Confocal Rheology Probes the Structure and Mechanics of Collagen through the Sol-Gel Transition.

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.

Longitudinally Controlled Modification of Cylindrical and Conical Track-Etched Poly(ethylene terephthalate) Pores Using an Electrochemically Assisted Click Reaction.

In this study, the longitudinally controlled modification of the inner surfaces of poly(ethylene terephthalate) (PET) track-etched pores was explored using an electrochemically assisted Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click reaction. Cylindrical or conical PET track-etched pores were first decorated with ethynyl groups via the amidation of surface -COOH groups, filled with a solution containing Cu(II) and azide-tagged fluorescent dye, and then sandwiched between comb-shaped and planar gold electrodes. Cu(I) was produced at the comb-shaped working electrode by the reduction of Cu(II); it diffused along the pores toward the other electrode and catalyzed CuAAC between an azide-tagged fluorescent dye and a pore-tethered ethynyl group. The modification efficiency of cylindrical pores (ca. 1 µm in diameter) was assessed from planar and cross-sectional fluorescence microscope images of modified membranes. Planar images showed that pore modification took place only above the teeth of the comb-shaped electrode with a higher reaction yield for longer Cu(II) reduction times. Cross-sectional images revealed micrometer-scale gradient modification along the pore axis, which reflected a Cu(I) concentration profile within the pores, as supported by finite-element computer simulations. The reported approach was applicable to the asymmetric modification of cylindrical pores with two different fluorescent dyes in the opposite directions and also for the selective visualization of the tip and base openings of conical pores (ca. 3.5 µm in base diameter and ca. 1 µm in tip diameter). The method based on electrochemically assisted CuAAC provides a controlled means to fabricate asymmetrically modified nanoporous membranes and, in the future, will be applicable for chemical separations and the development of sequential catalytic reactors.

Fluorescence Recovery after Photobleaching and Single-Molecule Tracking Measurements of Anisotropic Diffusion within Identical Regions of a Cylinder-Forming Diblock Copolymer Film.

This work demonstrates ensemble and single-molecule diffusion measurements within identical regions of a cylinder-forming polystyrene-poly(ethylene oxide) diblock copolymer (PS-b-PEO) film using fluorescence recovery after photobleaching (FRAP) and single-molecule tracking (SMT). A PS-b-PEO film (∼4 µm thick) with aligned cylindrical PEO microdomains containing 10 µM sulforhodamine B (SRB) was prepared by directional solvent-vapor penetration (SVP) of 1,4-dioxane. The ensemble diffusion behavior of SRB in the microdomains was assessed in FRAP studies of circular photobleached regions (∼7 µm in diameter). The SRB concentration was subsequently reduced by additional photobleaching, and the diffusion of individual SRB molecules was explored using SMT in the identical area (∼16 × 16 µm(2)). The FRAP data showed anisotropic fluorescence recovery, yielding the average microdomain orientation. The extent of fluorescence recovery observed (∼90%) demonstrated long-range microdomain connectivity, while the recovery time dependence provided an ensemble measurement of the SRB diffusion coefficient within the cylindrical microdomains. The SMT data exhibited one-dimensional diffusion of individual SRB molecules along the SVP direction across the entire film thickness, as consistent with the FRAP results. Importantly, the average of the single-molecule diffusion coefficients was close to the value obtained from FRAP in the identical area. In some cases, SMT offered smaller diffusion coefficients than FRAP, possibly due to contributions from SRB molecules confined within short PEO microdomains. The implementation of FRAP and SMT measurements in identical areas provides complementary information on molecular diffusion with minimal influence of sample heterogeneity, permitting direct comparison of ensemble and single-molecule diffusion behavior.

Trajectory-profile-guided single molecule tracking for assignment of one-dimensional diffusion trajectories.

A variety of algorithms exist for optical single molecule tracking in two and three dimensions. One general class of algorithms employs cost-functionals to link the individual fluorescent spots, produced by a molecule in sequential video frames, into trajectories. This method has also been used to track one-dimensional (1D) molecular motions for relatively low diffusion rates (i.e., D < 1 µm(2)/s). At high diffusion rates, the cost-functional approach often fails to accurately reproduce 1D trajectories, particularly when the molecules are closely spaced. In this paper, we present a new algorithm called trajectory-profile-guided (TPG) tracking that is designed specifically for 1D trajectories. TPG tracking involves an initial search for one-dimensionally aligned fluorescent spots (i.e., candidate molecules). Qualifying candidates are subsequently identified and linked into trajectories based on several criteria. We test the TPG algorithm's accuracy and precision against cost-functional based tracking using both simulated and experimental video data. The results show that TPG tracking more accurately reproduces the actual 1D trajectories, particularly at higher diffusion rates. TPG tracking is also shown to produce longer trajectories and more accurate estimates of trajectory aspect ratios (i.e., their dimensionality), molecular diffusion coefficients, and order parameters for aligned 1D trajectories over a wide range of diffusion coefficients.

Single-Particle Tracking in Poly(Ethylene Glycol) Diacrylate: Probe Size Effect on the Diffusion Behaviors of Nanoparticles in Unentangled Polymer Solutions.

A thorough understanding of the relevant factors governing the transport of nanoparticles in poly(ethylene glycol) diacrylate (PEGDA) is crucial for many applications utilizing this polymer. Here, single-particle tracking (SPT) was used to systematically investigate the role of the probe size (3-200 nm) on the diffusion behaviors of individual fluorescent nanoparticles in semidilute and unentangled PEGDA solutions. The quantitative assessment of the SPT data via the recorded single-particle trajectories and diffusion coefficients (D) not only showed that the observed probe dynamics in PEGDA were temporally and spatially heterogeneous, but more importantly that the measured D were observed to be significantly reduced (vs in solvent) and strongly size-dependent. We explained these results based on a modified multiscale model for particle diffusion, built upon well-established hydrodynamics and obstruction theories. We furthermore showed that the presence of steric interactions and probe confinement effects in highly crowded, unentangled PEGDA microstructures can lead to deviations in the single-particle displacements from the expected Gaussian behavior, as revealed by the van Hove displacement distributions and the associated non-Gaussian parameters. This study has demonstrated the power of SPT methods in offering an advanced characterization of the transport characteristics in complex polymer structures, overcoming challenges posed by traditional characterization techniques.

Alkaline activation of endogenous latent TGFß1 by an injectable hydrogel directs cell homing for in situ complex tissue regeneration.

Utilization of the body's regenerative potential for tissue repair is known as in situ tissue regeneration. However, the use of exogenous growth factors requires delicate control of the dose and delivery strategies and may be accompanied by safety, efficacy and cost concerns. In this study, we developed, for the first time, a biomaterial-based strategy to activate endogenous transforming growth factor beta 1 (TGFß1) under alkaline conditions for effective in situ tissue regeneration. We demonstrated that alkaline-activated TGFß1 from blood serum, bone marrow fluids and soaking solutions of meniscus and tooth dentin was capable of increasing cell recruitment and early differentiation, implying its broad practicability. Furthermore, we engineered an injectable hydrogel (MS-Gel) consisting of gelatin microspheres for loading strong alkaline substances and a modified gelatin matrix for hydrogel click crosslinking. In vitro models showed that alkaline MS-Gel controllably and sustainably activated endogenous TGFß1 from tooth dentin for robust bone marrow stem cell migration. More importantly, infusion of in vivo porcine prepared root canals with alkaline MS-Gel promoted significant pulp-dentin regeneration with neurovascular stroma and mineralized tissue by endogenous proliferative cells. Therefore, this work offers a new bench-to-beside translation strategy using biomaterial-activated endogenous biomolecules to achieve in situ tissue regeneration without the need for cell or protein delivery.

Single-Molecule Tracking in Poly(Ethylene Oxide) Films: Revealing the Effects of Molecular Weight, Network Plasticization, and Thermal Annealing on Anionic Dye Diffusion.

This paper reports single-molecule tracking (SMT) measurements of the diffusion behaviors of individual, anionic sulforhodamine B (SRB) dye molecules in a series of poly(ethylene oxide) (PEO) films, aimed at clarifying the influences of the molecular weight, network plasticization, and thermal annealing on such dynamics. Micrometer-thick PEO films were prepared by drop-casting from its aqueous (0.2%, 1 nM SRB) solution, followed by drying in air and thermal annealing at 90 °C for 36 h. The diffusion of individual SRB occurring within the amorphous domains was recorded at different relative humidities (5-95%) to characterize the microscale domains' local aspect-ratio, orientation, and molecular permeability at high spatial resolution. The results revealed the involvement of crystalline phases in confining SRB diffusion to submicron distances and guiding longer-range diffusion along one-dimensional-like amorphous morphologies. Upon annealing, amorphous domains were wider, more continuous, and more permeable to SRB probes. The enhanced transport in plasticized PEO, as reflected by the higher SRB mobility and diffusivity, was linked to the polymer's higher chain and segmental mobilities and reduced hydrogen-bonding interactions. This work has demonstrated the usefulness of SMT for an advanced characterization of solid polymer electrolytic films, highly beneficial for the development of safer lithium-ion batteries.

A biomimetic gelatin-based platform elicits a pro-differentiation effect on podocytes through mechanotransduction.

Using a gelatin microbial transglutaminase (gelatin-mTG) cell culture platform tuned to exhibit stiffness spanning that of healthy and diseased glomeruli, we demonstrate that kidney podocytes show marked stiffness sensitivity. Podocyte-specific markers that are critical in the formation of the renal filtration barrier are found to be regulated in association with stiffness-mediated cellular behaviors. While podocytes typically de-differentiate in culture and show diminished physiological function in nephropathies characterized by altered tissue stiffness, we show that gelatin-mTG substrates with Young's modulus near that of healthy glomeruli elicit a pro-differentiation and maturation response in podocytes better than substrates either softer or stiffer. The pro-differentiation phenotype is characterized by upregulation of gene and protein expression associated with podocyte function, which is observed for podocytes cultured on gelatin-mTG gels of physiological stiffness independent of extracellular matrix coating type and density. Signaling pathways involved in stiffness-mediated podocyte behaviors are identified, revealing the interdependence of podocyte mechanotransduction and maintenance of their physiological function. This study also highlights the utility of the gelatin-mTG platform as an in vitro system with tunable stiffness over a range relevant for recapitulating mechanical properties of soft tissues, suggesting its potential impact on a wide range of research in cellular biophysics.

Single-Molecule Tracking Study of the Permeability and Transverse Width of Individual Cylindrical Microdomains in Solvent-Swollen Polystyrene-block-poly(ethylene oxide) Films.

Understanding the properties of solvent-swollen block copolymer (BCP) microdomains is important for better solvent-based control of microdomain morphology, orientation, and permeability. In this study, single-molecule tracking (SMT) was explored to assess the permeability and transverse width of individual cylindrical microdomains in solvent-swollen polystyrene-block-poly(ethylene oxide) (PS-b-PEO) films. PS-b-PEO films comprising shear-elongated cylindrical PEO microdomains were prepared by sandwiching its benzene or tetrahydrofuran (THF) solution between two glass substrates. SMT measurements were performed at different drying times to investigate the effects of solvent evaporation on the microdomain properties. SMT data showed one-dimensional (1D) motions of single fluorescent molecules (sulforhodamine B) based on their diffusion within the cylindrical microdomains. Microdomain permeability and transverse width were assessed from the single-molecule diffusion coefficients (DSMT) and transverse variance of the 1D trajectories (σδ2), respectively. The DSMT and σδ2 values from individual 1D trajectories were widely distributed with no evidence of correlation on a single molecule basis, possibly because the individual microdomains in a film were swollen to different extents. On average, microdomain permeability (D) and effective radius (r) gradually decreased within the first 3 days of drying due to solvent evaporation, and changed negligibly thereafter. PS-b-PEO films prepared from THF solutions exhibited larger changes in D and r as compared with those from benzene solutions due to the better swelling of the PEO microdomains by THF. Importantly, changes in D were more prominent than those in r, suggesting that the permeability of the PEO microdomains is very susceptible to the presence of solvent. These results reveal the unique capability of SMT to assess the properties of individual cylindrical microdomains in a solvent-swollen BCP film.

Single-Molecule Investigations of Morphology and Mass Transport Dynamics in Nanostructured Materials.

Nanostructured materials such as mesoporous metal oxides and phase-separated block copolymers form the basis for new monolith, membrane, and thin film technologies having applications in energy storage, chemical catalysis, and separations. Mass transport plays an integral role in governing the application-specific performance characteristics of many such materials. The majority of methods employed in their characterization provide only ensemble data, often masking the nanoscale, molecular-level details of materials morphology and mass transport. Single-molecule fluorescence methods offer direct routes to probing these characteristics on a single-molecule/single-nanostructure basis. This article provides a review of single-molecule studies focused on measurements of anisotropic diffusion, adsorption, partitioning, and confinement in nanostructured materials. Experimental methods covered include confocal and wide-field fluorescence microscopy. The results obtained promise to deepen our understanding of mass transport mechanisms in nanostructures, thus aiding in the realization of advanced materials systems.

Single-molecule tracking studies of flow-induced microdomain alignment in cylinder-forming polystyrene-poly(ethylene oxide) diblock copolymer films.

Flow-based approaches are promising routes to preparation of aligned block copolymer microdomains within confined spaces. An in-depth characterization of such nanoscale morphologies within macroscopically nonuniform materials under ambient conditions is, however, often challenging. In this study, single-molecule tracking (SMT) methods were employed to probe the flow-induced alignment of cylindrical microdomains (ca. 22 nm in diameter) in polystyrene-poly(ethylene oxide) diblock copolymer (PS-b-PEO) films. Films of micrometer-scale thicknesses were prepared by overlaying a benzene solution droplet on a glass coverslip with a rectangular glass plate, followed by solvent evaporation under a nitrogen atmosphere. The microdomain alignment was quantitatively assessed from SMT data exhibiting the diffusional motions of individual sulforhodamine B fluorescent probes that preferentially partitioned into cylindrical PEO microdomains. Better overall microdomain orientation along the flow direction was observed near the substrate interface in films prepared at a higher flow rate, suggesting that the microdomain alignment was primarily induced by shear flow. The SMT data also revealed the presence of micrometer-scale grains consisting of highly ordered microdomains with coherent orientation. The results of this study provide insights into shear-based preparation of aligned cylindrical microdomains in block copolymer films from solutions within confined spaces.