Perspective

Text (author) [Times New Roman, No. 4].

【】【】【】

Case 1

Case 2

Case 3

Case 4

Case 5

Academic research

Research question

How does matrix elasticity affect the lineage differentiation of mesenchymal stem cells (MSCs)?

How do cells perceive groove structures at the micro-nanoscale (spatially confined and adhesion-induced) and by what mechanisms lead to cell polarization, and how does this polarization affect cell function (e.g., osteogenic differentiation)?

What is the effect of basal stiffness on the growth and differentiation of naïve mesenchymal stem cells (MSCs) and the role of non-muscle myosin II in this process

aim

To clearify the complex mechanism behind the topography-induced cellular response

To reveal the mechanism of induced cell polarization by spatial limitation and adhesion induction and its downstream effects on cell function

To clarify the influence of matrix elasticity on stem cell lineage differentiation, and to reveal the function of non-muscle myosin II

methods

novelty

The construction of the innovative materials platform has developed a hydrogel system with surface roughness from nanometer to micron scale and controllable stiffness prepared by soft lithography technology, which successfully integrates the two key factors of surface roughness and matrix stiffness, which are difficult to achieve precise control of these two factors in the same model in previous studies.

Unraveling a novel mechanism of cellular response Systematically revealing the mechanism by which cells respond to synergistic stimuli of surface roughness and matrix stiffness, it was found that cells sense and conduct this synergistic stimulation through the integrin-pFAK-RhoA/ROCK-actomyosin-nuclear cytoskeleton-YAP/TAZ signaling pathway, a finding that fills a gap in the understanding of how cells integrate multiple physical signaling mechanisms.

3. Discover the conditions to optimize the cell response It was determined that the high roughness (1.08 μm for \(R_{q}\)) on the soft substrate (3.8 kPa) and the medium roughness (645nm for \(R_{q}\)) on the hard substrate (31.3 kPa) could maximize the mechanical sensing and functional response of the cells, and the level of osteogenic differentiation of the cells on the soft substrate was comparable to or even better than that of the hard matrix under the condition of high roughness. This result provides a key parameter basis for the design of biomaterials, which is helpful for the development of tissue engineering and regenerative medicine.

1. Constructing a material model: The development of orthogonal intersecting micro-nano groove structures can clearly distinguish the effects of spatial confinement (micron grooves) and adhesion induction (nanogrooves) on cell polarization in the same material system, which provides a powerful tool for in-depth study of cell polarization mechanisms, which were difficult to clearly demonstrate the differences of different mechanisms inducing cell polarization in the same model.

2. Revealing different polarization mechanisms: It is elaborated that spatial restriction mainly promotes cell polarization by activating pseudopodia-related signaling pathways (Rac-Arp2/3 and CDC42-Formin signaling pathways), while adhesion induction relies on focal adhesion and RhoA/ROCK pathways, and adhesion-induced polarization can enhance cell cohesion and promote stem cell osteogenic differentiation, deepening the understanding of the signal transduction mechanism of cell polarization. It provides a new target for the regulation of cell behavior.

3. Emphasizing the role of surface morphology: Experiments have proved that the surface morphology characteristics at the nano and micro scales have a significant impact on the polarization direction, function and nuclear state of cells, highlighting the importance of surface morphology as a physical signal in the regulation of cell behavior, and providing a new idea for the surface engineering design of biomaterials to guide the orderly arrangement and function of cells in tissue engineering and regenerative medicine

1. Discovery of the key influence of matrix elasticity on stem cell differentiation: For the first time, it has been clearly demonstrated that matrix elasticity plays a decisive role in stem cell lineage differentiation, that is, matrix with different hardness can directly guide mesenchymal stem cells to differentiate into different lineages such as nerves, muscles or bones, such as soft matrix promotes neurogenic differentiation, and hard matrix is conducive to osteogenic differentiation, which breaks through the previous limitation of only focusing on the influence of soluble factors on stem cell differentiation.

2. Unraveling the role of non-muscle myosin II in stem cell differentiation: By using the inhibitor blebbistatin, it was found that non-muscle myosin II is involved in the process of stem cell perception and response to matrix elasticity, and its activity is crucial for the morphological changes and lineage differentiation of stem cells, providing a new perspective for in-depth understanding of the molecular mechanism of stem cell differentiation.

3. Propose a new regulatory model of stem cell differentiation: It emphasizes matrix elasticity as a physical signal, which interacts with soluble inducible factors in the process of stem cell differentiation and has a dominant role, which changes the previous understanding of stem cell differentiation regulatory factors, provides a new theoretical basis for stem cell fate regulation, and has an important impact on the research direction in the field of stem cell therapy and regenerative medicine.

results

In this study, hydrogels with surface roughness ranging from nanometer to micrometer scale and controllable stiffness were prepared by soft lithography, which provided an effective platform for studying the synergistic response of cells to surface roughness and matrix stiffness. It was found that the response of mesenchymal stem cells (MSCs) to surface roughness depended on matrix stiffness, and on soft substrates (3.8 kPa), high roughness enhanced cell mechanical sensing. On a hard substrate (31.3 kPa), moderate roughness (645 nm for ) optimizes cell response. Cells sense synergistic roughness and stiffness stimulation through the integrin-pFAK-RhoA/ROCK-actomyosin-nuclear cytoskeleton-YAP/TAZ signaling pathway, and this synergistic effect promotes the osteogenic differentiation of stem cells, and the level of osteogenic differentiation of cells under high roughness conditions on soft substrates is comparable to or even better than that of hard substrates.

1. Mechanism of cell polarization: The orthogonal crossover micro-nano groove structure constructed in this study successfully distinguished two modes of inducing cell polarization: sterically confined (micron groove) and adhesion-induced (nanogroove). On micron grooves, steric confinement inhibits intracellular force generation, polarizing cells by activating the Rac-Arp2/3 and CDC42-Formin signaling pathways to form pseudopodia; On the nanogrooves, adhesion induction enhances intracellular forces, polarizes cells by promoting focal adhesion formation and activating the RhoA/ROCK signaling pathway, which is sufficient to enable cells to overcome the spatial limitations of microgrooves.

2. Effect on cell functionThe two polarization modes have different effects on cell function, and the cell polarization caused by adhesion induction reduces the degree of chromatin condensation in the nucleus, which is conducive to the expression of osteogenic genes, thereby promoting the osteogenic differentiation of stem cells, while the osteogenic differentiation ability of cells is weak under space constraints

1. Cell morphology indicates that lineage specialization is guided by matrix stiffness and dependent on non-muscle myosin II

Cytoskeletal markers and transcription factors also affect lineage specificity

Academic writing

style

voice

Active

Active

Active

structure

IMRaD

IMRaD

IMRaD

Perspective

Analysis (reader)

Academic research

1. Research content

Several publications have focused on the topic of cell-biomaterial interactions. Refs 1 - 3 focus on how cells perceive and respond to the physical properties of biomaterials, such as matrix elasticity, surface roughness, and topography, particularly with regard to stem cell differentiation and cell polarization. All three studies explored cell differentiation and growth behavior through cell-secreted proteins, and all three studies used mouse bone marrow stem cells. Caase1 (MSC cells) combined the effects of basal stiffness and roughness on cell differentiation (the synergistic effects of the two had not been previously studied) on the basis of previous studies, and was finally discovered by preparing hydrogels with different surface morphologies and stiffness, adhesion to cells, and observation of protein secretionCellular mechanosensing and osteogenic differentiation of MSCs are surprisingly enhanced to levels comparable to or even better than those on smooth rigid substrates. At the same time, the optimized roughness and stiffness result in an increase of more than 3-fold in the osteogenic differentiation of cells compared to a smooth rigid substrate. Case 2 focused on the spatial confinement of the substrate on cells (spatially restricting cell stretching) and the downstream effects of adhesion induction (directing adhesion growth) on cell function. Here, sterically confined and adhesion induction of guided cell polarization were achieved by basal groove arrays of different sizes, respectively. The experiment found that although the morphology of polarized cells was similar in both structures, the signaling pathways and downstream functions that induced cell polarization were significantly different. Adhesion induction (nanogrooves) leads to the formation of focal adhesion and activates the RhoA/ROCK pathway to enhance myosin-based intracellular forces, while spatial confinement (microgrooves) only activates the formation of pseudopodia. The enhancement of intracellular forces caused by adhesion induction inhibits chromatin condensation, thereby promoting the osteogenic differentiation of stem cells. Case 3 explores the effect of matrix stiffness on stem cell differentiation and the role of non-muscle myosin II in stem cell differentiation, the synergistic effect of soluble inducible factors and matrix stiffness in stem cell differentiation. It was found that the soft substrate (mimic the brain) tended to be neurogenic, the harder substrate (mimic muscle) tended to be myogenic, and the harder substrate (mimic collagen bone) tended to be osteogenic. Inhibition of non-muscle myosin II blocks all elasticity-guided lineage specificity without strongly interfering with other aspects of cellular function and shape.

2. Complementarity of research methods

Documents 1 - 3 mainly use experimental methods to prepare materials with specific physical properties (such as different elastic matrices, roughness gradient hydrogels, micro and nano groove structures), observe the changes of cell behavior through cell culture experiments, and use a variety of cellular and molecular biology techniques (such as immunofluorescence staining, gene expression analysis, etc.) to explore the mechanism of cell response.

The results of the research corroborate and complement each other

1. Effect of surface properties: All three papers highlight the influence of surface properties (roughness, grooves) on cell behavior, but the specific mechanisms and results vary.

2. Stiffness and cell fate: The second paper specifically emphasizes the role of stiffness in cell polarization, while the third paper explores the effect of stiffness on stem cell lineage specificity more comprehensively.

3. Signaling pathways: The second paper explores in detail the role of different mechanosensing pathways (RhoA/ROCK) in cell polarization, while the third paper focuses more on the role of non-muscle myosin II in sensing basal stiffness.

Academic writing

Several papers have a lot in common in terms of writing, and they basically adopt the IMRaD writing structure, regardless of the length of the article