Papers by Roberto C Andresen Eguiluz
Biomaterials, 2015
Fibronectin (Fn) forms a fibrillar network that controls cell behavior in both physiological and ... more Fibronectin (Fn) forms a fibrillar network that controls cell behavior in both physiological and diseased conditions including cancer. Indeed, breast cancer-associated stromal cells not only increase the quantity of deposited Fn but also modify its conformation. However, (i) the interplay between mechanical and conformational properties of early tumor-associated Fn networks and (ii) its effect on tumor vascularization remain unclear. Here, we first used the Surface Forces Apparatus to reveal that 3T3-L1 preadipocytes exposed to tumor-secreted factors generate a stiffer Fn matrix relative to control cells. We then show that this early matrix stiffening correlates with increased molecular unfolding in Fn fibers, as determined by Förster Resonance Energy Transfer. Finally, we assessed the resulting changes in adhesion and proangiogenic factor (VEGF) secretion of newly seeded 3T3-L1s, and we examined altered integrin specificity as a potential mechanism of modified cell-matrix interactions through integrin blockers. Our data indicate that tumorconditioned Fn decreases adhesion while enhancing VEGF secretion by preadipocytes, and that an integrin switch is responsible for such changes. Collectively, our findings suggest that simultaneous stiffening and unfolding of initially deposited tumor-conditioned Fn alters both adhesion and proangiogenic behavior of surrounding stromal cells, likely promoting vascularization and growth of the breast tumor. This work enhances our knowledge of cell-Fn matrix interactions that may be exploited for other biomaterials-based applications, including advanced tissue engineering approaches.
The adhesion force between individual human hair fibers in a crosshair geometry was measured by o... more The adhesion force between individual human hair fibers in a crosshair geometry was measured by observing their natural bending and adhesive jumps out of contact, using optical video microscopy. The hair fibers' natural elastic responses, calibrated by measuring their natural resonant frequencies, were used to measure the forces. Using a custom-designed, automated apparatus to measure thousands of individual hair−hair contacts along millimeter length scales of hair, it was found that a broad, yet characteristic, spatially variant distribution in adhesion force is measured on the 1 to 1000 nN scale for both clean and conditioner-treated hair fibers. Comparison between the measured adhesion forces and adhesion forces modeled from the hairs' surface topography (measured using confocal laser profilometry) shows they have a good order-of-magnitude agreement and have similar breadth and shape. The agreement between the measurements and the model suggests, perhaps unsurprisingly, that hair−hair adhesion is governed, to a first approximation, by the unique surface structure of the hairs' cuticles and, therefore, the large distribution in local mean curvature at the various individual contact points along the hairs' lengths. We posit that haircare products could best control the surface properties (or at least the adhesive properties) between hairs by directly modifying the hair surface microstructure.
Time-Dependent Physicochemical Changes of Carbonate Surfaces from SmartWater (Diluted Seawater) Flooding Processes for Improved Oil Recovery, 2018
Over the past few decades, field-and laboratory-scale studies have shown enhancements in oil reco... more Over the past few decades, field-and laboratory-scale studies have shown enhancements in oil recovery when reservoirs, which contain high-salinity formation water (FW), are waterflooded with modified-salinity salt water (widely referred to as the low-salinity, dilution, or SmartWater effect for improved oil recovery). In this study, we investigated the time dependence of the physicochemical processes that occur during diluted seawater (i.e., SmartWater) waterflooding processes of specific relevance to carbonate oil reservoirs. We measured the changes to oil/water/rock wettability, surface roughness, and surface chemical composition during SmartWater flooding using 10-fold-diluted seawater under mimicked oil reservoir conditions with calcite and carbonate reservoir rocks. Distinct effects due to SmartWater flooding were observed and found to occur on two different timescales: (1) a rapid (<15 min) increase in the colloidal electrostatic double-layer repulsion between the rock and oil across the SmartWater, leading to a decreased oil/ water/rock adhesion energy and thus increased water wetness and (2) slower (>12 h to complete) physicochemical changes of the calcite and carbonate reservoir rock surfaces, including surface roughening via the dissolution of rock and the reprecipitation of dissolved carbonate species after exchanging key ions (Ca 2+ , Mg 2+ , CO 3 2− , and SO 4 2− in carbonates) with those in the flooding SmartWater. Our experiments using crude oil from a carbonate reservoir reveal that these reservoir rock surfaces are covered with organic−ionic preadsorbed films (ad-layers), which the SmartWater removes (detaches) as flakes. Removal of the organic−ionic ad-layers by SmartWater flooding enhances oil release from the surfaces, which was found to be critical to increasing the water wetness and significantly improving oil removal from carbonates. Additionally, the increase in water wetness is further enhanced by roughening of the rock surfaces, which decreases the effective contact (interaction) area between the oil and rock interfaces. Furthermore, we found that the rate of these slower physicochemical changes to the carbonate rock surfaces increases with increasing temperature (at least up to an experimental temperature of 75 °C). Our results suggest that the effectiveness of improved oil recovery from SmartWater flooding depends strongly on the formation of the organic−ionic ad-layers. In oil reservoirs where the ad-layer is fully developed and robust, injecting SmartWater would lead to significant removal of the ad-layer and improved oil recovery.
Lubricin (LUB), a major mucinous glycoprotein of mammalian synovial fluids, is believed to provid... more Lubricin (LUB), a major mucinous glycoprotein of mammalian synovial fluids, is believed to provide excellent lubrication to cartilage surfaces. Consequently, when joint disease or replacement leads to increased friction and surface damage in the joint, robust synthetic LUB alternatives that could be used therapeutically to improve lubrication and surface protection are needed. Here, we report the characterization of a lubricating multiblock bottlebrush polymer whose architecture was inspired by LUB, and we investigate the role of fibronectin (FN), a glycoprotein found in the superficial zone of cartilage, in mediating the tribological properties of the polymer upon shear between mica surfaces. Our surface forces apparatus (SFA) normal force measurements indicate that the lubricin-mimetic (mimLUB) could be kept anchored between mica surfaces, even under high contact pressures, when an intermediate layer of FN was present. Additional SFA friction measurements show that FN would also extend the wearless friction regime of the polymer up to pressures of 3.4 MPa while ensuring stable friction coefficients (μ ≈ 0.28). These results demonstrate synergistic interactions between mimLUB and FN in assisting the lubrication and wear protection of ideal (mica) substrates upon shear. Collectively, these findings suggest that our proposed mimLUB might be a promising alternative to LUB, as similar mechanisms could potentially facilitate the interaction between the polymer and cartilage surfaces in articular joints and prosthetic implants in vivo.
The vascular endothelium is subject to diverse mechanical cues that regulate vascular endothelial... more The vascular endothelium is subject to diverse mechanical cues that regulate vascular endothelial barrier function. In addition to rigidity sensing through integrin adhesions, mechanical perturbations such as changes in fluid shear stress can also activate force transduction signals at intercellular junctions. This study investigated how extracellular matrix rigidity and intercellular force transduction, activated by vascular endothelial cadherin, coordinate to regulate the integrity of endothelial monolayers. Studies used complementary mechanical measurements of endothelial monolayers grown on patterned substrates of variable stiffness. Specifically perturbing VE-cadherin receptors activated intercellular force transduction signals that increased integrin-dependent cell contractility and disrupted cell-cell and cell-matrix adhesions. Further investigations of the impact of substrate rigidity on force transduction signaling demonstrated how cells integrate extracellular mechanics cues and intercellular force transduction signals, to regulate endothelial integrity and global tissue mechanics. VE-cadherin specific signaling increased focal adhesion remodeling and cell contractility, while sustaining the overall mechanical equilibrium at the mesoscale. Conversely, increased substrate rigidity exacerbates the disruptive effects of intercellular force transduction signals, by increasing heterogeneity in monolayer stress distributions. The results provide new insights into how substrate stiffness and intercellular force transduction coordinate to regulate endothelial monolayer integrity.
The glycoprotein lubricin is the primary boundary lubricant of articular cartilage. Its boundary ... more The glycoprotein lubricin is the primary boundary lubricant of articular cartilage. Its boundary lubricating abilities arise from two key structural features: i) a dense mucin-like domain consisting of hydrophilic oligosaccharides and ii) an end terminus that anchors the molecule to articulating surfaces. When bound, lubricin molecules attract and trap water near a surface, reducing friction and facilitating glide. Synthetic analogues were previously created to mimic lubricin using thiol-terminated polyacrylic acid-graft-polyethylene glycol (pAA-g-PEG) brush copolymers. The PEG moiety was designed to mimic the mucin-like domain of lubricin and the thiol-terminus was designed to anchor the molecules to cartilage surfaces, mimicking the binding domain. In this study, these synthetic lubricin-mimetics were bound to gold-coated surfaces to characterize the relationship between the polymers' molecular architecture and their lubricating capacity. A library of nine copolymer brushes was synthesized using different sizes of pAA and PEG. Larger molecular weight polymers created smoother, more densely covered surfaces (p < 0.05). Additionally, the hydrodynamic sizes of the polymers in solution were correlated with their lubricating abilities (p < 0.05). Friction coefficients of cartilage against polymer-treated gold surfaces were lower than cartilage against untreated surfaces (Δμeq = − 0.065 ± 0.050 to − 0.093 ± 0.045, p < 0.05).
Pre-adipocytes exposed to tumor soluble factors
derived from MDA-MB-231 breast cancer cells, an i... more Pre-adipocytes exposed to tumor soluble factors
derived from MDA-MB-231 breast cancer cells, an in vitro model
of cancer-activated fibroblasts, deposited an initial extracellular
matrix comprised primarily of fibronectin (Fn). This tumorassociated
Fn was measured to be 87% more unfolded than that
of Fn deposited by control cells and correlated to being 62%
stiffer by the Förster Resonance Energy Transfer and Surface
Forces Apparatus techniques, respectively. The initial
dysregulated ECM lead to the rapid deposition of thick type I
collagen (Col I) fibers. Our findings suggest that initial unfolded
and stiff tumor-associated Fn ECMs promote altered cell-matrix
interactions to fuel tumor progression.
The need for effective biomedical glues spans a wide range of applications, from woundhealing sea... more The need for effective biomedical glues spans a wide range of applications, from woundhealing sealants to dental implants and restorations. While most conventional adhesives lose their bridging abilities in the aqueous, saline environment of the body, nature has developed efficient solutions to this problem. Marine mussels are able to make strong, permanent attachments to nearly any available surface in their turbulent ocean habitat. The mussel uses an assembly of polyphenolic proteins rich in the amino acid 3,4dihydroxyphenyl-alanine (DOPA), and single-molecule experiments have demonstrated high attachment strengths resulting from this moiety in isolation 1 . The largest of these proteins, mussel foot protein-1 (mfp-1), contains a high DOPA content, yet in its native state, it is unable to bridge surfaces together. This suggests that the mere presence of DOPA is not sufficient to formulate an effective glue. The conformation of the protein must work in concert with its chemical components to enable its function as an adhesive. The dynamic response of mfp-1 to mechanical stresses can provide clues as to the adhesive properties of the protein in its turbulent environment. Recent research 2 using the surface forces apparatus (SFA) indicates that shearing the exposed side of mfp-1 films can promote adhesion in the films. Using an Atomic Force Microscope (AFM) in liquid, we have investigated in detail this phenomenon over varying parameters such as protein film thickness and shearing conditions. By doing so, we have identified the relevant factors which determine the magnitude and limitations of the adhesive bridging capability that can be induced in mfp-1. Methods: Powders of mfp-1 were purified in the lab of Dr. J.H. Waite at UCSB according to published methods 3 . They were dissolved in a 0.1M sodium acetate, 0.25M potassium nitrate buffer (pH 5.5). Solutions of mfp-1 were deposited onto freshly-cleaved mica substrates and rinsed with buffer after various adsorption times. The topography of mfp-1 films was characterized using TappingMode® AFM in buffer (Agilent Technologies, Santa Clara CA). Shearing of the surface of mfp-1 was performed in the AFM by using a large, spherical silica bead (radius 300nm) -terminated tip (Novascan Technologies, Ames IA). During shearing, the friction between the silica tip and the mfp-1 film was measured through lateral force microscopy. Following each shearing cycle, adhesion spectroscopy was performed by recording normal force-distance profiles at the surface of the sheared film. All AFM experiments were performed in buffer. Results: Globally, our results show an increase in adhesion resulting from shearing the mfp-1 films with a bare silica tip. Changes in adhesion were found to depend on both the shearing parameters and the sample preparation. More specifically, adhesion was affected by the number of shearing cycles and the thickness of the adsorbed mfp-1 films, which was modulated by the adsorption time of mfp-1 on the substrate. In thick films, the adhesion increased with initial shearing cycles, and was followed by a drop to a steady-state adhesive plateau, which was independent of shear ( . This phenomenon has been attributed to irreversible damage of the protein film, leading to removal of mfp-1 molecules from the film and their likely attachment to the AFM tip. Shearing the film with an mfp-1-covered AFM probe had almost no effect on adhesion ( and led to low and stable friction (data not shown). In thin films of mfp-1, the adhesion measured was significantly higher than in the thicker films, and monotonically increased with no drop in adhesion observed, even after prolonged shearing (data not shown).
Obesity and extracellular matrix (ECM) density are considered independent risk and prognostic fac... more Obesity and extracellular matrix (ECM) density are considered independent risk and prognostic factors for breast cancer. Whether they are functionally linked is uncertain. We investigated the hypothesis that obesity enhances local myofibroblast content in mammary adipose tissue and that these stromal changes increase malignant potential by enhancing interstitial ECM stiffness. Indeed, mammary fat of both diet- and genetically induced mouse models of obesity were enriched for myofibroblasts and stiffness-promoting ECM components. These differences were related to varied adipose stromal cell (ASC) characteristics because ASCs isolated from obese mice contained more myofibroblasts and deposited denser and stiffer ECMs relative to ASCs from lean control mice. Accordingly, decellularized matrices from obese ASCs stimulated mechanosignaling and thereby the malignant potential of breast cancer cells. Finally, the clinical relevance and translational potential of our findings were supported by analysis of patient specimens and the observation that caloric restriction in a mouse model reduces myofibroblast content in mammary fat. Collectively, these findings suggest that obesity-induced interstitial fibrosis promotes breast tumorigenesis by altering mammary ECM mechanics with important potential implications for anticancer therapies.
Fibronectin (FN) is a glycoprotein found in the superficial
zone of cartilage; however, its role ... more Fibronectin (FN) is a glycoprotein found in the superficial
zone of cartilage; however, its role in the lubrication and the wear protection
of articular joints is unknown. In this work, we have investigated the molecular interactions between FN and various components of the synovial fluid such as lubricin (LUB), hyaluronan (HA), and serum albumin (SA), which are all believed to contribute to joint lubrication. Using a Surface Forces Apparatus, we have measured the normal (adhesion/repulsion) and lateral (friction) forces across layers of individual synovial fluid components physisorbed onto FN-coated mica substrates. Our chief findings are (i) FN strongly tethers LUB and HA to mica, as indicated by high and reversible long-range repulsive normal interactions between surfaces, and (ii) FN and LUB synergistically enhance wear protection of surfaces during shear, as suggested by the structural robustness of FN+LUB layers under pressures up to about 4 MPa. These findings provide new insights into the role of FN in the lubricating properties of synovial fluid components sheared between ideal substrates and represent a significant step forward in our understanding of cartilage damage involved in diseases such as osteoarthritis.
PLOS Biology, 2007
Fibronectin (Fn) forms a fibrillar network that controls cell behavior in both physiological and ... more Fibronectin (Fn) forms a fibrillar network that controls cell behavior in both physiological and diseased conditions including cancer. Indeed, breast cancer-associated stromal cells not only increase the quantity of deposited Fn but also modify its conformation. However, (i) the interplay between mechanical and conformational properties of early tumor-associated Fn networks and (ii) its effect on tumor vascularization remain unclear. Here, we first used the Surface Forces Apparatus to reveal that 3T3-L1 preadipocytes exposed to tumor-secreted factors generate a stiffer Fn matrix relative to control cells. We then show that this early matrix stiffening correlates with increased molecular unfolding in Fn fibers, as determined by F€ orster Resonance Energy Transfer. Finally, we assessed the resulting changes in adhesion and proangiogenic factor (VEGF) secretion of newly seeded 3T3-L1s, and we examined altered integrin specificity as a potential mechanism of modified cellematrix interactions through integrin blockers. Our data indicate that tumor-conditioned Fn decreases adhesion while enhancing VEGF secretion by preadipocytes, and that an integrin switch is responsible for such changes. Collectively, our findings suggest that simultaneous stiffening and unfolding of initially deposited tumor-conditioned Fn alters both adhesion and proangiogenic behavior of surrounding stromal cells, likely promoting vascularization and growth of the breast tumor. This work enhances our knowledge of cell e Fn matrix interactions that may be exploited for other biomaterials-based applications, including advanced tissue engineering approaches.
Fibronectin (Fn) forms a fibrillar network that controls cell behavior in both physiological and ... more Fibronectin (Fn) forms a fibrillar network that controls cell behavior in both physiological and diseased conditions including cancer. Indeed, breast cancer-associated stromal cells not only increase the quantity of deposited Fn but also modify its conformation. However, (i) the interplay between mechanical and conformational properties of early tumor-associated Fn networks and (ii) its effect on tumor vascularization remain unclear. Here, we first used the Surface Forces Apparatus to reveal that 3T3-L1 preadipocytes exposed to tumor-secreted factors generate a stiffer Fn matrix relative to control cells. We then show that this early matrix stiffening correlates with increased molecular unfolding in Fn fibers, as determined by Förster Resonance Energy Transfer. Finally, we assessed the resulting changes in adhesion and proangiogenic factor (VEGF) secretion of newly seeded 3T3-L1s, and we examined altered integrin specificity as a potential mechanism of modified cell–matrix interactions through integrin blockers. Our data indicate that tumor-conditioned Fn decreases adhesion while enhancing VEGF secretion by preadipocytes, and that an integrin switch is responsible for such changes. Collectively, our findings suggest that simultaneous stiffening and unfolding of initially deposited tumor-conditioned Fn alters both adhesion and proangiogenic behavior of surrounding stromal cells, likely promoting vascularization and growth of the breast tumor. This work enhances our knowledge of cell – Fn matrix interactions that may be exploited for other biomaterials-based applications, including advanced tissue engineering approaches.
Multipotent adipose-derived stem cells (ASCs) are increasingly used for regenerative purposes suc... more Multipotent adipose-derived stem cells (ASCs) are increasingly used for regenerative purposes such as soft tissue reconstruction following mastectomy; however, the ability of tumors to commandeer ASC functions to advance tumor progression is not well understood. Through the integration of physical sciences and oncology approaches we investigated the capability of tumor-derived chemical and mechanical cues to enhance ASC-mediated contributions to tumor stroma formation. Our results indicate that soluble factors from breast cancer cells inhibit adipogenic differentiation while increasing proliferation, proangiogenic factor secretion, and myofibroblastic differentiation of ASCs. This altered ASC phenotype led to varied extracellular matrix (ECM) deposition and contraction thereby enhancing tissue stiffness, a characteristic feature of breast tumors. Increased stiffness, in turn, facilitated changes in ASC behavior similar to those observed with tumor-derived chemical cues. Orthotopic mouse studies further confirmed the pathological relevance of ASCs in tumor progression and stiffness in vivo. In summary, altered ASC behavior can promote tumorigenesis and, thus, their implementation for regenerative therapy should be carefully considered in patients previously treated for cancer. mechanobiology | desmoplasia | angiogenesis | tissue engineering
Whether mechanically unfolded fibronectin (Fn) is present within native extracellular matrix fibr... more Whether mechanically unfolded fibronectin (Fn) is present within native extracellular matrix fibrils is controversial. Fn extensibility under the influence of cell traction forces has been proposed to originate either from the force-induced lengthening of an initially compact, folded quaternary structure as is found in solution (quaternary structure model, where the dimeric arms of Fn cross each other), or from the force-induced unfolding of type III modules (unfolding model). Clarification of this issue is central to our understanding of the structural arrangement of Fn within fibrils, the mechanism of fibrillogenesis, and whether cryptic sites, which are exposed by partial protein unfolding, can be exposed by cell-derived force. In order to differentiate between these two models, two fluorescence resonance energy transfer schemes to label plasma Fn were applied, with sensitivity to either compact-to-extended conformation (arm separation) without loss of secondary structure or compact-to-unfolded conformation. Fluorescence resonance energy transfer studies revealed that a significant fraction of fibrillar Fn within a three-dimensional human fibroblast matrix is partially unfolded. Complete relaxation of Fn fibrils led to a refolding of Fn. The compactly folded quaternary structure with crossed Fn arms, however, was never detected within extracellular matrix fibrils. We conclude that the resting state of Fn fibrils does not contain Fn molecules with crossed-over arms, and that the several-fold extensibility of Fn fibrils involves the unfolding of type III modules. This could imply that Fn might play a significant role in mechanotransduction processes. Citation: Smith ML, Gourdon D, Little WC, Kubow KE, Eguiluz RA, et al (2007) Force-induced unfolding of fibronectin in the extracellular matrix of living cells. PLoS Biol 5(10): e268.
Fecha de recepción: 08-06-09 Fecha de aceptación: 25-07-09
Theses by Roberto C Andresen Eguiluz
Surfaces in biology are present everywhere, and how biomacromolecules and cells interact with the... more Surfaces in biology are present everywhere, and how biomacromolecules and cells interact with them to form interfaces is crucial in cell biology, biotechnology and medicine. For example, biosensors are based on the specific recognition of sugars such as glucose in the glucose-sensors used by diabetic patients. Another example is found in cancer, where breast cancer cells secrete factors that interact with peripheral stromal cells (the interface) and alter their behavior. Another interesting example can be found in diarthrodial joints, such as the knee or the hip joint, where two opposing surfaces need efficient and durable interfacing as they slide against each other over the lifespan of a healthy person.
In the first part of this thesis, I present a direct quantitative correlation between the mechanics of the fibronectin extracellular matrix at the cellular scale and the conformation of fibronectin constituting the matrix at the molecular scale. Additionally, I analyze the effects of mechanics and conformation on cell behavior (adhesion and secretion) in a model system that represents the interface with the tumor.
Next, I present a phenomenological study of the molecular interactions between fibronectin (present in the superficial zone of cartilage) and synovial fluid components. I do this by examining their synergistic performance under confinement and shear to provide efficient lubrication. These results are compared with the tribological performance of a mimetic lubricin developed by my coworkers.
Combined, these results have important implications for our understanding of (i) tumor development and vascularization (ii) cell-matrix interactions (by providing new insights into the structure-mechanics relationship of protein networks), and (iii) the molecular mechanisms of boundary lubrication and wear
protection of articular surfaces. The findings presented in this work may be applied to future treatments of diseases such as breast cancer and osteoarthritis.
The microstructural change experienced by the surfaces of ductile tribologic alloys is of special... more The microstructural change experienced by the surfaces of ductile tribologic alloys is of special interest to achieve a reduction in the friction coef ficient and limits the wear of mechanical components such as journal bearings, extending their working life and consequently diminishing energy-related as well as economic costs. The present work analyses the tribolayer formed in a SAE 783 alloy (Al-20% Sn-1% Cu-0.6% Fe,% weight), used as the functional part in the mass production of small combustion engine journal bearings.
The alloy was subjected to several load and testing time conditions, employing a coaxial tribometer without lubrication and using AISI 9840 steel (Fe-0.4% C-0.25% Si-0.8% Mn-1% Ni-0.25% Mo,% weight) as the tribological counterpart. This was done to establish the effects of the already test conditions on the mechanical and microstructural properties of the new layer. Scanning electron microscopy and instrumented microindentation were used for analysis, revealing the existence of metastable microstructures and higher hardness, as a result of the high contact pressures achieved.
Two models to obtain the friction coefficient of the tribopair are presented. The first one, an analytical model, was developed from a force analysis and can be used to perform real time monitoring of the friction coeffi cient evolution. The second one, is based on parametric Finite Element Method simulations. Both models present enough arguments to continue being explored, so that good correlation with experimental results can be achived.
A Mami y Omi A Sol y Juan A la gran familia que me rodea, que a pesar de no tener un mismo nombre... more A Mami y Omi A Sol y Juan A la gran familia que me rodea, que a pesar de no tener un mismo nombre lo es, A mis cómplices, que durante la carrera logramos alivianar lo que se veía imposible, Rodrigo, Hipácatelas, Goyas, La Mafia, Luis, Demian. A Hugo, David y el Agus, por alivianar el rato en Recubrimientos.
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Papers by Roberto C Andresen Eguiluz
derived from MDA-MB-231 breast cancer cells, an in vitro model
of cancer-activated fibroblasts, deposited an initial extracellular
matrix comprised primarily of fibronectin (Fn). This tumorassociated
Fn was measured to be 87% more unfolded than that
of Fn deposited by control cells and correlated to being 62%
stiffer by the Förster Resonance Energy Transfer and Surface
Forces Apparatus techniques, respectively. The initial
dysregulated ECM lead to the rapid deposition of thick type I
collagen (Col I) fibers. Our findings suggest that initial unfolded
and stiff tumor-associated Fn ECMs promote altered cell-matrix
interactions to fuel tumor progression.
zone of cartilage; however, its role in the lubrication and the wear protection
of articular joints is unknown. In this work, we have investigated the molecular interactions between FN and various components of the synovial fluid such as lubricin (LUB), hyaluronan (HA), and serum albumin (SA), which are all believed to contribute to joint lubrication. Using a Surface Forces Apparatus, we have measured the normal (adhesion/repulsion) and lateral (friction) forces across layers of individual synovial fluid components physisorbed onto FN-coated mica substrates. Our chief findings are (i) FN strongly tethers LUB and HA to mica, as indicated by high and reversible long-range repulsive normal interactions between surfaces, and (ii) FN and LUB synergistically enhance wear protection of surfaces during shear, as suggested by the structural robustness of FN+LUB layers under pressures up to about 4 MPa. These findings provide new insights into the role of FN in the lubricating properties of synovial fluid components sheared between ideal substrates and represent a significant step forward in our understanding of cartilage damage involved in diseases such as osteoarthritis.
Theses by Roberto C Andresen Eguiluz
In the first part of this thesis, I present a direct quantitative correlation between the mechanics of the fibronectin extracellular matrix at the cellular scale and the conformation of fibronectin constituting the matrix at the molecular scale. Additionally, I analyze the effects of mechanics and conformation on cell behavior (adhesion and secretion) in a model system that represents the interface with the tumor.
Next, I present a phenomenological study of the molecular interactions between fibronectin (present in the superficial zone of cartilage) and synovial fluid components. I do this by examining their synergistic performance under confinement and shear to provide efficient lubrication. These results are compared with the tribological performance of a mimetic lubricin developed by my coworkers.
Combined, these results have important implications for our understanding of (i) tumor development and vascularization (ii) cell-matrix interactions (by providing new insights into the structure-mechanics relationship of protein networks), and (iii) the molecular mechanisms of boundary lubrication and wear
protection of articular surfaces. The findings presented in this work may be applied to future treatments of diseases such as breast cancer and osteoarthritis.
The alloy was subjected to several load and testing time conditions, employing a coaxial tribometer without lubrication and using AISI 9840 steel (Fe-0.4% C-0.25% Si-0.8% Mn-1% Ni-0.25% Mo,% weight) as the tribological counterpart. This was done to establish the effects of the already test conditions on the mechanical and microstructural properties of the new layer. Scanning electron microscopy and instrumented microindentation were used for analysis, revealing the existence of metastable microstructures and higher hardness, as a result of the high contact pressures achieved.
Two models to obtain the friction coefficient of the tribopair are presented. The first one, an analytical model, was developed from a force analysis and can be used to perform real time monitoring of the friction coeffi cient evolution. The second one, is based on parametric Finite Element Method simulations. Both models present enough arguments to continue being explored, so that good correlation with experimental results can be achived.
derived from MDA-MB-231 breast cancer cells, an in vitro model
of cancer-activated fibroblasts, deposited an initial extracellular
matrix comprised primarily of fibronectin (Fn). This tumorassociated
Fn was measured to be 87% more unfolded than that
of Fn deposited by control cells and correlated to being 62%
stiffer by the Förster Resonance Energy Transfer and Surface
Forces Apparatus techniques, respectively. The initial
dysregulated ECM lead to the rapid deposition of thick type I
collagen (Col I) fibers. Our findings suggest that initial unfolded
and stiff tumor-associated Fn ECMs promote altered cell-matrix
interactions to fuel tumor progression.
zone of cartilage; however, its role in the lubrication and the wear protection
of articular joints is unknown. In this work, we have investigated the molecular interactions between FN and various components of the synovial fluid such as lubricin (LUB), hyaluronan (HA), and serum albumin (SA), which are all believed to contribute to joint lubrication. Using a Surface Forces Apparatus, we have measured the normal (adhesion/repulsion) and lateral (friction) forces across layers of individual synovial fluid components physisorbed onto FN-coated mica substrates. Our chief findings are (i) FN strongly tethers LUB and HA to mica, as indicated by high and reversible long-range repulsive normal interactions between surfaces, and (ii) FN and LUB synergistically enhance wear protection of surfaces during shear, as suggested by the structural robustness of FN+LUB layers under pressures up to about 4 MPa. These findings provide new insights into the role of FN in the lubricating properties of synovial fluid components sheared between ideal substrates and represent a significant step forward in our understanding of cartilage damage involved in diseases such as osteoarthritis.
In the first part of this thesis, I present a direct quantitative correlation between the mechanics of the fibronectin extracellular matrix at the cellular scale and the conformation of fibronectin constituting the matrix at the molecular scale. Additionally, I analyze the effects of mechanics and conformation on cell behavior (adhesion and secretion) in a model system that represents the interface with the tumor.
Next, I present a phenomenological study of the molecular interactions between fibronectin (present in the superficial zone of cartilage) and synovial fluid components. I do this by examining their synergistic performance under confinement and shear to provide efficient lubrication. These results are compared with the tribological performance of a mimetic lubricin developed by my coworkers.
Combined, these results have important implications for our understanding of (i) tumor development and vascularization (ii) cell-matrix interactions (by providing new insights into the structure-mechanics relationship of protein networks), and (iii) the molecular mechanisms of boundary lubrication and wear
protection of articular surfaces. The findings presented in this work may be applied to future treatments of diseases such as breast cancer and osteoarthritis.
The alloy was subjected to several load and testing time conditions, employing a coaxial tribometer without lubrication and using AISI 9840 steel (Fe-0.4% C-0.25% Si-0.8% Mn-1% Ni-0.25% Mo,% weight) as the tribological counterpart. This was done to establish the effects of the already test conditions on the mechanical and microstructural properties of the new layer. Scanning electron microscopy and instrumented microindentation were used for analysis, revealing the existence of metastable microstructures and higher hardness, as a result of the high contact pressures achieved.
Two models to obtain the friction coefficient of the tribopair are presented. The first one, an analytical model, was developed from a force analysis and can be used to perform real time monitoring of the friction coeffi cient evolution. The second one, is based on parametric Finite Element Method simulations. Both models present enough arguments to continue being explored, so that good correlation with experimental results can be achived.
In this study, we use Atomic force microscopy (AFM) and the Surface Forces Apparatus (SFA) to characterize the molecular characteristics, and the normal and friction forces of a lubricin-mimetic pAA-graft-PEG copolymer (pAA-62kDa, PEG-2kDa abbreviated pAA-g-PEG) interacting with fibronectin (Fn), a structural protein found in the superficial zone of cartilage[3],[4]. The normal and the lateral (friction) forces of pAA-g-PEG polymers were recorded using the SFA, in the presence and absence of Fn coating.
Collectively, these results have allowed for molecular and microscopic characterization of the pAA-g-PEG interactions with surfaces, gaining insight inot both the lubrication mechanisms and the interactions of the biomimetic polymer with tissue through Fn, indicating that our proposed lubricin-mimetic lubricant might be a promising affordable alternative to lubricin.
Materials and Methods: Poly(acrylic acid) was synthesized by RAFT polymerization using acrylic acid (AA). The pAA-graft-PEG (pAA-g-PEG) copolymer was synthesized by polymer analogous conjugation of monoamine-functionalized PEG to the pAA backbone.
AFM measurements were performed in air using a commercial AFM. Pyramidal SiO2 probes were used for intermittent contact mode imaging.
Normal and friction forces between two mimLUB-coated mica surfaces were measured using the SFA using well-established procedures[5].
Results and Discussion: Our AFM data indicated that the pAA-g-PEG molecules had an average contour length and a diameter of 72nm and 10nm, respectively, as indicated in Figure 1.
Our SFA data showed that the pAA-PEG polymer was only weakly adsorbed onto (negatively charged) bare mica surfaces but became firmly attached when Fn was added as a polymer linker, as presented in Figure 2.
All our friction data exhibited (i) low friction coefficients (µ ≈ 0.25) up to applied pressures of 3 MPa and (ii) Amonton's like behavior, as shown in Figure 3.
Conclusion: We report the design and characterization of a biomimetic lubricant (mimLUB) whose molecular architecture was inspired by lubricin, and the role of fibronectin in mediating mimLUB lubrication of ideal mica substrates. Although poor lubrication was achieved with mimLUB alone, enhanced wearless friction and surface protection were measured up to contact pressures of 4MPa when mimLUB was combined with FN. This effect was attributed to FN-mediated stronger binding of mimLUB to mica, which facilitates the formation (and retention) of a dense, robust, and highly hydrated (through PEG) repulsive brush that prevents polymer chain interpenetration when surfaces are sheared past each other, even under high pressures. As FN is abundant in the superficial zone of articular cartilage, our findings suggest that mimLUB could be a potential effective alternative to natural lubricin for the treatment of damaged cartilage surfaces or artificial joints, such as in the knee or the hip.
NSF under award DMR-1352299; CONACYT under award 308671; NIH under award 1R01AR066667-01; NIH Award Number 1S10RR023748-01
References:
[1] Swann, D. a; Sotman, S.; Dixon, M.; Brooks, C. The Isolation and Partial Characterization of the Major Glycoprotein (LGP-I) from the Articular Lubricating Fraction from Bovine Synovial Fluid. Biochem. J. 1977, 161, 473–485.
[2] Swann, D. a; Slayter, H. S.; Silver, F. H. The Molecular Structure of Lubricating Glycoprotein-I, the Boundary Lubricant for Articular Cartilage. J. Biol. Chem. 1981, 256, 5921–5925.
[3] Balazs, E. a. The Role of Hyaluronan in the Structure and Function of the Biomatrix of Connective Tissues. Struct. Chem. 2009, 20, 233–243.
[4] Chevalier, X. Fibronectin, Cartilage, and Osteoarthritis. Semin. Arthritis Rheum. 1993, 22, 307–318.
[5] Gourdon, D.; Yasa, M.; Alig, A. R. G.; Li, Y.; Safinya, C. R.; Israelachvili, J. N. Mechanical and Structural Properties of BaCrO 4 Nanorod Films under Confinement and Shear. Adv. Funct. Mater. 2004, 14, 239–242