Regional Fibronectin and Collagen Fibril Co-Assembly Directs Cell Proliferation and Microtissue Morphology

Sevilla, Carlos A.; Dalecki, Diane; Hocking, Denise C.

doi:10.1371/journal.pone.0077316

Cited by 35 publications

(43 citation statements)

References 46 publications

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“…The growth-promoting site in FNIII1 is not exposed in soluble fibronectin, but becomes unmasked when cells exert force on polymerized fibronectin fibrils (Gui et al, 2006; Hocking et al, 2008; Zhong et al, 1998). Cell-mediated extension of fibronectin fibrils has recently been correlated with induction of osteogenic differentiation of mesenchymal stem cells (Li et al) and cell proliferation in response to ECM fibronectin (Sevilla et al, 2013). Hence, exposure of the FNIII1 site in ECM fibronectin may be reduced on vitronectin-adherent cells compared to collagen-adherent cells.…”

Section: Resultsmentioning

confidence: 99%

Opposing effects of collagen I and vitronectin on fibronectin fibril structure and function

et al. 2014

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Extracellular matrix fibronectin fibrils serve as passive structural supports for the organization of cells into tissues, yet can also actively stimulate a variety of cell and tissue functions, including cell proliferation. Factors that control and coordinate the functional activities of fibronectin fibrils are not known. Here, we compared effects of cell adhesion to vitronectin versus type I collagen on the assembly of and response to, extracellular matrix fibronectin fibrils. The amount of insoluble fibronectin matrix fibrils assembled by fibronectin-null mouse embryonic fibroblasts adherent to collagen- or vitronectin-coated substrates was not significantly different 20 h after fibronectin addition. However, the fibronectin matrix produced by vitronectin-adherent cells was ~10-fold less effective at enhancing cell proliferation than that of collagen-adherent cells. Increasing insoluble fibronectin levels with the fibronectin fragment, anastellin did not increase cell proliferation. Rather, native fibronectin fibrils polymerized by collagen- and vitronectin-adherent cells exhibited conformational differences in the growth-promoting, III-1 region of fibronectin, with collagen-adherent cells producing fibronectin fibrils in a more extended conformation. Fibronectin matrix assembly on either substrate was mediated by α5β1 integrins. However, on vitronectin-adherent cells, α5β1 integrins functioned in a lower activation state, characterized by reduced 9EG7 binding and decreased talin association. The inhibitory effect of vitronectin on fibronectin-mediated cell proliferation was localized to the cell-binding domain, but was not a general property of αvβ3 integrin-binding substrates. These data suggest that adhesion to vitronectin allows for the uncoupling of fibronectin fibril formation from downstream signaling events by reducing α5β1 integrin activation and fibronectin fibril extension.

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Section: Resultsmentioning

confidence: 99%

Opposing effects of collagen I and vitronectin on fibronectin fibril structure and function

et al. 2014

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“…To further comprehend the extracellular matrix organization, we analysed the distribution of Fibronectin and Collagen, whose interaction is necessary for ECM architecture and functionality (Sevilla et al 2013). As shown in Fig.…”

Section: Morphological Features Assessmentmentioning

confidence: 99%

Long term morphological characterization of mesenchymal stromal cells 3D spheroids built with a rapid method based on entry-level equipment

et al. 2016

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Three-dimensional (3D) spheroids of mesenchymal stromal cells (MSC) have been demonstrated to improve a wide range of MSC features, such as multilineage potential, secretion of therapeutic factors, and resistance against hypoxic condition. Accordingly, they represent a promising tool in regenerative medicine for several biological and clinical applications. Many approaches have been proposed to generate MSC spheroids. They usually require specific generation systems, such as rotatory bioreactors or low-attachment plates, and each approach has its own disadvantages. Furthermore, an over-time analysis of morphological homogeneity and architectural stability of the spheroids generated is rarely provided. In this work we adapted the ''pellet culture'' method to obtain homogenous spheroids of MSC and maintain them in vitro for long term studies. We analysed their outer and inner structure over a 2-month period to provide morphological and architectural information regarding the spheroids generated. Quantitative and qualitative data were obtained using brightfield and confocal microscope imaging coupled to a computational analysis to estimate volume, sphericity, and jagging degree. In addition, histological evaluation was performed to more thoroughly assess the cellular composition and the internal architecture of the 3D spheroids. The results provided show that MSC spheroids generated with the proposed approach are homogeneous and stable, from both morphological and architectural points of view, for a period of at least 15 days, approximately between day 15 and day 30 after their generation. Accordingly, the approach proposed serves as a rapid, cost-effective, and efficient method to generate and maintain MSC spheroids using common entry-level laboratory equipment only.Keywords Mesenchymal stromal/stem cells Á 3D multicellular spheroids Á Pellet culture method Á Morphological analysis Á Microscopy Á Computational analysis Chiara Bellotti and Serena Duchi contributed equally to this work.

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“…How different structural or conformational forms of fibrillar fibronectin influence cell and tissue function is only beginning to be understood. Recent studies provide evidence that relaxed fibronectin fibrils are associated with reduced α5β1 integrin activation [28], decreased cell proliferation [28], and increased cell cohesion [29]. In contrast, stretched/unfolded fibronectin fibrils are associated with enhanced cell proliferation [28] and migration [30], decreased adhesion [31], and enhanced growth factor secretion [31].…”

Section: Introductionmentioning

confidence: 99%

Cooperative effects of fibronectin matrix assembly and initial cell–substrate adhesion strength in cellular self-assembly

Brennan

Hocking

2016

Acta Biomaterialia

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The cell-dependent polymerization of intercellular fibronectin fibrils can stimulate cells to self-assemble into multicellular structures. The local physical cues that support fibronectin-mediated cellular self-assembly are largely unknown. Here, fibronectin matrix analogs were used as synthetic adhesive substrates to model cell-matrix fibronectin fibrils having different integrin-binding specificity, affinity, and/or density. We utilized this model to quantitatively assess the relationship between adhesive forces derived from cell-substrate interactions and the ability of fibronectin fibril assembly to induce cellular self-assembly. Results indicate that the strength of initial, rather than mature, cell-substrate attachments correlates with the ability of substrates to support fibronectin-mediated cellular self-assembly. The cellular response to soluble fibronectin was bimodal and independent of the integrin-binding specificity of the substrate; increasing soluble fibronectin levels above a critical threshold increased aggregate cohesion on permissive substrates. Once aggregates formed, continuous fibronectin polymerization was necessary to maintain cohesion. During self-assembly, soluble fibronectin decreased cell-substrate adhesion strength and induced aggregate cohesion via a Rho-dependent mechanism, suggesting that the balance of contractile forces derived from fibronectin fibrils within cell-cell versus cell-substrate adhesions controls self-assembly and aggregate cohesion. Thus, initial cell-substrate attachment strength may provide a quantitative basis with which to build predictive models of fibronectin-mediated microtissue fabrication on a variety of substrates.

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Regional Fibronectin and Collagen Fibril Co-Assembly Directs Cell Proliferation and Microtissue Morphology

Cited by 35 publications

References 46 publications

Opposing effects of collagen I and vitronectin on fibronectin fibril structure and function

Opposing effects of collagen I and vitronectin on fibronectin fibril structure and function

Long term morphological characterization of mesenchymal stromal cells 3D spheroids built with a rapid method based on entry-level equipment

Cooperative effects of fibronectin matrix assembly and initial cell–substrate adhesion strength in cellular self-assembly

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