One-Year Changes in Collagen Type in Semitendinosus Tendons: A Case Study Using Tissues Obtained From a Growing Patient.

Recently, there have been concerns about the high postoperative re-injury rate associated with the use of the semitendinosus tendon (ST) as an autograft for anterior cruciate ligament reconstruction in adolescent patients before the closure of the epiphyseal line. Our previous studies showed that this high re-injury is related to the histological and mechanical immaturity of ST in adolescent patients. Moreover, the overall structure of collagen fibers is strengthened with the application of traction force to tendon tissue. Therefore, it is assumed that, in vivo, bone growth and increased height increase the traction force applied to tendon tissue and the percentage of type I collagen, which has a remarkable physical strength. The present study aimed to investigate the changes in the content of ST's type I collagen in an adolescent patient over one year. The patient was an 11-year-old male with bilateral patellar dislocations. The orthopedic surgeon performed medial patellofemoral ligament reconstruction on the left knee using an ST graft, followed by a similar procedure on the right knee one year later. ST tissue that would have been discarded during each procedure was harvested and used. The bone of the patient's legs grew approximately 8 cm during the one-year period. The obtained tissues were immunostained and microscopically observed to evaluate the area content of type I and III collagen. The area content of type I collagen in STs collected from the patient was 66%. The area content of type I collagen increased rapidly to 95% one year later. A comparison of the two STs obtained from the patient in the first half of their 10th year showed that the type I collagen content of the STs increased rapidly over one year. This fact may provide a preliminary insight into the prevention of re-injury when selecting the autograft for anterior cruciate ligament (ACL) reconstruction in adolescent patients.

Interrogating the Molecular Clutch in Neuronal Growth Cones: Measuring Traction Forces, F-actin Retrograde Flow, and Point Contact Demographics.

Growth cone-dependent outgrowth of neuronal processes is essential for the development, plasticity, and regenerative capacity of the nervous system. This process involves the attachment of the growth cone to the substrate and the cyclical engagement/disengagement of the molecular clutch at the sites of adhesive contact. In this chapter, we describe protocols for traction force microscopy, measurement of F-actin retrograde flow velocities, and the assessment of adhesive point contacts by immunofluorescence. These complementary techniques collectively facilitate investigations into the regulation of the molecular clutch in neuronal growth cones.

Multimodal Mapping of Electrical and Mechanical Latency of Human-Induced Pluripotent Stem Cell-Derived Cardiomyocyte Layers.

The synchronization of the electrical and mechanical coupling assures the physiological pump function of the heart, but life-threatening pathologies may jeopardize this equilibrium. Recently, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have emerged as a model for personalized investigation because they can recapitulate human diseased traits, such as compromised electrical capacity or mechanical circuit disruption. This research avails the model of hiPSC-CMs and showcases innovative techniques to study the electrical and mechanical properties as well as their modulation due to inherited cardiomyopathies. In this work, hiPSC-CMs carrying either Brugada syndrome (BRU) or dilated cardiomyopathy (DCM), were organized in a bilayer configuration to first validate the experimental methods and second mimic the physiological environment. High-density CMOS-based microelectrode arrays (HD-MEA) have been employed to study the electrical activity. Furthermore, mechanical function was investigated via quantitative video-based evaluation, upon stimulation with a ß-adrenergic agonist. This study introduces two experimental methods. First, high-throughput mechanical measurements in the hiPSC-CM layers (xy-inspection) are obtained using both a recently developed optical tracker (OPT) and confocal reference-free traction force microscopy (cTFM) aimed to quantify cardiac kinematics. Second, atomic force microscopy (AFM) with FluidFM probes, combined with the xy-inspection methods, supplemented a three-dimensional understanding of cell-cell mechanical coupling (xyz-inspection). This particular combination represents a multi-technique approach to detecting electrical and mechanical latency among the cell layers, examining differences and possible implications following inherited cardiomyopathies. It can not only detect disease characteristics in the proposed in vitro model but also quantitatively assess its response to drugs, thereby demonstrating its feasibility as a scalable tool for clinical and pharmacological studies.

Exploration of Curvature and Stiffness Dual-Regulated Breast Cancer Cell Motility by a Motor-Clutch Model and Cell Traction Force Characterization.

The migration of breast cancer cells is the main cause of death and significantly regulated by physical factors of the extracellular matrix (ECM). To be specific, the curvature and stiffness of the ECM were discovered to effectively guide cell migration in velocity and direction. However, it is not clear what the extent of effect is when these dual-physical factors regulate cell migration. Moreover, the mechanobiology mechanism of breast cancer cell migration in the molecular level and analysis of cell traction force (CTF) are also important, but there is a lack of systematic investigation. Therefore, we employed a microfluidic platform to construct hydrogel microspheres with an independently adjustable curvature and stiffness as a three-dimensional substrate for breast cancer cell migration. We found that the cell migration velocity was negatively correlated to curvature and positively correlated to stiffness. In addition, curvature was investigated to influence the focal adhesion expression as well as the assignment of F-actin at the molecular level. Further, with the help of a motor-clutch mathematical model and hydrogel microsphere stress sensors, it was concluded that cells perceived physical factors (curvature and stiffness) to cause changes in CTF, which ultimately regulated cell motility. In summary, we employed a theoretical model (motor-clutch) and experimental strategy (stress sensors) to understand the mechanism of curvature and stiffness regulating breast cancer cell motility. These results provide evidence of force driven cancer cell migration by ECM physical factors and explain the mechanism from the perspective of mechanobiology.

Cell-substrate distance fluctuations of confluent cells enable fast and coherent collective migration.

Collective cell migration is an emergent phenomenon, with long-range cell-cell communication influenced by various factors, including transmission of forces, viscoelasticity of individual cells, substrate interactions, and mechanotransduction. We investigate how alterations in cell-substrate distance fluctuations, cell-substrate adhesion, and traction forces impact the average velocity and temporal-spatial correlation of confluent monolayers formed by either wild-type (WT) MDCKII cells or zonula occludens (ZO)-1/2-depleted MDCKII cells (double knockdown [dKD]) representing highly contractile cells. The data indicate that confluent dKD monolayers exhibit decreased average velocity compared to less contractile WT cells concomitant with increased substrate adhesion, reduced traction forces, a more compact shape, diminished cell-cell interactions, and reduced cell-substrate distance fluctuations. Depletion of basal actin and myosin further supports the notion that short-range cell-substrate interactions, particularly fluctuations driven by basal actomyosin, significantly influence the migration speed of the monolayer on a larger length scale.

The characterization of cell traction force on nonflat surfaces with different curvature by elastic hydrogel microspheres.

It is of great importance to study the detachment/attachment behaviors of cells (cancer cell, immune cell, and epithelial cell), as they are closely related with tumor metastasis, immunoreaction, and tissue development at variety scales. To characterize the detachment/attachment during the interaction between cells and substrate, some researchers proposed using cell traction force (CTF) as the indicator. To date, various strategies have been developed to measure the CTF. However, these methods only realize the measurements of cell passive forces on flat cases. To quantify the active CTF on nonflat surfaces, which can better mimic the in vivo case, we employed elastic hydrogel microspheres as a force sensor. The microspheres were fabricated by microfluidic chips with controllable size and mechanical properties to mimic substrate. Cells were cultured on microsphere and the CTF led to the deformation of microsphere. By detecting the morphology information, the CTF exerted by attached cells can be calculated by the in-house numerical code. Using these microspheres, the CTF of various cells (including tumor cell, immunological cell, and epithelium cell) were successfully obtained on nonflat surfaces with different curvature radii. The proposed method provides a versatile platform to measure the CTF with high precision and to understand the detachment/attachment behaviors during physiology processes.

Development and evaluation of vesicourethral anastomosis bench-top model for measurement of traction force on urethra in robotic surgery.

PURPOSE: In vesicourethral anastomosis (VUA), which is part of robot-assisted radical prostatectomy, surgeons must proceed carefully to avoid urethral damage. We developed and evaluated a VUA bench-top model that can measure the traction force on the urethra during robotic surgery. MATERIALS AND METHODS: The VUA model included the urethra, bladder, pelvic bones, and a small force sensor that was capable of measuring the traction force on the urethra. Eight skilled and eight novice urologists performed a VUA task in robotic surgery. The skilled surgeons assessed the model's realism and usefulness as a training tool using a 5-point Likert scale. The evaluation items [task time, maximum force, force volume, and length of time that specific excessive forces were applied to the urethra (2, 3, 4, and ≥ 5 N)] were compared between the skilled and novice surgeons using the Mann-Whitney U test. Measurements were conducted in four directions with respect to the maximum force on the urethra: 11-1, 2-4, 5-7, and 8-10 o'clock. RESULTS: The quality of the model was scored 3.7 to 4.9 points for all 16 items in 4 domains except for "Usefulness compared with animal models." There were differences in the task time and almost all force parameters between the skilled and novice surgeons. CONCLUSION: We developed a relatively high-quality VUA bench-top model that measures traction force on the urethra, and we have revealed differences in the forces of action on the urethra in two groups of surgeons with different skill levels.

Quantitative atlas of collagen hydrogels reveals mesenchymal cancer cell traction adaptation to the matrix nanoarchitecture.

Collagen-based hydrogels are commonly used in mechanobiology to mimic the extracellular matrix. A quantitative analysis of the influence of collagen concentration and properties on the structure and mechanics of the hydrogels is essential for tailored design adjustments for specific in vitro conditions. We combined focused ion beam scanning electron microscopy and rheology to provide a detailed quantitative atlas of the mechanical and nanoscale three-dimensional structural alterations that occur when manipulating different hydrogel's physicochemistry. Moreover, we study the effects of such alterations on the phenotype of breast cancer cells and their mechanical interactions with the extracellular matrix. Regardless of the microenvironment's pore size, porosity or mechanical properties, cancer cells are able to reach a stable mesenchymal-like morphology. Additionally, employing 3D traction force microscopy, a positive correlation between cellular tractions and ECM mechanics is observed up to a critical threshold, beyond which tractions plateau. This suggests that cancer cells in a stable mesenchymal state calibrate their mechanical interactions with the ECM to keep their migration and invasiveness capacities unaltered. STATEMENT OF SIGNIFICANCE: The paper presents a thorough study on the mechanical microenvironment in breast cancer cells during their interaction with collagen based hydrogels of different compositions. The hydrogels' microstructure were obtained using state-of-the-art 3D microscopy, namely focused ion beam-scanning electron microscope (FIB-SEM). FIB-SEM was originally applied in this work to reconstruct complex fibered collagen microstructures within the nanometer range, to obtain key microarchitectural parameters. The mechanical microenvironment of cells was recovered using Traction Force Microscopy (TFM). The obtained results suggest that cells calibrate tractions such that they depend on mechanical, microstructural and physicochemical characteristics of the hydrogels, hence revealing a steric hindrance. We hypothesize that cancer cells studied in this paper tune their mechanical state to keep their migration and invasiveness capacities unaltered.

Cellular Traction Force Holds the Potential as a Drug Testing Readout for In Vitro Cancer Metastasis.

Introduction: Metastasis is responsible for 90% of cancer-related deaths worldwide. However, the potential inhibitory effects of metastasis by various anticancer drugs have been left largely unexplored. Existing preclinical models primarily focus on antiproliferative agents on the primary tumor to halt the cancer growth but not in metastasis. Unlike primary tumors, metastasis requires cancer cells to exert sufficient cellular traction force through the actomyosin machinery to migrate away from the primary tumor site. Therefore, we seek to explore the potential of cellular traction force as a novel readout for screening drugs that target cancer metastasis. Methods: In vitro models of invasive and non-invasive breast cancer were first established using MDA-MB-231 and MCF-7 cell lines, respectively. Cellular morphology was characterized, revealing spindle-like morphology in MDA-MB-231 and spherical morphology in MCF-7 cells. The baseline cellular traction force was quantified using the Traction force Microscopy technique. Cisplatin, a paradigm antimetastatic drug, and 5-Fluorouracil (5FU), a non-antimetastatic drug, were selected to evaluate the potential of cellular traction force as a drug testing readout for the in vitro cancer metastasis. Results: MDA-MB-231 cells exhibited significantly higher baseline cellular traction force compared to MCF-7 cells. Treatment with Cisplatin, an antimetastatic drug, and 5-Fluorouracil (5FU), a non-antimetastatic drug, demonstrated distinct effects on cellular traction force in MDA-MB-231 but not in MCF-7 cells. These findings correlate with the invasive potential observed in the two models. Conclusion: Cellular traction force emerges as a promising metric for evaluating drug efficacy in inhibiting cancer metastasis using in vitro models. This approach could enhance the screening and development of novel anti-metastatic therapies, addressing a critical gap in current anticancer drug research.

Trusting the forces of our cell lines.

Cells isolated from their native tissues and cultured in vitro face different selection pressures than those cultured in vivo. These pressures induce a profound transformation that reshapes the cell, alters its genome, and transforms the way it senses and generates forces. In this perspective, we focus on the evidence that cells cultured on conventional polystyrene substrates display a fundamentally different mechanobiology than their in vivo counterparts. We explore the role of adhesion reinforcement in this transformation and to what extent it is reversible. We argue that this mechanoadaptation is often understood as a mechanical memory. We propose some strategies to mitigate the effects of on-plastic culture on mechanobiology, such as organoid-inspired protocols or mechanical priming. While isolating cells from their native tissues and culturing them on artificial substrates has revolutionized biomedical research, it has also transformed cellular forces. Only by understanding and controlling them, we can improve their truthfulness and validity.

Calpain Small Subunit Mediated Secretion of Galectin-3 Regulates Traction Stress.

The complex regulation of traction forces (TF) produced during cellular migration remains poorly understood. We have previously found that calpain 4 (Capn4), the small non-catalytic subunit of the calpain 1 and 2 proteases, regulates the production of TF independent of the proteolytic activity of the larger subunits. Capn4 was later found to facilitate tyrosine phosphorylation and secretion of the lectin-binding protein galectin-3 (Gal3). In this study, recombinant Gal3 (rGal3) was added to the media-enhanced TF generated by capn4-/- mouse embryonic fibroblasts (MEFs). Extracellular Gal3 also rescued defects in the distribution, morphology, and adhesive strength of focal adhesions present in capn4-/- MEF cells. Surprisingly, extracellular Gal3 does not influence mechanosensing. c-Abl kinase was found to affect Gal3 secretion and the production of TF through phosphorylation of Y107 on Gal3. Our study also suggests that Gal3-mediated regulation of TF occurs through signaling pathways triggered by ß1 integrin but not by focal adhesion kinase (FAK) Y397 autophosphorylation. Our findings provide insights into the signaling mechanism by which Capn4 and secreted Gal3 regulate cell migration through the modulation of TF distinctly independent from a mechanosensing mechanism.

Molecular Force Sensors for Biological Application.

The mechanical forces exerted by cells on their surrounding microenvironment are known as cellular traction forces. These forces play crucial roles in various biological processes, such as tissue development, wound healing and cell functions. However, it is hard for traditional techniques to measure cellular traction forces accurately because their magnitude (from pN to nN) and the length scales over which they occur (from nm to µm) are extremely small. In order to fully understand mechanotransduction, highly sensitive tools for measuring cellular forces are needed. Current powerful techniques for measuring traction forces include traction force microscopy (TFM) and fluorescent molecular force sensors (FMFS). In this review, we elucidate the force imaging principles of TFM and FMFS. Then we highlight the application of FMFS in a variety of biological processes and offer our perspectives and insights into the potential applications of FMFS.

Massive field-of-view sub-cellular traction force videography enabled by Single-Pixel Optical Tracers (SPOT).

We report a massive field-of-view and high-speed videography platform for measuring the sub-cellular traction forces of more than 10,000 biological cells over 13 mm2 at 83 frames per second. Our Single-Pixel Optical Tracers (SPOT) tool uses 2-dimensional diffraction gratings embedded into a soft substrate to convert cells' mechanical traction force into optical colors detectable by a video camera. The platform measures the sub-cellular traction forces of diverse cell types, including tightly connected tissue sheets and near isolated cells. We used this platform to explore the mechanical wave propagation in a tightly connected sheet of Neonatal Rat Ventricular Myocytes (NRVMs) and discovered that the activation time of some tissue regions are heterogeneous from the overall spiral wave behavior of the cardiac wave.

Confinement induces internal flows in adherent cell aggregates.

During mesenchymal migration, F-actin protrusion at the leading edge and actomyosin contraction determine the retrograde flow of F-actin within the lamella. The coupling of this flow to integrin-based adhesions determines the force transmitted to the extracellular matrix and the net motion of the cell. In tissues, motion may also arise from convection, driven by gradients in tissue-scale surface tensions and pressures. However, how migration coordinates with convection to determine the net motion of cellular ensembles is unclear. To explore this, we study the spreading of cell aggregates on adhesive micropatterns on compliant substrates. During spreading, a cell monolayer expands from the aggregate towards the adhesive boundary. However, cells are unable to stabilize the protrusion beyond the adhesive boundary, resulting in retraction of the protrusion and detachment of cells from the matrix. Subsequently, the cells move upwards and rearwards, yielding a bulk convective flow towards the centre of the aggregate. The process is cyclic, yielding a steady-state balance between outward (protrusive) migration along the surface, and 'retrograde' (contractile) flows above the surface. Modelling the cell aggregates as confined active droplets, we demonstrate that the interplay between surface tension-driven flows within the aggregate, radially outward monolayer flow and conservation of mass leads to an internal circulation.

Dynamics of centipede locomotion revealed by large-scale traction force microscopy.

We present a novel approach to traction force microscopy (TFM) for studying the locomotion of 10 cm long walking centipedes on soft substrates. Leveraging the remarkable elasticity and ductility of kudzu starch gels, we use them as a deformable gel substrate, providing resilience against the centipedes' sharp leg tips. By optimizing fiducial marker size and density and fine-tuning imaging conditions, we enhance measurement accuracy. Our TFM investigation reveals traction forces along the centipede's longitudinal axis that effectively counterbalance inertial forces within the 0-10 mN range, providing the first report of non-vanishing inertia forces in TFM studies. Interestingly, we observe waves of forces propagating from the head to the tail of the centipede, corresponding to its locomotion speed. Furthermore, we discover a characteristic cycle of leg clusters engaging with the substrate: forward force (friction) upon leg tip contact, backward force (traction) as the leg pulls the substrate while stationary, and subsequent forward force as the leg tip detaches to reposition itself in the anterior direction. This work opens perspectives for TFM applications in ethology, tribology and robotics.

Spatial confinement modulates endothelial cell behavior and traction force in 3D hydrogel microgrooves.

The mechanical environment of vascular endothelial cells (ECs) encompasses a wide range of curvatures due to variations in blood vessel diameters. Integrins, key mediators of cell-matrix interactions, establish connections between the extracellular matrix and the actin cytoskeleton, influencing diverse cellular behaviors. In this study, we explored the impact of spatial confinement on human umbilical vein ECs (HUVECs) cultured within three-dimensional hydrogel microgrooves of varying curvatures and the underlying role of integrins in mediating cellular responses. Employing maskless lithography, we successfully fabricated precise and wall curvatures-controlled hydrogel microgrooves, conferring spatial constraints on the cells. Our investigations revealed substantial alterations in HUVEC behavior within the hydrogel microgrooves with varying sidewall curvatures, marked by reduced cell size, enhanced orientation, and increased apoptosis. Interestingly, microgroove curvature emerged as a crucial factor influencing cell orientation and apoptosis, with rectangular microgrooves eliciting distinct changes in cell orientation, while ring-form microgrooves exhibited higher apoptosis rates. The side-wall effect in the 20 µm region near the microgroove wall had the greatest influence on cell orientation and apoptosis. HUVECs within the microgrooves exhibited elevated integrin expression, and inhibition of αV-integrin by cilengitide significantly curtailed cell apoptosis without affecting proliferation. Additionally, integrin-mediated cell traction force closely correlated with the spatial confinement effect. Cilengitide not only reduced integrin and focal adhesion expression but also attenuated cell traction force and cytoskeletal actin filament alignment. Overall, our findings elucidate the spatial confinement of ECs in hydrogel microgrooves and underscores the pivotal role of integrins, particularly αV-integrin, in mediating cell traction force and apoptosis within this microenvironment.

Field Guide to Traction Force Microscopy.

Introduction: Traction force microscopy (TFM) is a widely used technique to measure cell contractility on compliant substrates that mimic the stiffness of human tissues. For every step in a TFM workflow, users make choices which impact the quantitative results, yet many times the rationales and consequences for making these decisions are unclear. We have found few papers which show the complete experimental and mathematical steps of TFM, thus obfuscating the full effects of these decisions on the final output. Methods: Therefore, we present this "Field Guide" with the goal to explain the mathematical basis of common TFM methods to practitioners in an accessible way. We specifically focus on how errors propagate in TFM workflows given specific experimental design and analytical choices. Results: We cover important assumptions and considerations in TFM substrate manufacturing, substrate mechanical properties, imaging techniques, image processing methods, approaches and parameters used in calculating traction stress, and data-reporting strategies. Conclusions: By presenting a conceptual review and analysis of TFM-focused research articles published over the last two decades, we provide researchers in the field with a better understanding of their options to make more informed choices when creating TFM workflows depending on the type of cell being studied. With this review, we aim to empower experimentalists to quantify cell contractility with confidence. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-024-00801-6.

Mechanical Constraints in Tumor Guide Emergent Spatial Patterns of Glioblastoma Cancer Stem Cells.

The mechanical constraints in the overcrowding glioblastoma (GBM) microenvironment have been implicated in the regulation of tumor heterogeneity and disease progression. Especially, such mechanical cues can alter cellular DNA transcription and give rise to a subpopulation of tumor cells called cancer stem cells (CSCs). These CSCs with stem-like properties are critical drivers of tumorigenesis, metastasis, and treatment resistance. Yet, the biophysical and molecular machinery underlying the emergence of CSCs in tumor remained unexplored. This work employed a two-dimensional micropatterned multicellular model to examine the impact of mechanical constraints arisen from geometric confinement on the emergence and spatial patterning of CSCs in GBM tumor. Our study identified distinct spatial distributions of GBM CSCs in different geometric patterns, where CSCs mostly emerged in the peripheral regions. The spatial pattern of CSCs was found to correspond to the gradients of mechanical stresses resulted from the interplay between the cell-ECM and cell-cell interactions within the confined environment. Further mechanistic study highlighted a Piezo1-RhoA-focal adhesion signaling axis in regulating GBM cell mechanosensing and the subsequent CSC phenotypic transformation. These findings provide new insights into the biophysical origin of the unique spatial pattern of CSCs in GBM tumor and offer potential avenues for targeted therapeutic interventions.

An Assessment of the Mechanophysical and Hormonal Impact on Human Endometrial Epithelium Mechanics and Receptivity.

The endometrial epithelium and underlying stroma undergo profound changes to support and limit embryo adhesion and invasion, which occur in the secretory phase of the menstrual cycle during the window of implantation. This coincides with a peak in progesterone and estradiol production. We hypothesized that the interplay between hormone-induced changes in the mechanical properties of the endometrial epithelium and stroma supports this process. To study it, we used hormone-responsive endometrial adenocarcinoma-derived Ishikawa cells growing on substrates of different stiffness. We showed that Ishikawa monolayers on soft substrates are more tightly clustered and uniform than on stiff substrates. Probing for mechanical alterations, we found accelerated stress-relaxation after apical nanoindentation in hormone-stimulated monolayers on stiff substrates. Traction force microscopy furthermore revealed an increased number of foci with high traction in the presence of estradiol and progesterone on soft substrates. The detection of single cells and small cell clusters positive for the intermediate filament protein vimentin and the progesterone receptor further underscored monolayer heterogeneity. Finally, adhesion assays with trophoblast-derived AC-1M-88 spheroids were used to examine the effects of substrate stiffness and steroid hormones on endometrial receptivity. We conclude that the extracellular matrix and hormones act together to determine mechanical properties and, ultimately, embryo implantation.

Insect attachment on waxy plant surfaces: the effect of pad contamination by different waxes.

This study focuses on experimental testing of the contamination hypothesis and examines how the contamination of insect adhesive pads with three-dimensional epicuticular waxes of different plant species contributes to the reduction of insect attachment. We measured traction forces of tethered Chrysolina fastuosa male beetles having hairy adhesive pads on nine wax-bearing plant surfaces differing in both shape and dimensions of the wax structures and examined insect adhesive organs after they have contacted waxy substrates. For comparison, we performed the experiments with the same beetle individuals on a clean glass sample just before (gl1) and immediately after (gl2) the test on a plant surface. The tested insects showed a strong reduction of the maximum traction force on all waxy plant surfaces compared to the reference experiment on glass (gl1). After beetles have walked on waxy plant substrates, their adhesive pads were contaminated with wax material, however, to different extents depending on the plant species. The insects demonstrated significantly lower values of both the maximum traction force and the first peak of the traction force and needed significantly longer time to reach the maximum force value in the gl2 test than in the gl1 test. These effects were especially pronounced in cases of the plant surfaces covered with wax projections having higher aspect ratios. The data obtained clearly indicated the impact of waxy plant surfaces on the insect ability to subsequently attach to the clean smooth surface. This effect is caused by the contamination of adhesive pads and experimentally supports the contamination hypothesis.