Type I collagen gels are routinely used in biophysical studies and bioengineering applications. The structural and mechanical properties of these fibrillar matrices depend on the conditions under which collagen fibrillogenesis proceeds, and developing a fuller understanding of this process will enhance control over gel properties. Turbidity measurements have long been the method of choice for monitoring developing gels, whereas imaging methods are regularly used to visualize fully developed gels. In this study, turbidity and confocal reflectance microscopy (CRM) were simultaneously employed to track collagen fibrillogenesis and reconcile the information reported by the two techniques, with confocal fluorescence microscopy (CFM) used to supplement information about early events in fibrillogenesis. Time-lapse images of 0.5 mg/ml, 1.0 mg/ml, and 2.0 mg/ml acid-solubilized collagen I gels forming at 27°C, 32°C, and 37°C were collected. It was found that in situ turbidity measured in a scanning transmittance configuration was interchangeable with traditional turbidity measurements using a spectrophotometer. CRM and CFM were employed to reveal the structures responsible for the turbidity that develops during collagen self-assembly. Information from CRM and transmittance images was collapsed into straightforward single variables; total intensity in CRM images tracked turbidity development closely for all collagen gels investigated, and the two techniques were similarly sensitive to fibril number and dimension. Complementary CRM, CFM, and in situ turbidity measurements revealed that fibril and network formation occurred before substantial turbidity was present, and the majority of increasing turbidity during collagen self-assembly was due to increasing fibril thickness.

Rationale: Sepsis and acute lung injury (ALI) have devastatingly high mortality rates. Both are associated with increased vascular leak, a process regulated by complex molecular mechanisms. Objectives: We hypothesized that integrin avb3 could be an important determinant of vascular leak and endothelial permeability in sepsis and ALI. Methods: b3 subunit knockout mice were tested for lung vascular leak after endotracheal LPS, and systemic vascular leak and mortality after intraperitoneal LPS and cecal ligation and puncture. Possible contributory effects of b3 deficiency in platelets and other hematopoietic cells were excluded by bone marrow reconstitution experiments. Endothelial cells treated with avb3 antibodies were evaluated for sphingosine-1 phosphate (S1P)-mediated alterations in barrier function, cytoskeletal arrangement, and integrin localization. Measurements and Main Results: b3 knockout mice had increased vascular leak and pulmonary edema formation after endotracheal LPS, and increased vascular leak and mortality after intraperitoneal LPS and cecal ligation and puncture. In endothelial cells, avb3 antibodies inhibited barrier-enhancing and cortical actin responses to S1P. Furthermore, S1P induced translocation of avb3 from discrete focal adhesions to cortically distributed sites through Gi-and Rac1-mediated pathways. Cortical avb3 localization after S1P was decreased by avb3 antibodies, suggesting that ligation of the avb3 with its extracellular matrix ligands is required to stabilize cortical avb3 focal adhesions. Conclusions: Our studies identify a novel mechanism by which avb3 mitigates increased vascular leak, a pathophysiologic function central to sepsis and ALI. These studies suggest that drugs designed to block avb3 may have the unexpected side effect of intensifying sepsis-and ALI-associated vascular endothelial leak.Keywords: vascular endothelium; sepsis; acute lung injury; integrin Sepsis and acute lung injury (ALI) are associated with high mortality rates and worldwide healthcare burden (1-4). Development of these syndromes requires complex host responses involving multiple cell types, inflammatory mediators, and coagulation factors. One pathophysiologic hallmark common to both sepsis and ALI is increased vascular leak. Increased vascular leak in sepsis leads to redistribution of intravascular fluid to extravascular compartments, hypovolemia, hemoconcentration, and stasis of blood flow. In ALI, alveolar spaces become flooded with pulmonary edema, resulting in impaired gas exchange, arterial hypoxemia, and respiratory failure (4-6).It is generally believed that increased paracellular passage of solutes through the vascular endothelium occurs during acute inflammatory states (7,8). Frequently cited models suggest that paracellular gaps form because of disrupted homeostasis between cytoskeletal, adhesive cell-cell, and cell-matrix forces (8-10). Integrins, a large family of heterodimeric glycoprotein receptors, are important mediators of these cellular functions and have been shown to participa...

To mimic the extracellular matrix surrounding high grade gliomas, composite matrices composed of either acid-solubilized (AS) or pepsin-treated (PT) collagen and the glycosaminoglycans chondroitin sulfate (CS) and hyaluronic acid (HA) are prepared and characterized. The structure and mechanical properties of collagen/CS and collagen/HA gels are studied via confocal reflectance microscopy (CRM) and rheology. CRM reveals that CS induces fibril bundling and increased mesh size in AS collagen but not PT collagen networks. The presence of CS also induces more substantial changes in the storage and loss moduli of AS gels than of PT gels, in accordance with expectation based on network structural parameters. The presence of HA significantly reduces mesh size in AS collagen but has a smaller effect on PT collagen networks. However, both AS and PT collagen network viscoelasticity is strongly affected by the presence of HA. The effects of CS and HA on glioma invasion is then studied in collagen/GAG matrices with network structure both similar to (PT collagen-based gels) and disparate from (AS collagen-based gels) those of the corresponding pure collagen matrices. It is shown that CS inhibits and HA has no significant effect on glioma invasion in 1.0 mg/ml collagen matrices over 3 days. The inhibitory effect of CS on glioma invasion is more apparent in AS than in PT collagen gels, suggesting invasive behavior in these environments is affected by both biochemical and network morphological changes induced by GAGs. This study is among the few efforts to differentiate structural, mechanical and biochemical effects of changes to matrix composition on cell motility in 3D.

No abstract

Fibrillar type I collagen-based hydrogels are commonly used in tissue engineering and as matrices for biophysical studies. Mechanical and structural properties of these gels are known to be governed by the conditions under which fibrillogenesis occurs, exhibiting variation as a function of protein concentration, temperature, pH, and ionic strength. Deeper understanding of how macroscopic structure affects viscoelastic properties of collagen gels over the course of fibrillogenesis provides fundamental insight into biopolymer gel properties and promises enhanced control over the properties of such gels. Here, we investigate type I collagen fibrillogenesis using confocal rheology-simultaneous confocal reflectance microscopy, confocal fluorescence microscopy, and rheology. The multimodal approach allows direct comparison of how viscoelastic properties track the structural evolution of the gel on fiber and network length scales. Quantitative assessment and comparison of each imaging modality and the simultaneously collected rheological measurements show that the presence of a system-spanning structure occurs at a time similar to rheological determinants of gelation. Although this and some rheological measures are consistent with critical gelation through percolation, additional rheological and structural properties of the gel are found to be inconsistent with this theory. This study clarifies how structure sets viscoelasticity during collagen fibrillogenesis and more broadly highlights the utility of multimodal measurements as critical test-beds for theoretical descriptions of complex systems.

The gap between graduate research and introductory undergraduate teaching laboratories is often wide, but the development of teaching activities rooted within the research environment offers an opportunity for undergraduate students to have first-hand experience with research currently being conducted and for graduate students to develop communication and pedagogical skills. We describe the development, evaluation, and redesign of a Microsoft Excel-based activity where data analysis from contemporary research was adapted into a data analysis training exercise, requiring undergraduate students to engage with the data by performing a series of analytical tasks guided by commentary from the researcher. In addition to learning common data analysis techniques, this exercise provided an opportunity for undergraduate students to practice problem solving and collaboratively work through trouble-shooting. Throughout the activity development, multiple iterations allowed the authors to explore the impact of different pedagogical approaches including, but not limited to, student-prepared questions for the graduate student author that were used as a starting point for an in-class discussion of the research findings, techniques, considerations, and future goals. This conversation between general chemistry students and the graduate student researcher was coupled with an in-person laboratory tour or video introduction by the researcher. These activities humanized the research enterprise for the undergraduate students and serve as a model for future collaborations between research and instructional laboratories.

High-damping acoustic composites with unique sound insulation feature is reported, demonstrating surprisingly stable soundproof properties over a wide bandwidth of frequency (63-6300 Hz) with good mechanical properties. The extraordinary acoustic properties are attributed to the multiscale synergy of lamellar and hollow structures by multifillers adding. The acoustic composites are designed and fabricated through a low-temperature one-time rubber mixing process using nitrile butadiene rubber(NBR), chloroprene rubber (CR) as matrix and mica powder (MP), hollow glass beads (HGB), and montmorillonite (MMT) as multifillers. The soundproof mechanism is discussed in detail. Results indicate that the synergism of damping and sound absorption behaviors in the composition has a considerable impact on the acoustic characteristics, which can be controlled by the blending ratio of NBR/CR matrixes and the multiscale structures of MP/HGB/MMT multifillers. The designed multifillers acoustic composites can adapt to broadband engineering noise control including walls in express train constructions, panels of vessel or aircraft cabins, and large transportation pipelines.

No abstract