Microscale mapping of extracellular matrix elasticity of mouse joint cartilage: an approach to extracting bulk elasticity of soft matter with surface roughness.
ABSTRACT: Cartilage is composed of cells and an extracellular matrix, the latter being a composite of a collagen mesh interpenetrated by proteoglycans responsible for tissue osmotic swelling. The matrix composition and structure vary through the tissue depth. Mapping such variability requires tissue sectioning to gain access. The resulting surface roughness, and concomitant proteoglycan loss contribute to large uncertainties in elastic modulus estimates. To extract elasticity values for the bulk matrix which are not obfuscated by the indeterminate surface layer, we developed a novel experimental and data analysis methodology. We analyzed the surface roughness to optimize the probe size, and performed high-resolution (1 ?m) elasticity mapping on thin (?12 ?m), epiphyseal newborn mouse cartilage sections cut parallel to the bone longitudinal axis or normal to the articular surface. Mild fixation prevented the major proteoglycan loss observed in unfixed specimens but not the stress release that resulted in thickness changes in the sectioned matrix. Our novel data analysis method introduces a virtual contact point as a fitting parameter for the Hertz model, to minimize the effects of surface roughness and corrects for the finite section thickness. Our estimates of cartilage elasticity converge with increasing indentation depth and, unlike previous data interpretations, are consistent with linearly elastic material. A high cell density that leaves narrow matrix septa between cells may cause the underestimation of elastic moduli, whereas fixation probably causes an overestimation. The proposed methodology has broader relevance to nano- and micro-indentation of soft materials with multiple length scales of organization and whenever surface effects (including roughness, electrostatics, van der Waals forces, etc.) become significant.
Project description:The indentation test is widely used to determine the in situ biomechanical properties of articular cartilage. The mechanical parameters estimated from the test depend on the constitutive model adopted to analyze the data. Similar to most connective tissues, the solid matrix of cartilage displays different mechanical properties under tension and compression, termed tension-compression nonlinearity (TCN). In this study, cartilage was modeled as a porous elastic material with either a conewise linear elastic matrix with cubic symmetry or a solid matrix reinforced by a continuous fiber distribution. Both models are commonly used to describe the TCN of cartilage. The roles of each mechanical property in determining the indentation response of cartilage were identified by finite element simulation. Under constant loading, the equilibrium deformation of cartilage is mainly dependent on the compressive modulus, while the initial transient creep behavior is largely regulated by the tensile stiffness. More importantly, altering the permeability does not change the shape of the indentation creep curves, but introduces a parallel shift along the horizontal direction on a logarithmic time scale. Based on these findings, a highly efficient curve-fitting algorithm was designed, which can uniquely determine the three major mechanical properties of cartilage (compressive modulus, tensile modulus, and permeability) from a single indentation test. The new technique was tested on adult bovine knee cartilage and compared with results from the classic biphasic linear elastic curve-fitting program.
Project description:Contact force-indentation depth measurements in contact experiments involving compliant materials, such as polymers and gels, show a hysteresis loop whose size depends on the maximum indentation depth. This depth-dependent hysteresis (DDH) is not explained by classical contact mechanics theories and was believed to be due to effects such as material viscoelasticity, plasticity, surface polymer interdigitation, and moisture. It has been observed that the DDH energy loss initially increases and then decreases with roughness. A mechanics model based on the occurrence of adhesion and roughness related small-scale instabilities was presented by one of the authors for explaining DDH. However, that model only applies in the regime of infinitesimally small surface roughness, and consequently it does not capture the decrease in energy loss with surface roughness at the large roughness regime. We present a new mechanics model that applies in the regime of large surface roughness based on the Maugis-Dugdale theory of adhesive elastic contacts and Nayak's theory of rough surfaces. The model captures the trend of decreasing energy loss with increasing roughness. It also captures the experimentally observed dependencies of energy loss on the maximum indentation depth, and material and surface properties.
Project description:Biomechanical cues such as shear stress, stretching, compression, and matrix elasticity are vital in the establishment of next generation physiological in vitro tissue models. Matrix elasticity, for instance, is known to guide stem cell differentiation, influence healing processes and modulate extracellular matrix (ECM) deposition needed for tissue development and maintenance. To better understand the biomechanical effect of matrix elasticity on the formation of articular cartilage analogs in vitro, this study aims at assessing the redifferentiation capacity of primary human chondrocytes in three different hydrogel matrices of predefined matrix elasticities. The hydrogel elasticities were chosen to represent a broad spectrum of tissue stiffness ranging from very soft tissues with a Young's modulus of 1 kPa up to elasticities of 30 kPa, representative of the perichondral-space. In addition, the interplay of matrix elasticity and transforming growth factor beta-3 (TGF-?3) on the redifferentiation of primary human articular chondrocytes was studied by analyzing both qualitative (viability, morphology, histology) and quantitative (RT-qPCR, sGAG, DNA) parameters, crucial to the chondrotypic phenotype. Results show that fibrin hydrogels of 30 kPa Young's modulus best guide chondrocyte redifferentiation resulting in a native-like morphology as well as induces the synthesis of physiologic ECM constituents such as glycosaminoglycans (sGAG) and collagen type II. This comprehensive study sheds light onto the mechanobiological impact of matrix elasticity on formation and maintenance of articular cartilage and thus represents a major step toward meeting the need for advanced in vitro tissue models to study both re- and degeneration of articular cartilage.
Project description:In osteoarthrosis, pathological features of articular cartilage are associated with degeneration and nanomechanical changes. The aim of this paper is to show that indentation-atomic force microscopy can monitor wear-related biomechanical changes in the hip joint of patients with osteoarthritis. Fifty patients (N = 50), aged 40 to 65, were included in the study. The mechanical properties and the submicron surface morphology of hyaline cartilage were investigated using atomic force microscopy. Measurements of the roughness parameters of cartilage surfaces were performed, including the arithmetic average of absolute values (Ra), the maximum peak height (Rp), and the mean spacing between local peaks (S). The arithmetic mean of the absolute values of the height of healthy cartilage was 86 nm, while wear began at Ra = 73 nm. The maximum changes of values of the roughness parameters differed from the healthy ones by 71%, 80%, and 51% for Ra, Rp, and S, respectively. Young's modulus for healthy cartilage surfaces ranged from 1.7 to 0.5 MPa. For the three stages of cartilage wear, Young's modulus increased, and then it approached the maximum value and decreased. AFM seems to be a powerful tool for surface analysis of biological samples as it enables indentation measurements in addition to imaging.
Project description:Background The mechanical properties of single living cells have proven to be a powerful marker of the cell physiological state. The use of nanoindentation-based single cell force spectroscopy provided a wealth of information on the elasticity of cells, which is still largely to be exploited. The simplest model to describe cell mechanics is to treat them as a homogeneous elastic material and describe it in terms of the Young’s modulus. Beside its simplicity, this approach proved to be extremely informative, allowing to assess the potential of this physical indicator towards high throughput phenotyping in diagnostic and prognostic applications. Results Here we propose an extension of this analysis to explicitly account for the properties of the actin cortex. We present a method, the Elasticity Spectra, to calculate the apparent stiffness of the cell as a function of the indentation depth and we suggest a simple phenomenological approach to measure the thickness and stiffness of the actin cortex, in addition to the standard Young’s modulus. Conclusions The Elasticity Spectra approach is tested and validated on a set of cells treated with cytoskeleton-affecting drugs, showing the potential to extend the current representation of cell mechanics, without introducing a detailed and complex description of the intracellular structure.
Project description:We developed a dynamic cell culture platform with dynamically tunable nano-roughness and elasticity. Temperature-responsive poly(?-caprolactone) (PCL) films were successfully prepared by crosslinking linear and tetra-branched PCL macromonomers. By optimizing the mixing ratios, the crystal-amorphous transition temperature (Tm) of the crosslinked film was adjusted to the biological relevant temperature (~33 °C). While the crosslinked films are relatively stiff (50 MPa) below the Tm, they suddenly become soft (1 MPa) above the Tm. Correspondingly, roughness of the surface was decreased from 63.4-12.4 nm. It is noted that the surface wettability was independent of temperature. To investigate the role of dynamic surface roughness and elasticity on cell adhesion, cells were seeded on PCL films at 32 °C. Interestingly, spread myoblasts on the film became rounded when temperature was suddenly increased to 37 °C, while significant changes in cell morphology were not observed for fibroblasts. These results indicate that cells can sense dynamic changes in the surrounding environment but the sensitivity depends on cell types.
Project description:The endothelial glycocalyx (EG), a sugar-rich layer that lines the luminal surface of blood vessels, is an important constituent of the vascular system. Although the chemical composition of the EG is fairly well known, there is no consensus regarding its ultrastructure. While previous experiments probed the properties of the layer at the continuum level, they did not provide sufficient insight into its molecular organisation. In this work, we investigate the EG mechanics using two simple brush and bush-like simulation models, and use these models to describe its molecular structure and elastic response to indentation. We analyse the relationship between the mechanical properties of the EG layer and several molecular parameters, including the filament bending rigidity, grafting density, and the type of ultrastructure . We show that variations in the glycan density determine the elasticity of the EG for small deformations, and that the normal stress may be effectively dampened by the EG layer, preventing the stress from being transferred to the cell membrane. Furthermore, our bush-like model allows us to evaluate the forces and energies required to overcome the mechanical resistance of the EG.
Project description:The current knowledge of bone marrow mechanics is limited to its viscous properties, neglecting the elastic contribution of the extracellular matrix. To get a more complete view of the mechanics of marrow, we characterized intact yellow porcine bone marrow using three different, but complementary techniques: rheology, indentation, and cavitation. Our analysis shows that bone marrow is elastic, and has a large amount of intra- and inter-sample heterogeneity, with an effective Young?s modulus ranging from 0.25 to 24.7 kPa at physiological temperature. Each testing method was consistent across matched tissue samples, and each provided unique benefits depending on user needs. We recommend bulk rheology to capture the effects of temperature on tissue elasticity and moduli, indentation for quantifying local tissue heterogeneity, and cavitation rheology for mitigating destructive sample preparation. We anticipate the knowledge of bone marrow elastic properties for building in vitro models will elucidate mechanisms involved in disease progression and regenerative medicine.
Project description:We report that macrophage elasticity plays a dominant role in bacterial phagocytosis, release of TNF-alpha, and production of reactive oxygen species. We show that macrophage elasticity is modulated by mechanical factors including substrate rigidity and substrate stretch. Changes in macrophage elasticity are dependent upon the degree of actin polymerization, and mediated in part through small rhoGTPase activity. Moreover, the functional effects of macrophage elasticity are not predicted by gene expression profiles. Murine RAW 267.4 macrophages were separately grown on 2 matrix stiffness levels (1200, 150000 Pascals) for 0, 2, 6, 18 hours with 3 replicate sample experiments per condition. Total RNA extracted from the cells and profiled by microarrays. Keywords: Murine RAW 267.4 macrophage, matrix stiffness, phagocytosis, cell elasticity.
Project description:Elastic properties of cells are mainly derived from the actin cytoskeleton. However, intermediate filaments are emerging as major contributors to the mechanical properties of cells. Using atomic force microscopy, we studied the elasticity of mouse myoblasts expressing a mutant form of the gene encoding for desmin intermediate filaments, p.D399Y. This variant produces desmin aggregates, the main pathological symptom of myofibrillar myopathies. Here we show that desmin-mutated cells display a 39% increased median elastic modulus compared to wild-type cells. Desmin-mutated cells required higher forces than wild-type cells to reach high indentation depths, where desmin intermediate filaments are typically located. In addition, heat-shock treatment increased the proportion of cells with aggregates and induced a secondary peak in the distribution of Young's moduli. By performing atomic force microscopy mechanical mapping combined with fluorescence microscopy, we show that higher Young's moduli were measured where desmin aggregates were located, indicating that desmin aggregates are rigid. Therefore, we provide evidence that p.D399Y stiffens mouse myoblasts. Based on these results, we suggest that p.D399Y-related myofibrillar myopathy is at least partly due to altered mechanical properties at the single-cell scale, which are propagated to the tissue scale.