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Dynamic imaging of fibrin network formation correlated with other measures of polymerization.

ABSTRACT: Using deconvolution microscopy, we visualized in real time fibrin network formation in the hydrated state. Individual mobile fibers were observed before the gel point determined by eye. After gelation, an initial fibrin network was seen, which evolved over time by addition of new fibers and elongation and branching of others. Furthermore, some fibers in the network moved for a time. We quantified network formation by number of branch points, and longitudinal and lateral growth of fibers. Eighty percent of branch points were formed, and 70% of all fibers reached their maximum length at the gel point. In contrast, at the gel point, fiber diameter, measured as fluorescence intensity, was less than 25% and turbidity was less than 15% of the maximum values of the fully formed clot. The cumulative percentage of fibers reaching their final length and the number of branch points attained maximum values at 60% of maximum turbidity. Lateral fiber growth reached a plateau at the same time as turbidity. Measurements of clot mechanical properties revealed that the clots achieved maximum stiffness and minimum plasticity after the structural parameters reached their maxima. These results provide new information on the relative time sequence of events during fibrin network formation.

SUBMITTER: Chernysh IN

PROVIDER: S-EPMC2384121 | biostudies-literature | 2008 May

REPOSITORIES: biostudies-literature

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Dynamic imaging of fibrin network formation correlated with other measures of polymerization.

Chernysh Irina N IN Weisel John W JW

Blood 20080213 10

Using deconvolution microscopy, we visualized in real time fibrin network formation in the hydrated state. Individual mobile fibers were observed before the gel point determined by eye. After gelation, an initial fibrin network was seen, which evolved over time by addition of new fibers and elongation and branching of others. Furthermore, some fibers in the network moved for a time. We quantified network formation by number of branch points, and longitudinal and lateral growth of fibers. Eighty ...[more]

PMID: 18272815

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Project description:PurposeWe previously reported that fibroblasts migrating within 3-D collagen matrices move independently, whereas fibroblasts within 3-D fibrin matrices form an interconnected network. Similar networks have been identified previously during in vivo corneal wound healing. In this study, we investigate the role of fibronectin in mediating this mechanism of collective cell spreading, migration and patterning.MethodsTo assess cell spreading, corneal fibroblasts were plated within fibrillar collagen or fibrin matrices. To assess migration, compacted cell-populated collagen matrices were nested inside cell-free fibrin matrices. Constructs were cultured in serum-free media containing PDGF, with or without RGD peptide, anti-α5 or anti-fibronectin blocking antibodies. In some experiments, LifeAct and fluorescent fibronectin were used to allow dynamic assessment of cell-induced fibronectin reorganization. 3-D and 4-D imaging were used to assess cell mechanical behavior, connectivity, F-actin, α5 integrin and fibronectin organization.ResultsCorneal fibroblasts within 3-D fibrin matrices formed an interconnected network that was lined with cell-secreted fibronectin. Live cell imaging demonstrated that fibronectin tracks were formed at the leading edge of spreading and migrating cells. Furthermore, fibroblasts preferentially migrated through fibronectin tracks laid down by other cells. Interfering with cell-fibronectin binding with RGD, anti α5 integrin or anti fibronectin antibodies inhibited cell spreading and migration through fibrin, but did not affect cell behavior in collagen.ConclusionsIn this study, a novel mode of cell patterning was identified in which corneal fibroblasts secrete and attach to fibronectin via α5β1 integrin to facilitate spreading and migration within 3-D fibrin matrices, resulting in the formation of localized fibronectin tracks. Other cells use these fibronectin tracks as conduits, resulting in an interconnected cell-fibronectin network.

Project description:Hydrogels are effective platforms for use as artificial extracellular matrices, cell carriers, and to present bioactive cues. Two common natural polymers, fibrin and alginate, are broadly used to form hydrogels and have numerous advantages over synthetic materials. Fibrin is a provisional matrix containing native adhesion motifs for cell engagement, yet the interplay between mechanical properties, degradation, and gelation rate is difficult to decouple. Conversely, alginate is highly tunable yet bioinert and requires modification to present necessary adhesion ligands. To address these challenges, we developed a fibrin-alginate interpenetrating network (IPN) hydrogel to combine the desirable adhesion and stimulatory characteristics of fibrin with the tunable mechanical properties of alginate. We tested its efficacy by examining capillary network formation with entrapped co-cultures of mesenchymal stromal cells (MSCs) and endothelial cells (ECs). We manipulated thrombin concentration and alginate crosslinking density independently to modulate the fibrin structure, mesh size, degradation, and biomechanical properties of these constructs. In IPNs of lower stiffness, we observed a significant increase in total cell area (1.7 × 105 ± 7.9 × 104 µm2) and decrease in circularity (0.56 ± 0.03) compared to cells encapsulated in stiffer IPNs (4.0 × 104 ± 1.5 × 104 µm2 and 0.77 ± 0.09, respectively). Fibrinogen content did not influence capillary network formation. However, higher fibrinogen content led to greater retention of these networks confirmed via increased spreading and presence of F-actin at 7 days. This is an elegant platform to decouple cell adhesion and hydrogel bulk stiffness that will be broadly useful for cell instruction and delivery. STATEMENT OF SIGNIFICANCE: Hydrogels are widely used as drug and cell delivery vehicles and as artificial extracellular matrices to study cellular responses. However, there are limited opportunities to simultaneously control mechanical properties and degradation while mimicking the complex native adhesion motifs and ligands known to encourage cell engagement with the hydrogel. In this study, we describe a fibrin-alginate interpenetrating network (IPN) hydrogel designed to balance the compliance and provisional qualities of fibrin with the mechanical stability and tunability of alginate to interrogate these contributions on cell response. We used clinically relevant cell sources, a co-culture of endothelial cells and mesenchymal stromal cells, to test its efficacy in supporting capillary formation in vitro. These data demonstrate the promise of this IPN for use in tissue engineering.

Project description:A modular tissue engineering approach may have advantages over current therapies in providing rapid and sustained revascularization of ischemic tissue. In this study, modular protein microbeads were prepared from pure fibrin (FIB) and collagen-fibrin composites (COL-FIB) using a simple water-in-oil emulsification technique. Human endothelial cells and fibroblasts were embedded directly in the microbead matrix. The resulting microbeads were generally spheroidal with a diameter of 100-200?m. Cell viability was high (75-80% viable) in microbeads, but was marginally lower than in bulk hydrogels of corresponding composition (85-90% viable). Cell proliferation was significantly greater in COL-FIB microbeads after two weeks in culture, compared to pure FIB microbeads. Upon embedding of microbeads in a surrounding fibrin hydrogel, endothelial cell networks formed inside the microbead matrix and extended into the surrounding matrix. The number of vessel segments, average segment length, and number of branch points was higher in FIB samples, compared to COL-FIB samples, resulting in significantly longer total vessel networks. Anastomosis of vessel networks from adjacent microbeads was also observed. These studies demonstrate that primitive vessel networks can be formed by modular protein microbeads containing embedded endothelial cells and fibroblasts. Such microbeads may find utility as prevascularized tissue modules that can be delivered minimally invasively as a therapy to restore blood flow to ischemic tissues.Vascularization is critically important for tissue engineering and regenerative medicine, and materials that support and/or promote neovascularization are of value both for translational applications and for mechanistic studies and discovery-based research. Therefore, we fabricated small modular microbeads formulated from pure fibrin (FIB) and collagen-fibrin (COL-FIB) containing endothelial cells and supportive fibroblasts. We explored how cells encapsulated within these materials form microvessel-like networks both within and outside of the microbeads when embedded in larger 3D matrices. FIB microbeads were found to initiate more extensive sprouting into the surrounding ECM in vitro. These results represent an important step towards our goal of developing injectable biomaterial modules containing preformed vascular units that can rapidly restore vascularization to an ischemic tissue in vivo.

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Dynamic imaging of fibrin network formation correlated with other measures of polymerization.

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Dynamic imaging of fibrin network formation correlated with other measures of polymerization.

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