constancy of in vitro platforms that embrace key ideas of mechanosensation. *
Yong Hwee Tan et al. needed to make use of the identical technique which is used to evaluate hydrogel stiffness, particularly atomic drive microscopy (AFM), to measure ASM stiffness.*
Nonetheless, nanoscale measurements of ASM strips by AFM proved to be troublesome resulting from an uneven tissue floor after de-epithelialization (Determine S1C, Supporting Info of the cited article), leading to false drive triggering.
To validate the interpretation of stiffness values measured from macroscale compression (ASM strips) to nanoscale indentation (AFM on hydrogels), Yong Hwee Tan et al. fabricated extra hydrogels of 4 completely different stiffnesses utilizing well-characterized polyacrylamide and in contrast the stiffness of hydrogels measured by uniaxial compression tester and atomic drive microscopy (Determine S2A, Supporting Info of the cited article).
The nanoscale stiffness of hydrogels was assessed utilizing an atomic drive microscope (AFM) with NanoWorld triangular Pyrex-Nitride PNP-TR AFM probes (the longer AFM cantilever beam – CB 2 – with 200 µm size was used).
These AFM suggestions probed hydrogels immersed in 1 × PBS with 2 nN, an strategy velocity of two µm s−1 and a retraction velocity of 10 µm s−1.
Younger’s modulus was decided from linear parts of contact-generated drive curves utilizing a custom-written code in Igor Professional.
All probe indentations had been made in triplicate and averaged for a stiffness measurement in kilopascals (kPa).
An instance drive curve is proven in Determine S2B, Supporting Info of the cited article. Validation of a linear stiffness gradient was achieved with eight indentations on the hydrogel, 2 mm away from each edges of the hydrogel and at 1 mm intervals alongside the stiffness gradient axis. Measurements had been plotted in opposition to displacement from the hydrogel edge (smooth to stiff) (Determine 2B of the cited article).
Linear stiffness gradient hydrogel fabrication. A) A schematic of a two-step polymerization course of. i) 120 µL of blended polyacrylamide (PA) answer (% acrylamide + % bis-acrylamide) was added to the first mildew and left to polymerize beneath ii) a methacrylated coverslip for 20 min. iii) Wedge-shaped 1° gel was eliminated and flipped for placement of a iv) secondary mildew earlier than v) addition of a second 120 µL PA answer and polymerized beneath a vi) dichlorodimethylsilane-coated coverslip for 20 min. vii) Removing of coverslip and mildew completes the fabrication of bi-layered stiffness gradient hydrogel. viii) the dotted arrow indicating the course of gradient and atomic drive microscopy (AFM) measurement. B) Younger’s moduli gradient measured by AFM. Twelve hydrogels had been chosen (one gel per batch) and assessed for stiffness, yielding a gradient of 4.0 kPa mm−1, with a spread of 1.7 ± 1.2 to 29.6 ± 4.3 kPa (R2 = 0.998, n = 8). Knowledge are introduced as imply ± 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.](https://www.nanoworld.com/blog/wp-content/uploads/2024/07/Fig-S2-from-Young-Hwee-Tan-2024-Stiffness-Mediated-Mechanosensation-of-Airway-Smooth-Muscle-Cells-on-Linear-Stiffness-Gradient-Hydrogels-NanoWorld-PNP-TR-AFM-probe.jpg)
(A) Correlation of Younger’s modulus from macroscale stiffness (UCT) evaluation with nanoindentation (AFM), was carried out utilizing cylindrical PA hydrogels of various 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). Knowledge are introduced as imply SEM. (B) An instance drive curve from atomic drive microscopy with an strategy velocity of two μm/s, till a 2 nN set off drive 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 Clean Muscle Cells on Linear Stiffness Gradient Hydrogels
Superior Healthcare Supplies 2024, 2304254
DOI: https://doi.org/10.1002/adhm.202304254
The article “Stiffness Mediated-Mechanosensation of Airway Clean 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 beneath a Inventive Commons Attribution 4.0 Worldwide License, which allows use, sharing, adaptation, distribution and copy in any medium or format, so long as you give applicable credit score to the unique creator(s) and the supply, present a hyperlink to the Inventive Commons license, and point out if adjustments had been made. The pictures or different third-party materials on this article are included within the article’s Inventive Commons license, until indicated in any other case in a credit score line to the fabric. If materials will not be included within the article’s Inventive Commons license and your supposed use will not be permitted by statutory regulation or exceeds the permitted use, you have to to acquire permission straight from the copyright holder. To view a duplicate of this license, go to https://creativecommons.org/licenses/by/4.0/.