Micropillars in Biomechanics: Role in Guiding Mesenchymal Stem Cells Differentiation and Bone Regeneration

Efficient bone repair relies on biomaterials that exhibit superior biocompatibility and enhanced osteogenic capabilities. In recent, micropatterning technology has emerged as a robust strategy for directing stem cell differentiation, offering a notable shift from traditional chemical induction or genetic modification methods by providing environmental cues to regulate cellular behavior. Among the various types of microstructures, micropillars have garnered significant attention due to their adjustable properties and high‐throughput experimental capabilities. These structures play a pivotal role in governing nuclear deformation and deciphering cellular responses to biomechanical cues. In this comprehensive review, the influence of micropillars on mesenchymal stem cells (MSCs), drawing upon two decades of research findings is critically assessed. The study comprehensively evaluates how micropillar substrates impact crucial cellular processes such as deformation, migration, adhesion, and ultimately, MSC fate determination toward an osteogenic lineage. By synthesizing and analyzing past studies, the potential regulatory mechanisms through which micropillars modulate MSC behavior are aimed to be elucidated. Furthermore, the utility of micropillars as a cutting‐edge biomechanical detection tool and platform for investigating cellular behaviors is explored. By projecting future applications, the review highlights the growing significance of micropillars in the dynamic field of biomechanics, underscoring their transformative potential in tissue engineering and regenerative medicine.


Introduction
Research in the field of mesenchymal stem cells (MSCs), a unique subset of non-hematopoietic stem cells primarily found in the bone marrow, has revealed their remarkable potential to differentiate into various cell types, specifically including DOI: 10.1002/admi.202300703osteoblasts, chondrocytes, and adipocytes. [1]The complex process of MSC differentiation is guided by a combination of biological, chemical, and physical stimuli present in the microenvironment.Various signaling pathways, such as tissue growth factor-/bone morphogenetic protein (TGF-/BMP), Wnt/-catenin, Hedgehog (Hh), Notch, and fibroblast growth factors (FGFs), play crucial roles in directing MSCs toward an osteogenic lineage. [2]BMP2, specifically, influences both the TGF- and Wnt/-catenin pathways, contributing to the intricate regulatory network governing MSC differentiation cascade. [3]hysical cues within the stem cell niche, including shear forces, tension, pressure, and substrate stiffness, also influence the fate of MSCs.Manipulating these physical cues can effectively guide MSCs toward osteogenic differentiation. [4,5]Understanding the interplay between signaling pathways and physical cues is vital for harnessing the therapeutic potential of MSCs in regenerative medicine and tissue engineering.
Precisely engineered substrate morphologies offer versatile platforms for influencing the osteogenic differentiation of MSCs.Larger micropatterns mainly affect cell clusters, while micropatterns at the scale of 0.1-100 μm primarily impact monolayer cells. [6]Micropatterning involves creating specific topologies and controlling of the specific spatial distributions of biochemical molecules on materials.The technique plays a crucial role in quantifying and explaining cell behavior, developing organ chips, simulating normal or pathological conditions, and contributing to regenerative medicine. [7]In osteogenic differentiation studies, three main types of micropatterns are commonly used: microgrooves, micropits and micropillars.Microgrooves are composed of linear arrangements of grooves and ridges, with groove spacing, ridge width, and ridge depth as primary parameters.Micropits consist of square or circular arrays with recessed surfaces, with pit depth, side length, and shape as main parameters.Larger micropits can be utilized for organoid culture.Micropillars are 3D structures perpendicular to the substrate surface, with parameters such as shape, side length (diameter), spacing, height, and stiffness.These micropillars are instrumental in investigating the mechanisms that drive microenvironment-induced osteogenic differentiation in MSCs.
The interplay between physical cues and gene expression changes during MSC differentiation involves the transduction of mechanical signals through various components of the cell, including the nucleus. [9]The cell's response to mechanical stimuli relies on molecules like integrins, calreticulin, cilia, and ion channels, which capture external mechanical signals and transmit them to the cytoskeleton. [10]Focal adhesion-mediated signaling cascades and junction proteins facilitate the transmission of internal forces from the cytoskeleton to the nucleus, with laminin A/C playing a key role in stabilizing the nuclear structure against mechanical stresses. [11]Migration of transcription factors within the cell and localization patterns of Yes-associated protein (YAP)/transcriptional co-activator with PDZ-binding motif (TAZ) are influenced by matrix stiffness and nuclear shape. [11]Moreover, the hierarchical organization of the nucleus, characterized by distinct chromatin borders, plays a critical role in regulating gene expression. [12]he impact of micro/nano-patterned cues on the nuclear compartment suggests a correlation between morphological cues and changes in gene expression.Chromatin rearrangement occurs in response to direct forces mediated by nuclear membrane proteins and fibrillar layers, protecting euchromatin located closer to the center of the nucleus. [13]Different cell lineages display variations in chromatin cohesion and the expression of laminin A/C.16] Despite progress in understanding the relationship between topographical cues and gene expression changes, there is still much to uncover.Further studies are needed to elucidate the complete signaling pathways responsible for cellular responses to substrate morphology.
Traditional techniques such as micropipette aspiration, atomic force microscopy, and parallel plate compression have been instrumental in studying nuclear mechanics.However, more sustained nuclear deformation can be achieved with micropillars, allowing precise control over physical parameters.19][20] This review aims to consolidate the findings from the past two decades regarding the regulation mechanisms enabled by micropillars.It explores their impact on MSC morphology, adhesion, migration behaviors, and potential implications for bone regeneration.Micropillars have become invaluable tools in mechanical testing, particularly traction force microscopy and biomechanical platforms.By shedding light on the influence of microenvironmental morphology on MSC behavior, this review contributes to advancements in tissue engineering and regenerative medicine applications while providing a foundation for the development of novel detection tools and platforms.

Modulation of MSC Behaviors by Micropillars
The studies conducted by various scientists, as we outlined below, illustrate the crucial role of topology and the physical cues exerted by micropillars or nanoneedles on cell morphogenesis, nuclear deformation, and the kinetics of cell differentiation (Figure 1).Cells respond to these physical impositions by adjusting their cytoskeleton, which consequently leads to changes in nuclear shape and positioning.

MSC Morphology and Nuclear Morphology
Sizes and nuclear properties are critical factors that influence the behavior of MSCs.The average diameter of mouse MSCs ranges from 30 to 50 μm, [21] while rat MSCs exhibit an average size of ≈32.0 ± 7.2 μm after 7 days in culture. [22]Human MSCs have a diameter range fluctuating between 15 and 50 μm. [23,24]It has been observed that MSCs increase in dimension with an increase in the number of passages. [25]Regarding nuclear characteristics, their area covers ≈160 μm 2 in mouse MSCs and ≈250 μm 2 for human MSCs. [26]These enlarged nucleus features, combined with inherent properties associated with their nuclear membrane, impose restrictions on MSC migration capabilities. [27]Moreover, primary human MSCs have exhibited slow growth patterns, characterized by a low nucleoplasmic ratio and a significantly large cell volume. [28]Interestingly, both cell dimension and the geometric attributes of the nucleus serve as viable markers indicating the existence of self-renewal capacities. [29]ucaro et al. conducted an in-depth investigation into the effects of different spacings between cylindrical silica nanopillars on cell morphology-related behaviors such as cell projection area, cell body polarization, pseudopod formation, and alignment with the nanopillar grid. [30]By designing nanopillars with different sizes (i.e., diameters within a range of 0.2-0.5 μm and heights from 5 to 10 μm) and inter-pillar spacings (spanning from 0.8 to 5 μm), they observed distinct morphological changes in cells.Cells maintained their typical spindle-shaped morphology on a flat silica wafer.When grew on nanopillars with inter-pillar spacing of 1.5-2.5 μm, cells developed elongated and narrow axonlike extensions.As the spacing increased to values ranging from 3.5 to 5 μm, cells exhibited a star-shaped morphology with more branching-like structures.Moreover, the spacing of nanopillar arrays had a stronger effect on nuclear deformation than the pillar radius, while the height of the nanopillars moderately influenced nuclear deformation. [31]asturk et al. investigated the behavior of human dental pulp MSCs (DPSCs) on PMMA micropatterned surfaces with square micropillars (width or inter-pillar gap ranging from 4 to 16 μm) with a height of 8 μm. [32]They found that reduced pillar width and spacing led to enhanced cell nuclear deformation.Certain micropillar configurations (4 μm width or inter-pillar gap, with oxygen plasma treatment) induced elongated cell morphology, with severe nuclear deformation occurring when the nuclei entered the inter-pillars. [32]In micropillars without plasma treatment, the cells crawled back to the pillar surface, did not experience significant nuclear deformation and junction formation between the pillars.The presence of surface protein coating may influence cell morphology by increasing the hydrophilicity of the micropillars.Nuclear deformation is closely connected to laminin A/C. [33]easurement of nuclear modulus of elasticity using PDMS micropillars uncovered differences in nuclear stiffness between cell types, with HeLa cells having lower nuclear modulus of elasticity than smooth muscle cells, attributed to defects in laminin A/C. [34]oolin et al. observed that MSCs infiltrated strongly into micropillar arrays and that MSC and nucleus morphology changed as micropillar spacing decreased. [35]The micropillars were designed with spacings of 5, 10, 20, and 50 μm, a height of 14 μm, and a diameter of 10 μm.The pillars were coated with Pluronic F127 on the surface and collagen type I elsewhere to ensure cells were only present between the pillars.The area covered by cells in the pillar arrays with 20 and 50 μm spacing was significantly larger than that in the pillar arrays with 5 μm spacing at 48 h.In the micropillar arrays with 50 μm spacing, cells exhibited typical basal spreading behavior, while in narrower pitches, cells showed a more spindle-shaped morphology, with most cells wrapping around the micropillars.However, in arrays with 5 or 10 μm spacing, cells remained mostly elongated.Nucleus shape is also aligned with cell body changes.Nuclei changes followed similar patterns as the cell bodies.Measurements of nuclear axes revealed that nuclei took on an elliptical shape in 10 μm spacing pillar arrays, while noticeable deformation occurred in the 5 μm pillar arrays, where the fitted ellipse exceeded the actual size of the nuclei.While no significant difference was found in the long axis of the nucleus among different micropillar arrays, the short axis of the nucleus decreased significantly in arrays with 5 μm spacing.The investigation of volumetric changes in cell nuclei due to topographical constraints has not been conducted.
In studies by Liu et al., they discovered that most of the cytoplasm in PLGA square micropillar arrays (height: 7 μm, pillar spacing: 6 μm, width: 3 μm) settled between the micropillars, resulting in severe nuclear deformation. [36]After 120 h, many nuclei were no longer located between the micropillars but had climbed to the top, displaying a diffuse stretching state.This indicated that nuclei, which initially underwent nuclear deformation, gradually regained their shapes over time on the micropillars.Moreover, with increasing micropillar height, nuclei transitioned from no obvious deformation to severe self-deformation. [37]Nuclei with a deformation index <0.8 were defined as deformed nuclei.The study revealed that the nuclei on smooth film and 0.8 μm high micropillars were hardly deformed, with nuclei appearing round or oval.However, when the height of the micropillars increased to 3.2 μm, approximately half of the nuclei became distorted into the spaces between pillars, resulting in significantly more deformed nuclei compared to micropillars with heights of 4.6, 5.3, and 6.4 μm.Addition of the ROCK inhibitor Y27632 resulted in a significant reduction in the number of MSCs undergoing nuclear deformation, suggesting the involvement of the RhoA/Rho ROCK signaling pathway in micropillar-induced nuclear self-deformation.
The effect of physical cues generated by micropillars on cell differentiation can be explained by the leap model proposed by Liu et al., [37] Using the shape index (SI) to quantify nuclear shape, they found that nuclear deformation on PLGA micropillars followed the physical "first-order jump" model.In this model, the critical pillar height for nuclear deformation was determined to be 3.2 μm, resulting in a clear bimodal curve.The critical pillar height for nuclear deformation may depend on the "natural" height of the nucleus, where exceeding the nucleus height leads to deformation in the form of a "puncture".In subsequent studies, they utilized PLGA micropillars to investigate the dynamics of nuclear deformation on topological biomaterials.They quantified the proportion of deformed nuclei and the average SI (<0.8 indicating nuclear deformation).The investigations revealed that MSC nuclear deformation was time-dependent, and the micropillar-induced response of nuclei was transient and reversible, with a rapid deformation process followed by a slow and incomplete recovery process.During the recovery phase, nuclei needed to break free from micropillar constraints with the assistance of actin filaments.It was hypothesized that the initial rapid and severe nuclear deformation in the first phase was primarily due to mechanical deformation rather than irreversible biological regulation.Their study further introduced the concept of "overshoot", a kinetic response process, to explain this phenomenon.Negative overshoot of the SI occurred when signals or functions from the nucleus were transmitted to the cytoskeletal network via the Linker of nucleoskeleton and cytoskeleton (LINC) complex.Actin filaments then exerted tensile forces on the deformed nucleus to pull it out of the micropillars' center, aiming to restore the spherical or ellipsoidal shape of the nucleus as much as possible.Thus, in media without osteogenic inducers, nuclear deformation depended on the initial deformation in the first phase and the relatively slow partial recovery in the second phase.In osteogenesis-inducing media, a third stage of nuclear deformation occurred, indicating further deformation likely caused by growth factors present in the media.
Pan et al. found that significant nuclear deformation only occurred when the height of PLGA micropillars exceeded a certain threshold. [38]No cells with significant nuclear deformation were observed in micropore arrays with a width of 3 μm, spacing of 6 μm, and depths of 0.2, 1, and 5 μm.In contrast, the micropillar group exhibited slow proliferation of bone marrow MSCs (BM-SCs) undergoing nuclear deformation during the first 3 days, followed by significant proliferation at later stages while maintaining nuclear deformation after 7 days.Due to the size of cultured cell nuclei being larger than the area between four neighboring micropillars, the nuclei extruded from the gaps between neighboring micropillars but did not climb up the pillar surface.The investigation of nuclear shape revealed a descending order of concave, protruding spherical, dumbbell-shaped, square, and triangular shapes, with triangular nuclei accounting for a negligible percentage.Therefore, when controlling nucleus shape, considerations should include micropillar density, relative size of the surrounding region compared to the nucleus, and the inherent stiffness of the nucleus.It is worth noting that gravity may not affect BMSC nuclear deformation as similar levels of nuclear deformation were observed even when PLGA membranes with 5 μm high micropillars and inverted configurations were used.
Grespan et al. [39] determined that micropillar-induced nuclear height deformation could impact the expression of important proteins such as laminin A/C during early blastoderm commitment or reorganization of nuclear membrane structure in endodermal cells.Human embryonic stem cells (hESCs) were cultured on PDMS square micropillar arrays with a width, height, and spacing of 7 μm.The study revealed highly deformed nuclei adapting to the basal morphology, penetrating into inter-pillar spaces, and exhibiting a branch-like distribution.Comparisons of deformability among different cell types, including human induced pluripotent stem cells (hiPSCs), cord blood MSCs (CBM-SCs), human epithelial renal cells (hERCs), and human osteosarcoma cells (SaOs-2), by quantifying the nuclear concavity (NCI).NCI values significantly differed between CBMSCs and hERCs compared to hESCs, hiPSCs, and SaOs-2.The latter three cell types exhibited NCI values exceeding 65%, indicating that at least two-thirds of analyzed nuclei displayed one or more concave structures.On the other hand, CBMSCs and hERCs had lower NCI values, with most CBMSCs cells lacking concavities or only having one concavity, while the nuclei of most hERCs remained rounded and did not fit into inter-pillar gaps.Cellular changes during three germline periods that occur in human pluripotent stem cells (hPSCs) after inoculation into micropillar arrays were also described.Early ectodermal cells exhibited nuclei located mostly at the top of the pillars, whereas nuclei of mesodermal and endodermal cells resided in the interstices between the micropillars.Endodermal cell nuclei showed much higher NCI than ectodermal cell nuclei.Deformation abilities of early ectodermal cells differentiating into ectodermal cells significantly decreased during differentiation, while the endoderm early germ layer spectrum proved particularly sensitive to nuclear shape changes.The maintenance of nuclear deformation relied on actin and nesprin, while laminin A/C and intermediate filaments conferred nuclear rigidity.Thus, it was hypothesized that micropillar-induced significant nuclear shape changes could impact the expression of important proteins like laminin A/C during reorganization of the endodermal early germ layer or nuclear envelope structure, ultimately influencing cell differentiation.

Adhesion
Fu et al. [41] found that cell shape, focal adhesion (FA) structure, and cytoskeleton (CSK) are interconnected systems involved in rigid sensing.Micrometer-scale rigid sensing can occur between FAs since the nanoscale mechanics at the top of individual PDMS micropillars remains constant.On rigid micropillars (1.556 nN μm −1 ), human MSCs (hMSCs) exhibit a welldistributed morphology with organized actin stress fibers and large focal adhesions, while on soft micropillars (1.556 nN μm −1 ), hMSCs appear rounded with prominent microvilli, unorganized actin filaments, and small focal complexes.The stress applied to inner FAs was significantly higher than that applied to peripheral FAs, suggesting an anisotropic distribution of FA stress due to increased internal traction and larger peripheral FAs.Average FA stress per cell increased with increasing rigidity of the mi-cropillars for both hMSCs and human umbilical vein endothelial cells (HUVECs).
Liu et al. [37] found that MSC on smooth PLGA surfaces and PLGA surfaces with micropillars up to 0.8 μm in height exhibited high expression of F-actin and formed mature focal adhension(FA) around the micropillars, while the nuclei maintained a natural shape.However, when the micropillar height exceeded 4.6 μm, MSCs displayed densely distributed FAs and actin around the micropillars, along with increased pseudopod formation.Vassey et al. [42] used the TopoChip platform, a polystyrene (PS) microarray with 2176 unique surface morphologies, to investigate the effect of micropillar morphology on macrophage adhesion.Micropillars with diameters of 5-10 μm were found to play a dominant role in driving macrophage adhesion.Macrophage adhesion was high on micropillars with diameters ≤5 μm, but significantly reduced on micropillars with diameters ≥10 μm.This relationship was derived from the interaction between the macrophage adhesion area and the total surface area, with the size of the attachment area categorized as high, medium, or low.The most pronounced change occurred at 10 μm in diameter.However, the critical micropillar size that is optimal for MSC adhesion remains unclear and requires further investigation.
He et al. [43] used micropillars with a diameter of 5 μm, a height of 10 μm, and spacings of 3 μm to study the adhesion of hMSCs.On small micropillars, hMSCs were strictly confined with low sphericity, while on large micropillars, cells loosely adhered to the bottom and side walls of the micropits with low sphericity.On micropillars of intermediate size, cells exhibited a highly spherical shape, while on flat surfaces, cells displayed the least spherical morphology.The number of micropillars encircling each micropit varied, but all cell types showed low sphericity due to high densification.However, because the height of the micropillars was lower than the cell diameter, cells could climb out of the micropits.Additionally, due to gaps between the micropillars, cells could shuttle between the micropits.
Kolind et al. [44] found that the inter-pillar gap size of p-type silicon wafer micropillars affected cell adhesion by influencing pseudopod formation from the cell body to the micropillar.Using a Biological Surface Structure Array (BSSA) containing various micropillar configurations, they found that DPSCs on pillars with an inter-pillar gap of 1 or 2 μm did not exhibit significant pseudopod extensions, while cells on pillars with a gap of 4 or 6 μm displayed elongated pseudopod extensions from the cells.The specific pillar shapes and arrangements in the array did not have a significant effect.Doolin et al. [35] found that cytoskeletal elements became more diffuse as PDMS micropillar restriction increased.In arrays of micropillars spaced at 5, 10, 20, and 50 μm, cells exhibited F-actin-rich protrusions wrapped around the micropillars, with focal adhesions anchoring them.In arrays with spacing of 20 and 50 μm, MSCs showed well-established linear focal adhesions with stress fibers, while in arrays with 5 and 10 μm spacing, punctate focal adhesions were observed, along with restricted microtubules within the F-actin region.These findings suggest that micropillars limit cytoskeletal elements and cellular adhesion patterns, potentially altering mechanosignaling pathways and cellular phenotypes.
Wang et al. [45] compared the effects of PLLA micropillars, microtubes, and micropits on cell adhesion and density using "stereo coverage" analysis.They found that projected cell area did not significantly differ between different micropatterned shape groups.However, HUVECs cell density was significantly higher on small micropillar arrays (6 μm height), large micropillar arrays (4.5 μm diameter), and microtube arrays (4.5 μm diameter) compared to small micropit arrays (1.5 μm diameter) and large micropit arrays (4.5 μm diameter).Cells tended to bypass the micropits rather than infiltrate them.Among micropatterns with the same height, projected cell area decreased with increasing height.Micropillar arrays consistently exhibited the largest projected cell areas for a given substrate morphology.Additionally, "stereo coverage" analysis revealed that HUVECs on micropillars with heights of 1 and 3 μm were in complete adhered to bottom, resulting in an adhesion depth equal to the height of the pillars.On micropillars with a height of 6 μm, the adhesion depth exceeded the predicted value of 3 μm, reaching 4 μm.Large micropillars displayed irregular top surfaces and increased hydrophobicity, leading to smaller adhesion depths compared to the predicted values.
Carthew et al. [46] found that micropillars made of OrmoComp, an organic-inorganic hybrid polymer, "indented" the nucleus, causing displacement of nuclear DNA around the micropillars while maintaining an intact nuclear membrane.It is hypothesized that the tension transmitted from the micropillar sur-face to the cell membrane is then transmitted through the cytoskeleton to the nuclear.This leads to a change in laminin A/C, resulting in the indentation of the nuclear membrane in response to micropillar tension.Myosin II can also induce nuclear indentation by regulating cytoskeletal tension and promoting MSC mineralization.Nuclear remodeling during this process was associated with decreased expression of H3K9 (a heterochromatin marker) and the methyltransferases DNM1 and DNMT3b, which are critical for establishing transcriptionally repressed chromatin during cytokinesis.These findings suggest that nuclear remodeling induces alterations in gene expression levels.
Hasturk et al. [32] found that oxygen plasma-modified PMMA micropillar arrays (increased wettability) promoted cell attachment.When interpillar spaces were completely occupied by cells, they started to relocate to the top of the pillars.In the unmodified group, cells anchored themselves at the top of the pillars using pillar walls. [47]he regulation of MSC adhesion by micropillar and the underlying mechanism are summarized in Table 2.

Migration
Bucaro et al. [30] proposed a mechanism for cell migration, suggesting that on high-density nanopillar arrays where the pillar spacing is much smaller than the critical distance allowing cell migration, cells can extend filamentous pseudopods in all directions to bridge the surface gaps.On medium-density nanopillar arrays, where the pillar spacing is roughly equal to the critical distance, cells can only extend pseudopods toward the lattice direction, while extensions in other directions cannot bridge gaps larger than the critical distance.On low-density nanopillar arrays, where the pillar spacing is much larger than the critical distance, cells are unable to bridge gaps between pillars in any direction, resulting in cell penetration beneath the substrate and extension along the bottom of the silica nanopillars.However, further research is needed to confirm whether spacing-induced morphological changes correspond to differentiation and proliferation, as well as the extent to which nanopillar surface geometry can be utilized to enhance stem cell differentiation.Doolin et al. [35] found that the migration rate of MSCs was reduced when the micropillar spacing narrowed.MSCs exhibited distinct migration behaviors in the presence of micropillars.In PDMS micropillar arrays with spacing of 5 and 10 μm, cells mostly remained elongated and tended to approach a pillar, probe on both sides, and choose one side to migrate toward.Occasionally, cells appeared to engulf a pillar completely and migrate around it.In arrays spaced 20 and 50 μm apart, cells displayed migration patterns similar to a 2D environment, including rapid directional migration or slower, more random migration.Sometimes, cells clung to one pillar and then migrated rapidly to another.As the pillar spacing decreased, cells seemed to migrate in a more "straight" path, while increased spacing resulted in more random migration.The speed of migration was reduced by narrower micropillar spacing.This phenomenon likewise manifests within non-stem cells, for instance, cell motility decreased with as micropillar spacing decreased, correlating with increased nuclear deformation in NIH 3T3 fibroblasts. [48]able 2. Adhesion of MSCs on Micropillars.

Mechanism
The "indentation" of the nucleus leads to complete displacement of nuclear DNA around the micropillar and subsequent gene expression alteration, which can be achieved by regulating cytoskeletal tension. [46]attern dimensions and hydrophilicity [32] A549 cells grown on polyacrylamide hydrogel micro-pillar arrays with 10 μm in height, 2 μm in diameter, and 16 μm in spacing exhibited faster closure of scratch wounds, indicating higher rates of migration and pillar invasion.However, in low micropillar arrays (2 μm), cells distributed on the pillar arrays did not experience severe nuclear deformation and did not bypass the pillar structures in an amoeba-like pattern.Knocking down CXCL16 increased the chances of cell migration and invasion when it was knocked down. [49]IH 3T3 cells cultured on PDMS micropillars with heights of 77.87 ± 7.86 μm and 4.07 ± 0.11 μm exhibited actin polarization toward the stiffer portion of the array due to unbalanced tractive forces induced by pillar elasticity.When cells grew parallel to the direction of elasticity, a contractile gradient increased to some extent, which may lead to cell migration.As cells entered the rigid region, they began to explore that part of the array, and some cells grew perpendicular to the rigidity gradient. [50]aez et al. [51] designed PDMS dense micropillar arrays with an elliptical cross-section.Varying the height of the micropillars created an anisotropic stiffness distribution ranging from 10-78 nN μm −1 .Epithelial cells were induced to migrate in the direction of maximum stiffness, and high traction stress and high deformation zones were mainly located at cell edges, which correlated with actin stress fiber orientation and adhesion patterns.Actin stress fibers aligned with the highest rigidity direction of the micropillar array, although the mitotic axis of islet cells did not display a preferred orientation, suggesting that the extension of cellular components in the direction of maximal stiffness could not be solely explained by cell division orientation.
Liu et al. [52] fabricated "Janus" micropillar arrays with asymmetric cell adhesion properties, allowing cells to sense asymmetric adhesion signals when adhering to microstructures on material surfaces.This asymmetric adhesion signal induced cell polarization, which provided the driving force for cell migration.Each cell made contact with multiple Janus micropillars, resulting in the generation of adhesion polarization and subsequent cell migration toward the gold side.
The cell adhesion and movement mechanisms were explained by Curtis and Wilkinson, [53] proposing that cells perceive surrounding structures through sensory elements such as adhesion patch complexes.If the physical cues are discontinuous and smaller than the distance between sensory elements, cells can exhibit continuous adhesion between the cues.However, if the distance between the cues exceeds the distance between sensory elements, cells will show discontinuous adhesion.The limitations of sensory element distances and their cell specificity are not fully understood, and further experimental studies are needed.
The invasion and migration of hMSCs within tight interstitial space in micropillar arrays were increased with reduced deformability and viscoelastic solids with passage. [54]he regulation of MSC migration by micropillar and the underlying mechanism are summarized in Table 3.
Moreover, micropillar arrays were used to study migration of individual and collectively epithelial mesenchymal transition (EMT)-activated tumor cells.Individual cells with a small number of neighboring cells exhibited rapid, straight trajectory spreading, while individual cells with multiple neighboring cells displaying the same epithelial markers migrated collectively. [55]This suggests a relationship between cell migration patterns and the manner in which cells migrate.

Characteristics of micropillars Alterations in migration & mechanism Micropillar stiffness
Anisotropic stiffness promotes scratch wound closure [49] Epithelial cell migration toward maximal stiffness [51] Micropillar spacing Narrower spacing reduces migration [35] Mechanism Actin stress fiber orientation [51] Adhesive plaque complexes [52] Viscoelastic solids deformation and deformability reduction (by passage) increase invasion and migration [54] et al. [61] developed an artificial bone marrow cavity using PDMS, filled with type I collagen gel containing osteoinductive demineralised bone powder (DBP) and bone morphogenetic proteins.The setup resulted in the formation of a bone marrow compartment within 4-8 weeks.Initially, adipocytes were predominantly, but sealing the cavity with a solid PDMS layer resulted in bonelike tissue formation with a predominatly haematopoietic cell population within 8 weeks.Micropillar arrays exert different effects on MSCs differentiation.Stiff micropillar arrays enhance osteogenic differentiation of MSCs, while soft micropillar arrays promote lipogenic differentiation. [62,63]The early cytoskeletal response during hM-SCs differentiation is important, but it remains unclear if the endogenous cytoskeletal contractility of individual hMSCs predicts differentiation outcomes. [41]asturk et al. [32] found that hydrophobic micropillars on PMMA surface can induce osteogenic differentiation of MSCs without chemical inducers.They prepared square PMMA micropillar arrays with an 8 μm height and different lateral dimensions (width or inter-pillar gap ranging from 4 to 16 μm).
DPSCs on hydrophobic micropillar arrays showed upregulated expression of the osteogenic factor Osterix (OSX) on the first day, increased expression on the 7th day, and decreased expression below baseline on the 28th day.In contrast, DPSCs on hydrophilic micropillar arrays (plasma-treatment) exhibited significantly lower expression of OSX, alkaline phosphatase (ALP), and osteocalcin (OC) compared to the non-plasma-treated group by the seventh day.Plasma treatment accelerated cell spreading and proliferation while reducing osteogenic differentiation and mineralization.Micropatterning restricted cell spreading, promoted nuclear deformation, limited cell proliferation, and enhanced osteogenic differentiation and mineralization.
Liu et al. [37] found that even though cells on higher micropillars had a reduced projected area, altered nuclear shape due to nuclear deformation played a predominant role in determining cytoskeletal-mediated tension and stem cell differentiation.MSCs placed on micropillars of 4.6 and 6.4 μm in height (with a side length and spacing of 3 and 6 μm, respectively) exhibited higher expression of osteogenic markers including ALP and runt-related transcription factor 2(Runx2) and lower lipogenic markers including low density lipoprotein (LDL) and peroxisome proliferator-activated receptor  (PPAR) than MSCs on smooth surface and height of 0.8 μm micropillars.This suggests that the altered nuclear shape led to changes in gene expression, affecting the expression of osteogenic and lipogenic genes.Nuclear shape has been shown to alter the gene expression and protein synthesis in primary rat osteoblasts on a multi-structural domain model. [64]Nuclear deformation has been shown to induce dramatic chromatin condensation and decrease cell proliferation in primary rat osteoblasts, as well as elongation of endothelial cells. [65]aivosoja et al. [66] found that MSC could grow in 3D within the space of 20 μm square micropillars.Osteogenic differentiation mainly occurred between 200 nm, 5 μm high TiO 2 -covered silica pillars, whereas 20 μm high silica pillars enhanced cytoskeletal elongation and ERK signaling but reduced cell density, leading to a decreased intercellular communication of osteoblasts through calreticulin and gap junctions, resulting in poor osteogenesis.RAS-induced ERK signaling is essential for maintaining cellular homeostasis and organismal development.The complexity arises from the wide range of effector substrates involved. [67]RK has been shown to phosphorylate and activate RUNX2 in osteoblasts, [68] thereby inducing an osteogenic gene expression pattern.
Kolind et al. [44] found that deposition of osteogenic markers increased significantly as interpillar distance increased by 1, 2, and 4 μm, but then decreased with a further increase to 6 μm.They hypothesized that the increase in interpillar distance forced cytoskeletal rearrangement, resulting in direct pulling of the nucleus and chromosomes, which was detrimental to the expression of osteogenic genes and related proteins.They also found that medium components had a large effect on topographical changes during induction of osteogenesis.Medium containing osteogenic inducers may enhance accumulation of extracellular matrix thereby limiting the effect of micropatterning on osteogenic response, so that DPSCs responded most significantly to topographical cues only during the initial stages of differentiation.The alternative explanation is that the effect of basal morphology on osteogenesis is regulated in part by controlling the expression level of cellular -conjugated proteins, which play key roles in the BMP and Wnt signaling pathways. [69]conjugated proteins are essential for differentiation of precursor cells into osteoblasts but not chondrocytes. [70]The Wnt signaling pathway actsas an upstream activator of BMP2 expression in osteoblasts. [71]mong different micropillars, the highest level of MSC mineralization was observed in the 5 × 5 μm 2 micropillar group, where 48% of the YAP proteins localized in the nucleus, probably due to the "indentation" of the nucleus by the micropillar and regulation of cytoskeletal tension by myosin II. [46]Silica nanopins reduced YAP nuclear accumulation, possibly due to focal points of tension caused by the "sharp" nature of nanopins, leading to a reduction in the YAP nuclear localization.This indicates that the degree of surface micropillar concavity and convexity is also an important factor influencing MSC differentiation into osteoblasts.YAP is highly responsive to mechanical stress.Mechanical signals

Soft Micropillar arrays
Enhance osteogenic differentiation

Osteogenic differentiation
Poor osteogenesis due to lower cell density and cell-cell communication

Patterning and Surface Modification
Anisotropic micropillars or curved microstructures Promote osteogenic differentiation via the RhoA / ROCK pathway [74] Hydrophobic micropillars on the PMMA surface Induce osteogenic differentiation independent on chemical inducers [ 32,44] transmitted through the cytoskeleton can regulate YAP nuclear entry including YAP phosphorylation induced by actin-myosin contraction, and alterations in YAP nuclear localization through cytoskeletal integrity. [72]YAP promotes osteogenesis and inhibits adipose differentiation by regulating -catenin signaling. [73]urved microstructures have also been reported to promote MSC osteogenesis through the RhoA / ROCK pathway. [74]This suggests that micropillar with varying curvatures or anisotropy could be introduced to explore the mechanism of osteogenic differentiation induced by physical cues.RhoA has been extensively involved in integrin-mediated signaling and mechanotransduction.The process through which ECM rigidity influences osteogenic differentiation might entail MAPK activation downstream of the RhoA-ROCK signaling pathway. [75]reparation of medical devices with micropillar morphology can promote bone regeneration through chromatin programming.Wang et al. [76] induced nuclear morphology changes through contact guidance in a micropillar model to investigate their effects on 3D chromatin conformation and transcriptional reprogramming of hMSCs.Implantation of hMSC-containing micropillar scaffolds into rodents promoted bone regeneration in a cranial defect model.Their study demonstrates that micropillarinduced nuclear deformation serves as a promising tool for chromatin engineering to promote transcriptional reprogramming in stem cells, thereby predictably controlling cell fate and specifically enhancing the bone regeneration potential of stem cells.
Micropillar arrays have the potential to improve the biocompatibility of grafted devices.Hauscwitz et al. [77] analyzed the laser induced periodic surface structure (LIPSS) and square micropillar arrays for improved biocompatibility.The combination of LIPSS and micropillars was found to produce good biocompatibility on stainless steel surfaces by giving precise cell orientation while providing a better environment for cell attachment and proliferation.Their pioneering combination of high energy ultrashort pulsed laser system with multi-beam and beamshaping technology enabled the high-speed generation of functional micropillar structures, which is expected to lead to the large-scale production of bone implants with integrative properties that control the growth of cellular tissues and improve the success rate of bone regeneration and grafting.Lauria et al. [78] fabricated micropillar arrays made of alumina ceramics with a column height of 40um and a pillar spacing of 100um or 300um by inkjet printing technology in order to improve the integrative capacity of inert highperformance ceramics for bone and found that micropatterning can mediate the generation of focal cell adhesion and stimulate osteogenic differentiation of cells.
In summary, micropillar size, stiffness, shape, and surface features can influence MSC differentiation and gene expression patterns (Table 4).Understanding these relationships can help design novel strategies for directing stem cell differentiation and advancing tissue engineering applications.The role of micropillars in osteogenic differentiation and associated mechanotransduction pathways are summarized in Figure 2.

Micropillars for Cellular Biomechanics Detection
Micropillar arrays offer unique tools to study cell-extracellular matrix interaction, cellular mechanical properties, and response of cells to physical cues, providing valuable insights into cell behavior in various contexts such as migration, mechanotransduction, and differentiation processes.Role of micropillars in osteogenic differentiation and associated mechanotransduction pathways.Micropillars play a crucial role in guiding the osteogenic differentiation of MSCs, primarily through the activation of Wnt/-catenin, and RhoA/ROCK signaling pathways.These pathways regulate the translocation of -catenin and YAP from the cytoplasmic to the nucleus, thereby influencing gene expression.Consequently, the levels of osteogenic marker such as alkaline phosphatase (ALP), osterix (OSX), osteocalcin (OC) and runt-related transcription factor 2 (Runx2) are enhanced.

Cell Traction Force Microscopy
Adherent cells deform the extracellular matrix through the intracellular skeleton, and this deformation can be measured using traction force microscopy (TFM) to quantify the cellular traction forces generated. [79]Two commonly used TFM methods include embedding fluorescent magnetic beads in an elastic polymer film to measure substrate displacement caused by cellular forces, and using PDMS micropillar arrays (Figure 3A) to assess cellular traction forces based on pillar deformation. [80]icropillar arrays can be categorized into micron and nanometer scale sizes, with nanoscale pillars offering higher spatial resolution of cell forces but exerting a greater effect on cells due to the higher number of pillars compared to micrometer-sized pillars.By varying pillar spacing, it has been observed that cells exert less force on regions of higher pillar density and more force on regions of lower density.Additionally, cells tend to migrate in the direction of increasing pillar density, with higher forces concentrated at the trailing edge and lower forces in the center. [81]dification of micropillars or application of external forces to the pillars, such as magnetic micropillars, vacuum stretching, or microfluidics, can mimic forces experienced by cells in their surrounding environment.Magnetic micropillars containing magnetic particles can alter the pillar spacing and induce changes in cell attachment area and migration.These external forces can influence cell behavior by transmitting information from the extracellular matrix to the cells through the cytoskeleton and mechanosensitive ion channels.However, careful control of these simulated external forces is crucial to avoid significant alterations in cell behavior. [81]DMS pillar arrays can not only assess cell-substrate forces but also indirectly respond to cell-cell forces.While some studies suggest that cell-cell forces are proportional to cellcell contact area, [82,83] other studies using TFM for epithelial cells did not find this correlation, [84] indicating that cell-cell forces are not solely dependent on contact area size.
Figure 3. Advancements in Frontier Detection and Technological Analysis using micropatterns.The micropatterns have emerged for utilization in quantifying and simulating cell-substrate forces, assessing the viscoelastic properties of cells, imparting physical cues including matrix stiffness, providing cellular stretching, and facilitating interactions between cells or organoids.A) Quantification of Cell Traction Force.The generation and calculation of cell traction force are achieved by precisely measuring the displacement of micropatterns.This quantitative analysis enables the precise calculation of forces exerted by cells, facilitating the study of cellular mechanics.B) Micropillar-Induced Cell Stretching: Micropillars serve as mechanical stimuli that induce cell stretching through the generation of pressure difference by vacuum.This mechanical stimulation plays a crucial role in modulating cellular behaviors.C) Organ-on-a-chip, for instance, micropillars have been employed to sort skeletal stem cells for bone regeneration (i).The critical radius, denoted as Rc, is a crucial parameter in this process and is determined by both the spacing of the columns and the angle at which the column array is oriented relative to the main flow direction.When it comes to separating cells with radii smaller than Rc, their behavior is primarily influenced by the flow pattern near the microcolumns.These smaller cells are not compelled to cross the flow lines but tend to follow the path defined by the microcolumns.When subjected to deformation due to gradient shear stresses, acceleration, and interactions with the microcolumns, more compliant particles with radii smaller than Rc can continue along their original flow trajectory without experiencing a net displacement.In another context, vascularization has been a crucial consideration in the development of bone-on-a-chip systems (ii).One such microfluidic device comprises four parallel channels separated by microcolumns with a 100 μm gap.In this setup, lung fibroblasts (FLs) serve as stromal cells, supplying essential vascular endothelial growth factors (VEGF) and extracellular matrix (ECM) proteins that promote angiogenesis of endothelial cells (ECs).To maintain the desired stiffness and rigidity of the bone ECM, hydroxyapatite particles are incorporated into fibronectin.The doping ratio of these particles can be adjusted to influence EC outgrowth and ensure the appropriate mechanical properties for bone tissue engineering.

Detection of Cellular Mechanical Properties
Micropillars can serve as tools to measure cellular viscoelastic parameters and assess cellular mechanical properties.On smooth surfaces, primary hMSCs were found to be softer than human fibroblasts, with hMSCs transitioning from a viscoelas-tic fluid to a viscoelastic solid during passaging, indicating an increase in cell stiffness. [54]In the presence of micropillar arrays, primary hMSCs tend to exhibit linear growth similar to a mechano-viscoelastic fluid, while primary human foreskin fibroblasts (hHFF) grow in branches, which may be related to their stiff skeleton and generation of leading edge forces.
Progenitor hMSCs with lower mechanical deformability have the highest probability of invading micropillars with small spacing, but excessively soft cells show poor deformability in narrow spaces. [54]Micropillar arrays resembling extracellular matrix structures can increase cell stiffness and contractility, providing driving forces for hMSC invasion through intracellular molecular mechanisms and the cytoskeleton. [54]The induced nuclear deformation (MIND) platform utilizes nuclear stiffness as a physical parameter to quantify the nuclear elasticity of adherent cell populations and individual adherent cells, as well as assess cancer cell species and lineage. [85]icropillars can be used to control cell elongation and alignment.For example, PDMS micropillar arrays with varying stiffness and geometry have been shown to effectively elongate and align hMSCs and cardiomyocytes. [86]icropillars with anisotropic stiffness and geometry resulted in increased elongation rates compared to 2D microenvironments. [86]icropillars can be used to assess cell membrane fluidity.Using traction microscopy, the membrane fluidity of BMSCs containing rhodamine B isothiocyanate (MNPs@SiO2 (RITC)) were assessed, also, biological functions related to membrane fluidity such as cell viability, reactive oxygen species (ROS) production, intracellular cytoskeleton, and the migratory capability could be then evaluated. [87]icropillars can also be used for measurement of ECM properties in bone.In osteogenesis imperfecta (OI), a condition characterized by increased bone brittleness, Indermaur et al. [88] used the micropillar pressurization method to compare the mechanical properties of OI bone with healthy bone.Surprisingly, they found that the compressive strength of OI bone did not decrease and even increased with the severity of OI.However, this study highlighted the importance of considering factors like porosity, as increased porosity can affect the mineralization of the ECM and eventually lead to a reversal in the observed mechanical properties.
It's important to note that the PDMS pillar array method creates a rough surface rather than a continuous flat plane, which differs from the physiological microenvironment in which cells reside.The physical cues provided by PDMS pillar arrays, such as stiffness, pillar surface area, roughness, and density, can influence cellular behavior.MSCs are capable of sensing matrix stiffness, pillar length, and concavity/convexity of pillar surfaces, leading to corresponding cellular changes.Lipogenic differentiation was found to be similar on long and soft substrates, while osteogenic differentiation was observed on short and stiff substrates.Elliptical-shaped pillars with higher stiffness in the long-axis direction were found to induce MSC growth and migration along the direction of higher stiffness. [89]Additionally, introducing protein coatings on the PDMS surface can also influence cell behavior depending on the type, coverage area, and cellular sensitivity to the proteins.For instance, covalently attaching collagen or fibronectin to the PDMS surface enables the maintenance of undifferentiated states in MSCs, [90] although restricting cell spreading area has been found to impact cellular traction and mechanotransduction effects within that area. [91]

Novel Cellular Biomechanics Experimental Platforms
The innovative platforms enable precise control over mechanical cues, differentiation induction, and organoid establishment.They offer new opportunities for studying cellular behavior and advancing tissue engineering and regenerative medicine applications.

Stretching Loading
Cyclic stretching has been shown to induce significant changes in the direction of cell spreading.Different groups of cells exhibit smaller differences in stabilized spreading values when subjected to the same cyclic stretching protocols.Micropillar density restricts cell spreading, and it takes longer for cells in the Small size group to reach equilibrium compared to other groups.Stretching also affects the diffusion rate within microwells, with the order being Medium size >Large size >Small size groups.Differences in cell roundness in microwells may impact cell adhesion to micropillars and cytoskeletal arrangement.Cells with higher roundness may experience attenuated adhesion due to stronger mechanical disturbances, making them more suitable for cell migration. [43]evealing the response of individual cells to mechanical cues is challenging without innovative material platforms to control mechanical stimuli at the single-cell scale.Micropillars fabricated on elastic polymer membranes integrated into microfluidic chips allow for controlled mechanical stimulation of individual cells (Figure 3B).Plasma treatment of PDMS membranes eliminates the effects of adhesion proteins that promote cell attachment. [43]

Organ-On-A-Chip
Microfluidic systems integrated with micropillar arrays enable valuable applications, such as screening suitable skeletal stem cells (SSC) for bone regeneration (Figure 3C).In a study by Xavier et al., [92] label-free sorting of SSCs based on cell size and stiffness was achieved using the deterministic lateral displacement (DLD) technique.The sorted SSCs remained viable and retained the ability to form clonogenic cultures (CFU-F).Additionally, the research showed that SSCs containing larger bone marrow cells exhibited greater CFU-F capacity.This approach holds promise for identifying and isolating specific cell populations for bone regeneration therapies.
Cellular interactions that occur between micropillars are of great importance.For example, in a study by Jusoh et al., [93] a vascularized bone-on-a-chip (Figure 3C), a microfluidic device with four parallel channels separated by fibronectin, and micropillars with a gap of 100 μm gap was used to mimic a bone tissue scaffold.This microfluidic platform featured specific channels for endothelial cell, bone cell, media, and stromal cells.The micropillar spacing facilitated the formation of blood vessels and allowed for paracrine communication between endothelial cells and stromal cells.The use of microfluidics induces gradient changes in matrix density and vascular endothelial growth factor (VEGF) concentration, while incorporating hydroxyapatite doped with fibrin to achieve a platform hardness and stiffness closer to that of real bone.This model provided a good representation of bone tissue microenvironment, supporting bone vascular sprouting.The vascularized 3D bone tissue generated in this model played a vital role in nutrient renewal within the tissue, transportation of metabolic waste products, distribution of tissue cytokines, and maintenance of the cellular niche in bone tissue.Such studies using micropillar-based microenvironments contribute to our understanding of tissue engineering and regenerative medicine.
While multilayer microfluidic chips incorporating PDMS micropillar arrays have been employed to cultivate specific brain region organoids from human induced pluripotent stem cells (hiPSCs), [94] this promising technique has not yet been applied in bone regeneration.

Conclusion and Prospective: Advancing Micropillar Technology for Precise Control of Stem Cell Behaviors in Tissue Engineering
Tissue engineering aims to control cell behavior for regenerative medicine applications.Patterns of chemical-induced differentiation have revealed certain benefits, but also pose risks due to potential oncogenicity resulting from excessive exposure to BMP and dexamethasone. [95]An alternative approach utilizing substrates with specific morphologic patterns for cellular incubation offers precise control over cell growth and development without the need for potentially harmful chemicals.Among these methods, micropillars have emerged as a promising strategy to modulate stem cell responses, influencing aspects such as morphology, adhesion, migration, and differentiation.Novel methods for cellular mechanical properties detection and analysis of cell-cell interactions are developed using micropattern (Figure 3).
While multilayer microfluidic chips featuring PDMS micropillar arrays have been successfully utilized for the development of specific brain region organoids from human induced pluripotent stem cells (hiPSCs), it's crucial to recognize that applying this technique to bone regeneration may pose unique challenges.Bone regeneration is a multifaceted process encompassing not only stem cell differentiation into osteoblasts but also the intricate formation of a functional extracellular matrix, vascularization, and seamless integration with the surrounding tissues.
Although micropillar arrays and microfluidic devices can offer valuable insights into cell behavior and tissue development, their direct application to bone regeneration may necessitate tailored adaptations and optimizations to address the distinct hurdles inherent to bone tissue engineering.Researchers in the field of bone regeneration typically explore a wide array of approaches, encompassing the utilization of biomaterial scaffolds, growth factors, mechanical stimulation, and various cell sources.
Micropillar arrays and microfluidic devices hold the potential to contribute significantly to the refinement of these approaches and the study of specific facets of bone tissue development.However, their seamless integration into bone regeneration strategies calls for further dedicated research and development efforts.Additionally, the curved membrane itself can act as a biochemical signal, participating in intracellular signaling through curvature-sensing proteins. [96,97]ombining various mechanical and biochemical stimuli with micropillar-based modulation creates sophisticated microenvironments, mimicking in vivo conditions.Investigating the combinatorial effects of multi-dimensional cues improves tissue engineering applications and regenerative therapies.11) Novel Micropillar Fabrication Techniques: Novel micropillar fabrication techniques represent a crucial area of advancement in harnessing the full potential of micropillars for various applications.While conventional methods like soft lithography and micromolding have been widely used to create micropillars, exploring new fabrication techniques can offer more precise control over micropillar properties and design parameters, such as shape, spacing, and height.This enhanced control can open up new opportunities for optimizing micropillar configurations and tailoring their properties for specific applications.Promising novel fabrication techniques include 3D printing, [98,99] nanofabrication, [100,101] and microfluidics-based methods. [102,103] summary, micropillars hold great promise in the field of stem cell research and tissue engineering.While the innovative techniques involving PDMS micropillar arrays and microfluidic chips have found success in other areas of regenerative medicine, their direct application to bone regeneration may necessitate further customization and exploration to effectively address the complex demands of bone tissue engineering.Their unique ability of micropillars to modulate MSC behavior and respond to mechanical cues offers exciting possibilities for various applications, including cell behavior modulation, biomechanical testing, and organoid development.To fully unlock their potential, it is essential to continue research into understanding the mechanotransduction pathways, investigating cell-cell interactions, optimizing micropillar designs, and exploring the interplay between physical and biochemical cues.Moreover, the development of advanced fabrication techniques will enable researchers to design and control micropillar properties with greater precision, expanding their applications in regenerative medicine and tissue engineering.By addressing these key aspects, we can harness the full potential of micropillars and pave the way for transformative advancements in biomedical research and clinical applications.

Figure 1 .
Figure 1.Effect of micropillars on cell behaviors.The behavior of MSCs were regulated by micropillars including cellular and nuclear deformation, adhesion, migration, and differentiation.When MSCs are situated on the micropillar, they can either lie flat on the surface of micropillars or become partially or completely trapped between the micropillars, resulting in cellular and the nucleus deformation.Focal adhesion primarily mediates the attachment of MSCs around the micropillars.The migration of MSCs in micropillars is influenced by the physical parameters of micropillars, including their size, shape, and spacing.Moreover, micropillars induce osteogenic differentiation of cells through the mechanotransduction signaling.

Figure 2 .
Figure2.Role of micropillars in osteogenic differentiation and associated mechanotransduction pathways.Micropillars play a crucial role in guiding the osteogenic differentiation of MSCs, primarily through the activation of Wnt/-catenin, and RhoA/ROCK signaling pathways.These pathways regulate the translocation of -catenin and YAP from the cytoplasmic to the nucleus, thereby influencing gene expression.Consequently, the levels of osteogenic marker such as alkaline phosphatase (ALP), osterix (OSX), osteocalcin (OC) and runt-related transcription factor 2 (Runx2) are enhanced.

1 ) 6 )
Mechanotransduction Mechanisms: The dimensions and arrangement of micropillar architectures play a crucial role in shaping MSC behavior, serving as valuable tools to measure cellular biomechanics.However, the underlying molecular and cellular mechanisms are not fully understood.Investigating specific signaling pathways, mechanotransduction mechanisms, and gene expression profiles involved in the cellular response to micropillars is essential.Integrating cutting-edge techniques like single-cell sequencing and chromatin accessibility profiling with micropillar assays can uncover novel gene regulatory networks and signaling pathways, shedding light on mechanotransduction processes.Additionally, testing whether micropillar hold the mastery to steer the multifarious differentiation trajectories of MSCs (such as osteogenesis, adipogenesis, neurogenesis, and angiogenesis), and studying different stem cell types' responses to micropillar-mediated modulation will advance our overall understanding in this field.2) Cell-Cell Interaction: While current research has focused on cell-matrix interactions, the influence of micropillars on cell-cell interactions is equally crucial.To comprehensively understand stem cell behavior, studying their responses within a multicellular microenvironment is imperative.Exploring how micropillars affect cell clustering, communication, and collective behaviors will provide insights into the role of physical cues in stem cell behavior.Particularly, investigating MSC responses in co-culture with other cell types like endothelial cells or immune cells will enhance our understanding of how physical cues modulate stem cell behaviors in complex biological environments.3) Microenvironmental Factors: The surrounding microenvironment significantly influences stem cell responses to micropillars.Understanding the interplay between soluble factors, neighboring cells, and the extracellular matrix composition is critical for precise prediction and control of stem cell fate.Investigating how microenvironmental factors modulate the effects of micropillars will offer a comprehensive understanding of the complex signaling networks involved in stem cell behavior, enabling the design of tailored micropillar-based platforms.4) Long-Term Effects and Tissue Integration: While micropillars have shown promise in guiding MSC differentiation toward specific lineages, understanding their long-term impact on MSC behavior is essential.Investigating the sustained effects, including stemness, self-renewal capacity, and multilineage differentiation potential, will optimize micropillar-based scaffold design.Long-term stability studies should assess induced cell fates during extended culture periods and conditions mimicking physiological remodeling processes, facilitating the development of functional tissues or organs.5) Combination with Biochemical Factors: Micropillars offer a unique advantage for targeted and controlled delivery of biomolecules and growth factors to MSCs.Exploring the synergistic interplay between physical cues provided by micropillars and biochemical factors is crucial in directing MSC behavior effectively.Functionalizing micropillar surfaces with bioactive molecules can enhance our understanding of the combined effects of physical and biochemical cues on stem cell behavior, advancing stem cell-based therapies.Mechanobiology of Stem Cell Aggregates: Stem cell aggregates, such as embryoid bodies or spheroids, offer a physiologically relevant model for studying stem cell behavior.Investigating how stem cell aggregates respond to micropillars provides valuable insights into collective stem cell behavior and their ability to sense and integrate mechanical cues.Understanding interactions within aggregates can inform the design of biomimetic strategies for engineering functional tissues and organs.7) Scaffold Optimization: Optimizing scaffolds is critical for maximizing the potential of micropillar-based approaches in tissue engineering.Further research is needed to determine the ideal parameters for micropillar-based scaffolds, facilitating cellular processes and efficient cell infiltration.Strategies for scalable production of large-scale micropillar-based scaffolds are crucial for clinical applications.8) Clinical Translation: Before clinical application, validating micropillar effects in animal models and primary human MSCs is essential.Assessing scalability, reproducibility, and safety of micropillar-based therapies through rigorous preclinical studies is vital.Incorporating biocompatible materials into micropillar fabrication ensures seamless integration with host tissues.9) Advanced Techniques: Leveraging advanced techniques such as microfluidics, live-cell imaging, and dynamic modulation of mechanical forces enhances our understanding of micropillar-based modulation and stem cell behavior.Highthroughput screening platforms can accelerate the discovery of materials and treatments, advancing stem cell-based therapies.10) Integration of Multi-Dimensional Cues: Various mechanical stimuli, such as hydrostatic pressure, tension loading, oscillatory fluid flow, compressive loading, vibration, and lowintensity pulsed ultrasound, have been shown to influence MSC differentiation.