Mind the Viscous Modulus: The Mechanotransductive Response to the Viscous Nature of Isoelastic Matrices Regulates Stem Cell Chondrogenesis.

The design of hydrogels as mimetics of tissues' matrices typically disregards the viscous nature of native tissues and focuses only on their elastic properties. In the case of stem cell chondrogenesis, this has led to contradictory results, likely due to unreported changes in the matrices' viscous modulus. Here, by employing isoelastic matrices with Young's modulus of ≈12 kPa, variations in viscous properties alone (i.e., loss tangent between 0.1 and 0.25) are demonstrated to be sufficient to drive efficient growth factor-free chondrogenesis of human mesenchymal stem cells, both in 2D and 3D cultures. The increase of the viscous component of RGD-functionalized polyacrylamide or polyethylene glycol maleimide hydrogels promotes a phenotype with reduced adhesion, alters mechanosensitive signaling, and boosts cell-cell contacts. In turn, this upregulates the chondrogenic transcription factor SOX9 and supports neocartilage formation, demonstrating that the mechanotransductive response to the viscous nature of the matrix can be harnessed to direct cell fate.

Collagen microarchitecture mechanically controls myofibroblast differentiation.

Altered microarchitecture of collagen type I is a hallmark of wound healing and cancer that is commonly attributed to myofibroblasts. However, it remains unknown which effect collagen microarchitecture has on myofibroblast differentiation. Here, we combined experimental and computational approaches to investigate the hypothesis that the microarchitecture of fibrillar collagen networks mechanically regulates myofibroblast differentiation of adipose stromal cells (ASCs) independent of bulk stiffness. Collagen gels with controlled fiber thickness and pore size were microfabricated by adjusting the gelation temperature while keeping their concentration constant. Rheological characterization and simulation data indicated that networks with thicker fibers and larger pores exhibited increased strain-stiffening relative to networks with thinner fibers and smaller pores. Accordingly, ASCs cultured in scaffolds with thicker fibers were more contractile, expressed myofibroblast markers, and deposited more extended fibronectin fibers. Consistent with elevated myofibroblast differentiation, ASCs in scaffolds with thicker fibers exhibited a more proangiogenic phenotype that promoted endothelial sprouting in a contractility-dependent manner. Our findings suggest that changes of collagen microarchitecture regulate myofibroblast differentiation and fibrosis independent of collagen quantity and bulk stiffness by locally modulating cellular mechanosignaling. These findings have implications for regenerative medicine and anticancer treatments.

Dynamics of Synovial Fluid Aggregation under Shear.

The synovial fluid (SF) that lubricates articular joints exhibits complex rheological and tribological properties due to the interactions and behaviors of its various molecular components. Under shear, SF films abruptly thicken by more than 300% and large, dense aggregates form within the fluid. In this study, we used the Surface Force Apparatus to elucidate which SF components are involved in this shear-induced transformation by (i) determining which (if any) of all major SF components replicate the behavior of SF under shear and (ii) observing the effect of removing implicated components from SF by enzymatic digestion. While most previous studies of SF have focused on the tribological roles of lubricin or hyaluronic acid, our results indicate that albumin is a key contributor to the formation of aggregates in SF under shear. Our results also suggest that SF aggregation is associated with efficient surface protection against wear. As our findings are based on experiments involving rigid, nonporous surfaces, they may be used to investigate shear-mediated aggregation mechanisms occurring during the lubrication of artificial joints, ultimately advancing our current vision of implant design.

Boundary mode lubrication of articular cartilage with a biomimetic diblock copolymer.

We report the design of a diblock copolymer with architecture and function inspired by the lubricating glycoprotein lubricin. This diblock copolymer, synthesized by sequential reversible addition-fragmentation chain-transfer polymerization, consists of a cationic cartilage-binding domain and a brush-lubricating domain. It reduces the coefficient of friction of articular cartilage under boundary mode conditions (0.088 ± 0.039) to a level equivalent to that provided by lubricin (0.093 ± 0.011). Additionally, both the EC50 (0.404 mg/mL) and cartilage-binding time constant (7.19 min) of the polymer are comparable to purified human and recombinant lubricin. Like lubricin, the tribological properties of this polymer are dependent on molecular architecture. When the same monomer composition was evaluated either as an AB diblock copolymer or as a random copolymer, the diblock effectively lubricated cartilage under boundary mode conditions whereas the random copolymer did not. Additionally, the individual polymer blocks did not lubricate independently, and lubrication could be competitively inhibited with an excess of binding domain. This diblock copolymer is an example of a synthetic polymer with lubrication properties equal to lubricin under boundary mode conditions, suggesting its potential utility as a therapy for joint pathologies like osteoarthritis.

Synergistic Interactions of a Synthetic Lubricin-Mimetic with Fibronectin for Enhanced Wear Protection.

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.

Breast cancer cells alter the dynamics of stromal fibronectin-collagen interactions.

Breast cancer cells recruit surrounding stromal cells, such as cancer-associated fibroblasts (CAFs), to remodel their extracellular matrix (ECM) and promote invasive tumor growth. Two major ECM components, fibronectin (Fn) and collagen I (Col I), are known to interact with each other to regulate cellular behavior. In this study, we seek to understand how Fn and Col I interplay and promote a dysregulated signaling pathway to facilitate tumor progression. Specifically, we investigated the evolution of tumor-conditioned stromal ECM composition, structure, and relaxation. Furthermore, we assessed how evolving Fn-Col I interactions gradually affected pro-angiogenic signaling. Our data first indicate that CAFs initially assembled a strained, viscous, and unfolded Fn matrix. This early altered Fn matrix was later remodeled into a thick Col I-rich matrix that was characteristic of a dense tumor mass. Next, our results suggest that this ECM remodeling was primarily mediated by matrix metalloproteinases (MMPs). This MMP activity caused profound structural and mechanical changes in the developing ECM, which then modified vascular endothelial growth factor (VEGF) secretion by CAFs and matrix sequestration. Collectively, these findings enhance our understanding of the mechanisms by which Fn and Col I synergistically interplay in promoting a sustained altered signaling cascade to remodel the breast tumor stroma for invasive breast tumor growth.

Protein-crystal interface mediates cell adhesion and proangiogenic secretion.

The nanoscale materials properties of bone apatite crystals have been implicated in breast cancer bone metastasis and their interactions with extracellular matrix proteins are likely involved. In this study, we used geologic hydroxyapatite (HAP, Ca10(PO4)6(OH)2), closely related to bone apatite, to investigate how HAP surface chemistry and nano/microscale topography individually influence the crystal-protein interface, and how the altered protein deposition impacts subsequent breast cancer cell activities. We first utilized Förster resonance energy transfer (FRET) to assess the molecular conformation of fibronectin (Fn), a major extracellular matrix protein upregulated in cancer, when it adsorbed onto HAP facets. Our analysis reveals that both low surface charge density and nanoscale roughness of HAP facets individually contributed to molecular unfolding of Fn. We next quantified cell adhesion and secretion on Fn-coated HAP facets using MDA-MB-231 breast cancer cells. Our data show elevated proangiogenic and proinflammatory secretions associated with more unfolded Fn adsorbed onto nano-rough HAP facets with low surface charge density. These findings not only deconvolute the roles of crystal surface chemistry and topography in interfacial protein deposition but also enhance our knowledge of protein-mediated breast cancer cell interactions with apatite, which may be implicated in tumor growth and bone metastasis.

Fibronectin Mechanobiology Regulates Tumorigenesis.

Fibronectin (Fn) is an essential extracellular matrix (ECM) glycoprotein involved in both physiological and pathological processes. The structure-function relationship of Fn has been and is still being studied, as changes in its molecular structure are integral in regulating (or dysregulating) its biological activities via its cell, matrix component, and growth factor binding sites. Fn comprises three types of repeating modules; among them, FnIII modules are mechanically unstable domains that may be extended/unfolded upon cell traction and either uncover cryptic binding sites or disrupt otherwise exposed binding sites. Cells assemble Fn into a fibrillar network; its conformational flexibility implicates Fn as a critical mechanoregulator of the ECM. Fn has been shown to contribute to altered stroma remodeling during tumorigenesis. This review will discuss (i) the significance of the structure-function relationship of Fn at both the molecular and the matrix scales, (ii) the role of Fn mechanobiology in the regulation of tumorigenesis, and (iii) Fn-related advances in cancer therapy development.

3D Conducting Polymer Platforms for Electrical Control of Protein Conformation and Cellular Functions.

We report the fabrication of three dimensional (3D) macroporous scaffolds made from poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) via an ice-templating method. The scaffolds offer tunable pore size and morphology, and are electrochemically active. When a potential is applied to the scaffolds, reversible changes take place in their electrical doping state, which in turn enables precise control over the conformation of adsorbed proteins (e.g., fibronectin). Additionally, the scaffolds support the growth of mouse fibroblasts (3T3-L1) for 7 days, and are able to electrically control cell adhesion and pro-angiogenic capability. These 3D matrix-mimicking platforms offer precise control of protein conformation and major cell functions, over large volumes and long cell culture times. As such, they represent a new tool for biological research with many potential applications in bioelectronics, tissue engineering, and regenerative medicine.

Mechanical forces regulate the interactions of fibronectin and collagen I in extracellular matrix.

Despite the crucial role of extracellular matrix (ECM) in directing cell fate in healthy and diseased tissues--particularly in development, wound healing, tissue regeneration and cancer--the mechanisms that direct the assembly and regulate hierarchical architectures of ECM are poorly understood. Collagen I matrix assembly in vivo requires active fibronectin (Fn) fibrillogenesis by cells. Here we exploit Fn-FRET probes as mechanical strain sensors and demonstrate that collagen I fibres preferentially co-localize with more-relaxed Fn fibrils in the ECM of fibroblasts in cell culture. Fibre stretch-assay studies reveal that collagen I's Fn-binding domain is responsible for the mechano-regulated interaction. Furthermore, we show that Fn-collagen interactions are reciprocal: relaxed Fn fibrils act as multivalent templates for collagen assembly, but once assembled, collagen fibres shield Fn fibres from being stretched by cellular traction forces. Thus, in addition to the well-recognized, force-regulated, cell-matrix interactions, forces also tune the interactions between different structural ECM components.

Obesity-dependent changes in interstitial ECM mechanics promote breast tumorigenesis.

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.

Effect of the Materials Properties of Hydroxyapatite Nanoparticles on Fibronectin Deposition and Conformation.

Hydroxyapatite (HAP, Ca10(PO4)6(OH)2) nanoparticles with controlled materials properties have been synthesized through a two-step hydrothermal aging method to investigate fibronectin (Fn) adsorption. Two distinct populations of HAP nanoparticles have been generated: HAP1 particles had smaller size, plate-like shape, lower crystallinity, and more negative ζ potential than HAP2 particles. We then developed two-dimensional platforms containing HAP and Fn and analyzed both the amount and the conformation of Fn via Förster resonance energy transfer (FRET) at various HAP concentrations. Our FRET analysis reveals that larger amounts of more compact Fn molecules were adsorbed onto HAP1 than onto HAP2 particles. Additionally, our data show that the amount of compact Fn adsorbed increased with increasing HAP concentration due to the formation of nanoparticle agglomerates. We propose that both the surface chemistry of single nanoparticles and the size and morphology of HAP agglomerates play significant roles in the interaction of Fn with HAP. Collectively, our findings suggest that the HAP-induced conformational changes of Fn, a critical mechanotransducer protein involved in the communication of cells with their environment, will ultimately affect downstream cellular behaviors. These results have important implications for our understanding of organic-inorganic interactions in physiological and pathological biomineralization processes such as HAP-related inflammation.

Fibronectin mediates enhanced wear protection of lubricin during shear.

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.

Stiffening and unfolding of early deposited-fibronectin increase proangiogenic factor secretion by breast cancer-associated stromal cells.

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.

Fibronectin conformation regulates the proangiogenic capability of tumor-associated adipogenic stromal cells.

BACKGROUND: Changes in fibronectin (Fn) matrix remodeling contribute to mammary tumor angiogenesis and are related to altered behavior of adipogenic stromal cells; yet, the underlying mechanisms remain unclear due in part to a lack of reductionist model systems that allow the inherent complexity of cell-derived extracellular matrices (ECMs) to be deciphered. In particular, breast cancer-associated adipogenic stromal cells not only enhance the composition, quantity, and rigidity of deposited Fn, but also partially unfold these matrices. However, the specific effect of Fn conformation on tumor angiogenesis is undefined. METHODS: Decellularized matrices and a conducting polymer device consisting of poly(3,4-ethylenedioxythiophene) doped with poly(styrenesulfonate) (PEDOT:PSS) were used to examine the effect of Fn conformation on the behavior of 3T3-L1 preadipocytes. Changes in cell adhesion and proangiogenic capability were tested via cell counting and by quantification of vascular endothelial growth factor (VEGF) secretion, respectively. Integrin-blocking antibodies were utilized to examine varied integrin specificity as a potential mechanism. RESULTS: Our findings suggest that tumor-associated partial unfolding of Fn decreases adhesion while enhancing VEGF secretion by breast cancer-associated adipogenic precursor cells, and that altered integrin specificity may underlie these changes. CONCLUSIONS AND GENERAL SIGNIFICANCE: These results not only have important implications for our understanding of tumorigenesis, but also enhance knowledge of cell-ECM interactions that may be harnessed for other applications including advanced tissue engineering approaches. This article is part of a Special Issue entitled Organic Bioelectronics - Novel Applications in Biomedicine.

Implanted adipose progenitor cells as physicochemical regulators of breast cancer.

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.

Electrical control of protein conformation.

Conducting polymer devices that enable precise control of fibronectin conformation over macroscopic areas are reported. Single conformations as well as conformation gradients are achieved by applying an appropriate potential. These surfaces remain biologically relevant and support cell culture; hence, they may serve as a model to understand and control cell-surface interactions, with applications in basic research, medical diagnostics, and tissue engineering.

Crosslinking of cell-derived 3D scaffolds up-regulates the stretching and unfolding of new extracellular matrix assembled by reseeded cells.

Elevated levels of tissue crosslinking are associated with numerous diseases (cancer stroma, organ fibrosis), and also eliminate the otherwise remarkable clinical successes of tissue-derived scaffolds, instead eliciting a foreign body reaction. Nevertheless, it is not well understood how the initial physical and biochemical properties of cellular microenvironments, stem cell niches, or of 3D tissue scaffolds guide the assembly and remodeling of new extracellular matrix (ECM) that is ultimately sensed by cells. Here, we incorporated FRET-based mechanical strain sensors, either into cell-derived ECM scaffolds or into the fibronectin (Fn) matrix assembled by reseeded fibroblasts, and demonstrated the following. Cell-generated tensile forces change the conformation of Fn in both 3D scaffolds and new matrix over time. The time course by which new matrix fibers are stretched by reseeded cells is accelerated by scaffold crosslinking. Importantly, stretching Fn fibers increases their elastic modulus (rigidity) and alters their biochemical display. Regulated by Fn fiber unfolding, more soluble Fn binds to the native than to the crosslinked scaffolds. Additionally, matrix assembly of fibroblasts is decreased by scaffold crosslinking. Taken together, scaffold crosslinking has a multifactorial impact on the microenvironment that reseeded cells assemble and respond to, with far-reaching implications for tissue engineering and disease physiology.

Fibronectin forms the most extensible biological fibers displaying switchable force-exposed cryptic binding sites.

Rather than maximizing toughness, as needed for silk and muscle titin fibers to withstand external impact, the much softer extracellular matrix fibers made from fibronectin (Fn) can be stretched by cell generated forces and display extraordinary extensibility. We show that Fn fibers can be extended more than 8-fold (>700% strain) before 50% of the fibers break. The Young's modulus of single fibers, given by the highly nonlinear slope of the stress-strain curve, changes orders of magnitude, up to MPa. Although many other materials plastically deform before they rupture, evidence is provided that the reversible breakage of force-bearing backbone hydrogen bonds enables the large strain. When tension is released, the nano-sized Fn domains first contract in the crowded environment of fibers within seconds into random coil conformations (molten globule states), before the force-bearing hydrogen bond networks that stabilize the domain's secondary structures are reestablished within minutes (double exponential). The exposure of cryptic binding sites on Fn type III modules increases steeply upon stretching. Thus fiber extension steadily up-regulates fiber rigidity and cryptic epitope exposure, both of which are known to differentially alter cell behavior. Finally, since stress-strain relationships cannot directly be measured in native extracellular matrix (ECM), the stress-strain curves were correlated with stretch-induced alterations of intramolecular fluorescence resonance energy transfer (FRET) obtained from trace amounts of Fn probes (mechanical strain sensors) that can be incorporated into native ECM. Physiological implications of the extraordinary extensibility of Fn fibers and contraction kinetics are discussed.

Stretched extracellular matrix proteins turn fouling and are functionally rescued by the chaperones albumin and casein.

While evidence is mounting that cells exploit protein unfolding for mechanochemical signal conversion (mechanotransduction), what mechanisms are in place to deal with the unwanted consequences of exposing hydrophobic residues upon force-induced protein unfolding? Here, we show that mechanical chaperones exist that can transiently bind to hydrophobic residues that are freshly exposed by mechanical force. The stretch-upregulated binding of albumin or casein to fibronectin fibers is reversible and does not inhibit fiber contraction once the tension is released.