Fibronectin Fibril Formation Research Articles

Differential regulation of fibronectin expression and fibrillogenesis by autocrine TGF-β1 signaling in malignant and benign mammary epithelial cells

Remodeling of the extracellular matrix (ECM) is a key hallmark of cancer progression. A critical component of ECM remodeling is the assembly of the glycoprotein fibronectin (FN) into insoluble fibrils, which provide a scaffold for invading vascular endothelial cells and escaping cancer cells, as well as a framework for collagen deposition and oncogenic cytokine tethering. FN fibril assembly is induced by Transforming Growth Factor-β1 (TGF-β1), which was originally identified for its role in malignant transformation. Addition of exogenous TGF-β1 drives FN fibril assembly while also upregulating endogenous TGF-β1 expression and autocrine signaling. In the current study, we sought to determine if autocrine TGF-β1 signaling plays a role in FN fibril formation in either MCF10A mammary epithelial cells, which behave similarly to healthy epithelia, or malignant MDA- MB-231 breast cancer cells. Our results show two interesting findings: first, malignant MDA-MB- 231 cells assemble less FN into fibrils, despite expressing and secreting more soluble FN; second, autocrine TGF-β1 signaling is required for FN fibril formation in MCF10A epithelial cells, even in the presence of exogenous, active TGF-β1. This suggests that autocrine TGF-β1 is signaling through distinct pathways from active exogenous TGF-β1. We hypothesized that this signaling was mediated by interactions between the TGF-β1 latency associated peptide (LAP) and αv integrins; indeed, incubating MCF10As with soluble LAP, even in the absence of the active TGF-β1 ligand, partially recovered FN fibril assembly. Taken together, these data suggests that autocrine TGF-β1 plays a critical role in FN fibril assembly, and this interaction is mediated by LAP-integrin signaling.

Heparan sulfate is necessary for the early formation of nascent fibronectin and collagen I fibrils at matrix assembly sites

Fibronectin (FN), an essential component of the extracellular matrix (ECM), is assembled via a cell-mediated process in which integrin receptors bind secreted FN and mediate its polymerization into fibrils that extend between cells, ultimately forming an insoluble matrix. Our previous work using mutant Chinese hamster ovary (CHO) cells identified the glycosaminoglycan heparan sulfate (HS) and its binding to FN as essential for the formation of insoluble FN fibrils. In this study, we investigated the contributions of HS at an early stage of the assembly process using knockdown of exostosin-1 (EXT1), one of the glycosyltransferases required for HS chain synthesis. NIH 3T3 fibroblasts with decreased EXT1 expression exhibited a significant reduction in both FN and type I collagen in the insoluble matrix. We show that FN fibril formation is initiated at matrix assembly sites, and while these sites were formed by cells with EXT1 knockdown, their growth was stunted compared with wild-type cells. The most severe defect observed was in the polymerization of nascent FN fibrils, which was reduced 2.5-fold upon EXT1 knockdown. This defect was rescued by the addition of exogenous soluble heparin chains long enough to simultaneously bind multiple FN molecules. The activity of soluble heparin in this process indicates that nascent fibril formation depends on HS more so than on the protein component of a specific HS proteoglycan. Together, our results suggest that heparin or HS is necessary for concentrating and localizing FN molecules at sites of early fibril assembly.

Different Conformations of Fibronectin Fibrils Formed Under Cell-Free Condition in Vitro display Distinct Effects on Cell Adhesion and Spreading

Introduction: Insoluble fibrils are the active form of fibronectin (FN) with exposed functional domains capable of interacting with a variety of biomolecules and cells to support many cellular processes. Recent studies show that the formation of FN fibrils can occur in cell-free systems. Currently, the characteristic of FN fibrils and their potential in wound healing application are not fully understood. Our project aims at (i) using urea to induce the formation of FN fibrils with different conformations and (ii) to characterize their conformation-dependent effect on cellular adhesion and spreading.Method: To induce FN fibrillogenesis, native plasma FN at various initial concentrations (0.25, 0.5, 0.75 and 1 mg/ml) was treated by dialysis in urea at concentrations of 1, 2, and 4 M for 16h at room temperature followed by dialysis in PBS buffer (to remove urea). Morphological analysis of formed FN fibrils was conducted bylight microscopy and scanning electron microscopy (SEM). To assess biological effects of FN fibrils on cells in relationto their morphologies, platelet and fibroblast cell adhesion assays were performed. Specifically, platelet rich plasma (5x107 platelets/ml) or fibroblasts (L929, 104 cells/ml) were incubated on surfaces pre-coated with 0.25 and 0.75 mg/ml native plasma FN or FN fibrils for 30 min at 370C.Results: Our cell-free FN fibrillogenesis experiments in vitro indicated that the optimal concentrations of FN and urea were 1 mg/ml and 2 M, respectively, to induce formation of FN fibrils with various conformations. FN only formed minuscule fibrils at 1 M urea while there was no fibril formation at 4 M urea. In contrast, FN fibrils with various conformations ranging from fibrillar FN to non-fibrillar FN (i.e., nodular FN aggregates) were formed when using 2 M urea. Fibril formation was shown to be dependent on the FN concentration. FN at low concentrations (0.25 or 0.5 mg/ml) formed a few insoluble minuscule fibrils or nodular FN aggregates with variation in size ranging from 10 to 50 µm. FN at higher concentrations (0.75 or 1 mg/ml) formed much more nodular FN and fibrillar FN with diameters mostly ranging from 10 to 30 µm that tended to interlace into the fibrillar matrix. SEM analysis documented morphological variations of fibrils formed under different conditions. For example, at low FN concentration (0.25 mg/ml) small fibrils with a smooth surface were formed. By contrast, larger fibrils with attached nodular FN and a resulting rough surface were formed at high FN concentration (≥ 0.75 mg/ml). Adhesion assays indicated that platelets adhered approximately 6 times more firmly on FN fibrils than on native plasma FN (n=3, p < 0.05).On immobilized native FN, adherent platelets were evenly distributed or formed small clusters, while fibrillar FN induced platelet accumulation on the matrix. Fibroblasts adhered both on non-fibrillar FN and matrix of small fibrils (diameter in range of 10-30 µm) but did not interact to larger fibrillar bundles (diameter > 292 µm). Investigations of FN-cell interaction by SEM revealed a structure-dependent effect of FN on fibroblast morphology after adhesion. Specifically, cells remained in spherical shape and separately adhered on nodular FN while extending their shape in multiple directions on the rough surface of FN fibrils.Conclusion: Treatment of native plasma FN by urea at a concentration of 2 M induces irreversible formation of heterogeneous FN fibrils, displaying a rough surface due to attachment of FN nodules. The conformational and compositional changes of the FN matrix can lead to firm adhesion of platelets and fibroblasts and may thus provide a future tool to improve wound healing upon local application. DisclosuresNo relevant conflicts of interest to declare.

Effects of substrate stiffness and actin velocity on in silico fibronectin fibril morphometry and mechanics.

Assembly of the extracellular matrix protein fibronectin (FN) into insoluble, viscoelastic fibrils is a critical step during embryonic development and wound healing; misregulation of FN fibril assembly has been implicated in many diseases, including fibrotic diseases and cancer. We have previously developed a computational model of FN fibril assembly that recapitulates the morphometry and mechanics of cell-derived FN fibrils. Here we use this model to probe two important questions: how is FN fibril formation affected by the contractile phenotype of the cell, and how is FN fibril formation affected by the stiffness of the surrounding tissue? We show that FN fibril formation depends strongly on the contractile phenotype of the cell, but only weakly on in vitro substrate stiffness, which is an analog for in vivo tissue stiffness. These results are consistent with previous experimental data and provide a better insight into conditions that promote FN fibril assembly. We have also investigated two distinct phenotypes of FN fibrils that we have previously identified; we show that the ratio of the two phenotypes depends on both substrate stiffness and contractile phenotype, with intermediate contractility and high substrate stiffness creating an optimal condition for stably stretched fibrils. Finally, we have investigated how re-stretch of a fibril affects cellular response. We probed how the contractile phenotype of the re-stretching cell affects the mechanics of the fibril; results indicate that the number of myosin motors only weakly affects the cellular response, but increasing actin velocity results in a decrease in the apparent stiffness of the fibril and a decrease in the stably-applied force to the fibril. Taken together, these results give novel insights into the combinatorial effects of substrate stiffness and cell contractility on FN fibril assembly.

JMJD6 Dysfunction Due to Iron Deficiency in Preeclampsia Disrupts Fibronectin Homeostasis Resulting in Diminished Trophoblast Migration.

The mechanisms contributing to excessive fibronectin in preeclampsia, a pregnancy-related disorder, remain unknown. Herein, we investigated the role of JMJD6, an O2- and Fe2+-dependent enzyme, in mediating placental fibronectin processing and function. MALDI-TOF identified fibronectin as a novel target of JMJD6-mediated lysyl hydroxylation, preceding fibronectin glycosylation, deposition, and degradation. In preeclamptic placentae, fibronectin accumulated primarily in lysosomes of the mesenchyme. Using primary placental mesenchymal cells (pMSCs), we found that fibronectin fibril formation and turnover were markedly impeded in preeclamptic pMSCs, partly due to impaired lysosomal degradation. JMJD6 knockdown in control pMSCs recapitulated the preeclamptic FN phenotype. Importantly, preeclamptic pMSCs had less total and labile Fe2+ and Hinokitiol treatment rescued fibronectin assembly and promoted lysosomal degradation. Time-lapse imaging demonstrated that defective ECM deposition by preeclamptic pMSCs impeded HTR-8/SVneo cell migration, which was rescued upon Hinokitiol exposure. Our findings reveal new Fe2+-dependent mechanisms controlling fibronectin homeostasis/function in the placenta that go awry in preeclampsia.

Fibronectin assembly regulates lumen formation in breast acini.

Fibronectin (FN) is an extracellular matrix (ECM) glycoprotein that self‐assembles into FN fibrils, forming a FN matrix contributing to the stiffness of the ECM. Stromal FN stiffness in cancer has been shown to impact epithelial functions such as migration, cancer metastasis, and epithelial‐to‐mesenchymal transition. The role of the FN matrix of epithelial cells in driving such processes remains less well understood and is the focus of this study. Hypoxia, defined by low oxygen tension (<5%) is one of the hallmarks of tumor microenvironments impacting fibril reorganization in stromal and epithelial cells. Here, using the MCF10 breast epithelial progression series of cell lines encompassing normal, preinvasive, and invasive states, we show that FN fibril formation decreases during hypoxia, coinciding with a decrease in migratory potential of these cells. Conversely, we find that FN fibril disruption during three‐dimensional acinar growth of normal breast cells resulted in acinar luminal filling. Our data also demonstrates that the luminal filling upon fibril disruption in untransformed MCF10A cells results in a loss of apicobasal polarity, characteristic of pre‐invasive and invasive breast cell lines MCF10AT and MCF10 DCIS.com. Overall this is the first study that relates fibril‐mediated changes in epithelial cells as critical players in lumen clearing of breast acini and maintenance of the untransformed growth characteristic.

Dermatopontin, A Novel Adipokine Promoting Adipose Tissue Extracellular Matrix Remodelling and Inflammation in Obesity.

Compelling evidence suggests that dermatopontin (DPT) regulates collagen and fibronectin fibril formation, the induction of cell adhesion and the prompting of wound healing. We aimed to evaluate the role of DPT on obesity and its associated metabolic alterations as well as its impact in visceral adipose tissue (VAT) inflammation and extracellular matrix (ECM) remodelling. Samples obtained from 54 subjects were used in a case-control study. Circulating and VAT expression levels of DPT as well as key ECM remodelling- and inflammation-related genes were analysed. The effect of pro- and anti-inflammatory mediators on the transcript levels of DPT in visceral adipocytes was explored. The impact of DPT on ECM remodelling and inflammation pathways was also evaluated in cultured adipocytes. We show that obesity and obesity-associated type 2 diabetes (T2D) increased (p < 0.05) circulating levels of DPT. In this line, DPT mRNA in VAT was increased (p < 0.05) in obese patients with and without T2D. Gene expression levels of DPT were enhanced (p < 0.05) in human visceral adipocytes after the treatment with lipopolysaccharide, tumour growth factor (TGF)-β and palmitic acid, whereas a downregulation (p < 0.05) was detected after the stimulation with interleukin (IL)-4 and IL-13, critical cytokines mediating anti-inflammatory pathways. Additionally, we revealed that DPT increased (p < 0.05) the expression of ECM- (COL6A3, ELN, MMP9, TNMD) and inflammation-related factors (IL6, IL8, TNF) in human visceral adipocytes. These findings provide, for the first time, evidence of a novel role of DPT in obesity and its associated comorbidities by influencing AT remodelling and inflammation.

Crosstalk with lung epithelial cells regulates Sfrp2-mediated latency in breast cancer dissemination.

The process of metastasis is complex1. In breast cancer, there are frequently long timespans between cells leaving the primary tumour and growth of overt metastases 2,3. Reasons for disease indolence and subsequent transitioning back to aggressive growth include interplay with myeloid and fibroblastic cells in the tumour microenvironment and ongoing immune surveillance 4–6. However, the signals causing actively growing cells to enter into an indolent state, and enabling them to survive for extended periods of time, are not well understood. We reveal how the behaviour of indolent breast cancer cells in the lung is determined by their interactions with alveolar epithelial cells, in particular AT1 cells. This promotes the formation of fibronectin (FN) fibrils by indolent cells that drive integrin-dependent pro-survival signals. Combined in vivo RNA sequencing and drop-out screening identified Secreted frizzled-related protein 2 (Sfrp2) as a key mediator of this interaction. Sfrp2 is induced in breast cancer cells by signals emanating from lung epithelial cells and promotes FN fibril formation and survival, while blockade of Sfrp2 expression reduces the burden of indolent disease.

Activation of αvβ3 Integrin Alters Fibronectin Fibril Formation in Human Trabecular Meshwork Cells in a ROCK-Independent Manner.

PurposeFibronectin fibrillogenesis is an integrin-mediated process that may contribute to the pathogenesis of primary open-angle glaucoma (POAG). Here, we examined the effects of αvβ3 integrins on fibrillogenesis in immortalized TM-1 cells and human trabecular meshwork (HTM) cells.MethodsTM-1 cells overexpressing wild-type β3 (WTβ3) or constitutively active β3 (CAβ3) integrin subunits were generated. Control cells were transduced with an empty vector (EV). Deoxycholic acid (DOC) extraction of monolayers, immunofluorescence microscopy, and On-cell western analyses were used to determine levels of fibronectin fibrillogenesis and fibronectin fibril composition (EDA+ and EDB+ fibronectins) and conformation. αvβ3 and α5β1 Integrin levels were determined using fluorescence-activated cell sorting (FACS). Cilengitide and an adenovirus vector expressing WTβ3 or CAβ3 integrin subunits were used to examine the role of αvβ3 integrin in HTM cells. The role of the canonical α5β1 integrin–mediated pathway in fibrillogenesis was determined using the fibronectin-binding peptide FUD, the β1 integrin function-blocking antibody 13, and the Rho kinase (ROCK) inhibitor Y27632.ResultsActivation of αvβ3 integrin enhanced the assembly of fibronectin into DOC-insoluble fibrils in both TM-1 and HTM cells. The formation of fibronectin fibrils was dependent on α5β1 integrin and could be inhibited by FUD. However, fibrillogenesis was unaffected by Y27632. Fibrils assembled by CAβ3 cells also contained high levels of EDA+ and EDB+ fibronectin and fibronectin that was stretched.Conclusionsαvβ3 Integrin signaling altered the deposition and structure of fibronectin fibrils using a β1 integrin/ROCK-independent mechanism. Thus, αvβ3 integrins could play a significant role in altering the function of fibronectin matrices in POAG.

Alterations in Nuclear Lamina and the Cytoskeleton of Bone Marrow-Derived Human Mesenchymal Stem Cells Cultured Under Simulated Microgravity Conditions

Cells sense and respond to environmental changes induced by gravity. Although reactions to conventional culture have been intensively studied, little is known about the cellular reaction to simulated microgravity conditions. Thus, in this study, we investigated the effects of simulated microgravity on human mesenchymal stem cells using a three-dimensional clinostat (Gravite®), a recently developed device used to generate simulated microgravity condition in vitro. Our time-lapse analysis shows that cells cultured under conventional culture conditions have a stretched morphology and undergo unidirectional migration, whereas cells cultured under simulated microgravity conditions undergo multidirectional migration with directional changes of cell movement. Furthermore, cells cultured under conventional culture conditions maintained their spindle shape through fibronectin fibril formation in their bodies and focal adhesion stabilization with enriched stress fibers. However, cells cultured under simulated microgravity conditions were partially contracted and the fibril structures were degraded in the cell bodies. Additionally, paxillin phosphorylation in the cells cultured under simulated microgravity conditions was more intense at the cell periphery in regions near the leading and trailing edges, but was less expressed in the cell bodies compared with that observed in cells cultured under conventional culture conditions. Furthermore, lamin A/C, a major component of the nuclear lamina, was mainly located on the apical side in cells cultured under conventional culture conditions, indicating basal-to-apical polarization. However, cells cultured under simulated microgravity conditions showed lamin A/C localization on both the apical and basal sides. Taken together, these results demonstrate that simulated microgravity-driven fibronectin assembly affects nuclear lamina organization through the spatial reorganization of the cytoskeleton.

Human kallikrein-related peptidase 12 stimulates endothelial cell migration by remodeling the fibronectin matrix

Kallikrein-related peptidase 12 (KLK12) is a kallikrein family peptidase involved in angiogenesis – a complex biological process in which the sprouting, migration and stabilization of endothelial cells requires extracellular matrix remodeling. To characterize the molecular mechanisms associated with KLK12′s proangiogenic activity, we evaluated its ability to hydrolyze various matrix proteins. Our results show that KLK12 efficiently cleaved the human extracellular matrix proteins fibronectin and tenascin, both of which are involved in the regulation of endothelial cell adhesion and migration. For fibronectin, the major proteolytic product generated by KLK12 was a 29 kDa fragment containing the amino-terminal domain and the first five type I fibronectin-domains, which are essential for regulating fibronectin assembly. We also demonstrated that KLK12-mediated fibronectin proteolysis antagonizes fibronectin polymerization and fibronectin fibril formation by endothelial cells, leading to an increase in cell migration. Furthermore, a polyclonal antibody raised against KLK12′s proteolytic cleavage site on fibronectin prevented the KLK12-dependent inhibition of fibronectin polymerization and the KLK12-mediated pro-migratory effect on endothelial cells. Taken as a whole, our results indicate that KLK12′s proangiogenic effect is mediated through several molecular mechanisms.

Abstract 80: Interplay Between Cell-cell and Cell-extracellular Matrix Forces Regulate Myocardial Proliferation

Changes in expression and distribution of integrin, fibronectin (FN), and N-cadherin in the postnatal heart accompany the switch from hyperplastic to hypertrophic growth. As FN decreases after birth, N-cadherin/catenin complex accumulates at the cell termini creating a specialized type of cell-cell contact called the intercalated disc (ID). Integrin-FN interactions promote cardiomyocyte and cardiac progenitor cell proliferation. However, little is known regarding the reciprocity between integrin and N-cadherin adhesions in the regulation of myocardial proliferation. Alpha-catenins function as mechanosensors and transduce the intercellular force from N-cadherin to the actin cytoskeleton. To investigate mechanotransduction in the heart, we generated cardiac-specific αE- and αT-catenins double knockout (DKO) mice. The relationship between N-cadherin, integrin, and FN was examined at postnatal day (P) 4, P7, P14, and P60. DKO hearts exhibited aberrant N-cadherin expression accompanied by increased expression of α5/β1 integrin, the primary receptor for FN. Normally found at the lateral membrane, α5 and FN accumulated at the ID along with N-cadherin in DKO hearts. FN-integrin binding leads to the formation of FN fibrils that are initially soluble in the detergent deoxycholate (DOC) but are gradually converted into a stable, DOC-insoluble form that comprises the mature matrix. Both α5 and FN were increased in the DOC-insoluble fraction consistent with enhanced matrix assembly in DKO hearts. Activation of focal adhesion kinase (FAK) and p130CAS were observed in the DKO hearts consistent with increased cell-extracellular matrix interactions. Complementary experiments performed with deformable substrata demonstrated that stiffness-mediated Yap nuclear accumulation was dependent on FAK activity. These data demonstrate that α-catenins regulate the balance between cell-cell and cell-matrix adhesions, which, in turn, controls Yap subcellular localization, thus providing a molecular explanation for loss of regenerative potential in the adult heart.

Mechanotransduction Dynamics at the Cell-Matrix Interface

The ability of cells to sense and respond to mechanical cues from the surrounding environment has been implicated as a key regulator of cell differentiation, migration, and proliferation. The extracellular matrix (ECM) is an oft-overlooked component of the interface between cells and their surroundings. Cells assemble soluble ECM proteins into insoluble fibrils with unique mechanical properties that can alter the mechanical cues a cell receives. In this study, we construct a model that predicts the dynamics of cellular traction force generation and subsequent assembly of fibrils of the ECM protein fibronectin (FN). FN fibrils are the primary component in primordial ECM and, as such, FN assembly is a critical component in the cellular mechanical response. The model consists of a network of Hookean springs, each representing an extensible domain within an assembling FN fibril. As actomyosin forces stretch the spring network, simulations predict the resulting traction force and FN fibril formation. The model accurately predicts FN fibril morphometry and demonstrates a mechanism by which FN fibril assembly regulates traction force dynamics in response to mechanical stimuli and varying surrounding substrate stiffness.

A Small Chimeric Fibronectin Fragment Accelerates Dermal Wound Repair in Diabetic Mice.

Objective: During wound repair, soluble fibronectin is converted into biologically active, insoluble fibrils via a cell-mediated process. This fibrillar, extracellular matrix (ECM) form of fibronectin stimulates cell processes critical to tissue repair. Nonhealing wounds show reduced levels of ECM fibronectin fibrils. The objective of this study was to produce a small, recombinant wound supplement with the biological activity of insoluble fibronectin fibrils. Approach: A chimeric fibronectin fragment was produced by inserting the integrin-binding Arg-Gly-Asp (RGD) loop from the tenth type III repeat of fibronectin (FNIII10) into the analogous site within the heparin-binding, bioactive fragment of the first type III repeat (FNIII1H). FNIII1HRGD was tested for its ability to support cell functions necessary for wound healing, and then evaluated for its capacity to accelerate healing of full-thickness dermal wounds in diabetic mice. Results:In vitro, FNIII1HRGD supported cell adhesion, proliferation, and ECM fibronectin deposition. Application of FNIII1HRGD to dermal wounds of diabetic mice significantly enhanced wound closure compared with controls (73.9% ±4.1% vs. 58.1% ±4.7% closure on day 9, respectively), and significantly increased granulation tissue thickness (2.88 ± 0.75-fold increase over controls on day 14). Innovation: Recombinant proteins designed to functionally mimic the ECM form of fibronectin provide a novel therapeutic approach to circumvent diminished fibronectin fibril formation by delivering ECM fibronectin signals in a soluble form to chronic wounds. Conclusion: A small, chimeric fibronectin protein was developed. FNIII1HRGD demonstrated enhanced bioactivity in vitro and stimulated wound repair in a murine model of chronic wounds.

Role of chemical crosslinking in material-driven assembly of fibronectin (nano)networks: 2D surfaces and 3D scaffolds

Poly(ethyl acrylate) (PEA) induces the formation of biomimetic fibronectin (FN) (nano)networks upon simple adsorption from solutions, a process referred to as material-driven FN fibrillogenesis. The ability of PEA to organize FN has been demonstrated in 2D and 2.5D environments, but not as yet in 3D scaffolds, which incorporate three-dimensionality and chemical crosslinkers that may influence its fibrillogenic potential. In this paper we show for the first time that while three-dimensionality does not interfere with PEA-induced FN fibrillogenesis, crosslinking does, and we determined the maximum amount of crosslinker that can be added to PEA to maintain FN fibrillogenesis. For this, we synthesised 2D substrates with different amounts of crosslinker (1–10% of ethylene glycol dimethacrylate) and studied the role of crosslinking in FN organization using AFM. The glass transition temperature was seen to increase with crosslinking density and, accordingly, polymer segmental mobility was reduced. The organization of FN after adsorption (formation of FN fibrils) and the availability of the FN cell-binding domain were found to be dependent on crosslinking density. Surface mobility was identified as a key parameter for FN supramolecular organization. PEA networks with up to 2% crosslinker organize the FN in a similar way to non-crosslinked PEA. Scaffolds prepared with 2% crosslinker also had FN (nano)networks assembled on their walls, showing PEA’s ability to induce FN fibrillogenesis in 3D environments as long as the amounts of crosslinker is low enough.

Fibronectin (nano)fibrils induced by materials: from 2D to 3D microenvironments

Event Abstract Back to Event Fibronectin (nano)fibrils induced by materials: from 2D to 3D microenvironments Laia León-Boigues1, Antonio Sánchez-Laosa1, Luis Gómez-Estrada2, José Luis Gómez Ribelles1, 3, Manuel Salmerón-Sánchez4, Gloria Gallego Ferrer1, 3 and Roser Sabater I Serra1 1 Universitat Politècnica de València, Center for Biomaterials and Tissue Engineering, Spain 2 Ikasia Technologies SL, Spain 3 Biomedical Research Networking Centre (CIBER), Spain 4 University of Glasgow, Division of Biomedical Engineering, School of Engineering, United Kingdom Introduction: Biomaterials play a key role in regenerative medicine, acting as a synthetic extracellular matrix. However, synthetic biomaterials, biologically inert, have to be functionalised to become bioactive. Adsorption of adhesive proteins, as fibronectin (FN), on the biomaterial surface is one of the strategies commonly used to enhance bioactivity[1],[2]. It have been shown that certain materials, such as poly(ethyl acrylate), PEA, are able to induce the organization of FN in a biomimetic way. A physiological-like FN nanonetwork is organised upon simple adsorption of FN from a protein solution as consequence of protein-material interactions[3]. The novelty of this work is to show the ability of PEA to induce the formation of fibronectin (nano)fibrils in 3D-like environments, as it has been shown only on 2D substrates. For this purpose, PEA scaffolds with interconnected pores larger than 300 µm were synthesised, to be afterwards 3D-coated with FN to promote vascularization and new bone formation[4]. The distribution of the adsorbed FN within the walls of the scaffold surface was studied by AFM. Experimental Methods: PEA scaffolds were obtained by radical polymerization (1 wt% of benzoyl peroxide initiator). 2 wt% ethylene glycol dimethacrylate, EGDMA, was used as crosslinker to improve PEA mechanical properties. Polyvinyl alcohol templates, obtained by rapid-prototyping, were used as porogen. The polymerization was carried out at 60 ºC for 24h. Then, the template was dissolved in water. 2D substrates (films) with 0 and 2 wt% EGDMA were also prepared to be used as 2D controls. Scaffold mechanical properties, morphology and porosity were studied by compression tests, SEM and a liquid displacement method, respectively. The distribution of FN (10 µg/mL PBS solution, 10 min) was investigated by AFM. Results and Discussion: PEA scaffolds show a homogeneous structure, with channels (ca. 350 µm of diameter) in orthogonal directions and distance between channels ca. 400 µm (Fig. 1); the overall porosity is ca. 50%. The elastic modulus is ~ 2.5 MPa. Figure 1. SEM images. Cross-section of PEA scaffolds (2 %wt EGDMA)) AFM images (Fig. 2) show FN distribution after adsorption on 3D substrates (Fig. 2c) and 2D uncrosslinked and crosskinked films (Fig. 2a and 2b respectively). Similar interconnected FN nanofibrils were found on both 2D surfaces, indicating that the process of material-driven FN assembly is unaffected by the crosslinking process. FN fibrils induced in 3D substrates show no significant differences with the ones induced on 2D PEA films. Figure 2. AFM phase (1 μm x 1 μm). a) film (0 wt% EGDMA), b) film (2 wt% EGDMA), c) scaffold (2 wt% EGDMA). Conclusion: We demonstrated that PEA is able to induce the formation of fibronectin (nano)fibrils in 3D environments. Bioactive scaffolds with homogeneous channels and suitable pore size for bone regeneration have been engineered. The support of the project MAT2012-38359-C03-01 (including the FEDER financial support) as well as CIBER is acknowledged. CIBER-BBN is an initiative funded by the VI National R&D&I Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with the assistance from the European Regional Development Fund.

Sulfated hyaluronan affects fibronectin matrix assembly by human bone marrow stromal cells

Event Abstract Back to Event Sulfated hyaluronan affects fibronectin matrix assembly by human bone marrow stromal cells Sarah Vogel1, Stephanie Moeller2, Simon Arnoldini3, Sergey Samsonov4, Matthias Schnabelrauch2, Viola Vogel3 and Ute Hempel1 1 TU Dresden, Institute of Physiological Chemistry, Carl Gustav Carus Faculty of Medicine, Germany 2 Innovent e. V, Biomaterials Department, Germany 3 ETH Zurich, Laboratory of Applied Mechanobiology, Department of Health Science and Technology, Switzerland 4 Tu Dresden, Structural Bioinformatics, Biotec, Germany Introduction: In the bone marrow niche, human bone marrow stromal cells (hBMSC) are surrounded by extracellular matrix (ECM) containing CaP, collagen I, fibronectin (FN) and sulfated glycosaminoglycans (sGAG). Synthetically sulfated hyaluronan (sHA1) has been shown to support osteogenesis of hBMSC[1] and to inhibit osteoclast formation[2]. Osteoblasts synthesize large amounts of FN and deposit the protein into the bone matrix. FN is secreted as a 440 kDa dimeric glycoprotein with binding domains for cells via integrins and several ECM components like collagen I and heparin. The assembly of FN fibrils is a cell-mediated process and depends on integrin binding[3]. In this study we describe the effects of sHA1 on FN matrix assembly by hBMSC in vitro. Materials and Methods: The sulfation of native HA was performed with SO3/DMF and resulted in sHA1with a sulfation degree of 1.3 and a molecular weight of 26,600 g/mol[4]. Fluorescence-labelled HA and sHA1 were obtained with Atto565-NH2 using side-on modification[2]. Human BMSC were cultured in DMEM with 10% FCS supplemented with 200 µg HA or sHA1 / ml[1]. For characterization of FN matrix at day 8, immunofluorescence staining was performed[1]. The influence of HA and sHA1 on FN matrix assembly was characterized using FRET-assay[5]. Results and Discussion: Immunofluorescence staining indicated a colocalization of Atto565-sHA1 and FN fibrils (figure 1C) but no colocalization of Atto565-HA and FN (figure 1B). In silico studies supported the electrostatic interaction between sHA1 and heparin binding sites which might affect growth factor binding and FN fibril formation. Figure 1: Immunofluorescence staining of hBMSC at day 8 (A) and after permanent treatment with Atto565-HA (B) or Atto565-sHA1 (C). FN (green), Nuclei (blue). It was found that sHA1 affected FN matrix assembly as more and thinner FN fibrils were formed in comparison to untreated control and HA (figure 1). The effect of sHA1 on FN matrix assembly was more detailed characterized by FRET-analysis which confirmed FN-stretching in the presence of sHA1 similar to heparin[5]. Besides its effects on FN assembly, sHA1 induced FN protein and influenced its remodeling by reducing activity of matrix metalloproteinases and increasing the formation of tissue inhibitor of metalloproteinase-3 (TIMP-3)[6],[7]. Conclusion: The present study shows one putative mechanism how sHA1 influences ECM assembly and remodelling. Our findings contribute to clarify the impact of sHA1 on molecular mechanisms in hBMSC. Deutsche Forschungsgemeinschaft TRR67 (projects B1, A7, Z3)

Shear-induced fibrillar-like supramolecule of plasma fibronectin: A new form of fibronectin with enhanced activity in platelet adhesion and aggregation

Background: We reported on the formation of fibronectin fibrils (FN fibrils) induced by shear rates.

Changes in human mesenchymal stem cell behaviors on dendrimer-immobilized surfaces due to mediation of fibronectin adsorption and assembly

Dynamic changes of morphologies in human mesenchymal stem cells (hMSCs) were investigated on dendrimer surfaces with different capacities for fibronectin adsorption by changing the polymeric generation numbers of first (G1), third (G3), and fifth (G5) generations. The amount of adsorbed fibronectin on dendrimer surfaces increased with the generation number. Time-lapse observations revealed that cells on the G1 surface maintained their shape with formation of fibronectin fibrils in the bodies, introducing to the stabilization of focal adhesion with enriched stress fibers. Cells on the G3 surface showed partial contraction with degradation of fibril structures in the trailing edge. Cells on the G5 surface changed the shape by active extension and strong contracting without stabilization of focal adhesion through the formation of fibronectin aggregates and immature stress fibers. In addition, the paxillin which is a focal adhesion protein at lamellipodia was phosphorylated, leading to active lamellipodium protrusions. These results indicate that the amount and structure of fibronectin affects dynamic hMSC behaviors through the formation of cytoskeletons and focal adhesions.

Inflammatory cell infiltration in experimental liver fibrosis is affected by inhibition of fibronectin matrix assembly

Background and Aims: The accumulation of inflammatory cells is an important step in liver fibrosis. Fibronectin has been shown to enhance tissue infiltration of various cell types including immune cells. Our aim was to determine how the loss of fibronectin in the liver might affect the inflammatory response to liver injury. In prior work by our group, deletion of fibronectin production by the myofibroblasts resulted in an increase in available TGF-β and hence an increase in fibrotic signals within the liver precluding an examination of the direct effect of fibronectin on inflammation in the response to fibrosis induction. We therefore instead inhibited fibronectin fibril formation pharmacologically and evaluated the various inflammatory cells in a toxic liver injury model.