Probing Flow-Induced Biomolecular Interactions With Micro-Extensional Rheology: Tau Protein Aggregation.

Biomolecules in solutions subjected to extensional strain can form aggregates, which may be important for our understanding of pathologies involving insoluble protein structures where mechanical forces are thought to be causative (e.g., tau fibers in chronic traumatic encephalopathy (CTE)). To examine the behavior of biomolecules in solution under mechanical strains requires applying rheological methods, often to very small sample volumes. There were two primary objectives in this investigation: (1) To probe flow-induced aggregation of proteins in microliter-sized samples and (2) To test the hypothesis that tau protein aggregates under extensional flow. Tau protein (isoform:3R 0 N; 36.7 kDa) was divided into 10 µl droplets and subjected to extensional strain in a modified tensiometer. Sixteen independent tests were performed where one test on a single droplet comprised three extensional events. To assess the rheological performance of the fluid/tau mixture, the diameter of the filament that formed during extension was tracked as function of time and analyzed for signs of aggregation (i.e., increased relaxation time). The results were compared to two molecules of similar and greater size (Polyethylene Oxide: PEO35, 35 kDa and PEO100, 100 kDa). Analysis showed that the tau protein solution and PEO35 are likely to have formed aggregates, albeit at relatively high extensional strain rates (∼10 kHz). The investigation demonstrates an extensional rheological method capable of determining the properties of protein solutions in µl volumes and that tau protein can aggregate when exposed to a single extensional strain with potentially significant biological implications.

Simulated remodeling of loaded collagen networks via strain-dependent enzymatic degradation and constant-rate fiber growth.

Recent work has demonstrated that enzymatic degradation of collagen fibers exhibits strain-dependent kinetics. Conceptualizing how the strain dependence affects remodeling of collagenous tissues is vital to our understanding of collagen management in native and bioengineered tissues. As a first step towards this goal, the current study puts forward a multiscale model for enzymatic degradation and remodeling of collagen networks for two sample geometries we routinely use in experiments as model tissues. The multiscale model, driven by microstructural data from an enzymatic decay experiment, includes an exponential strain-dependent kinetic relation for degradation and constant growth. For a dogbone sample under uniaxial load, the model predicted that the distribution of fiber diameters would spread over the course of degradation because of variation in individual fiber load. In a cross-shaped sample, the central region, which experiences smaller, more isotropic loads, showed more decay and less spread in fiber diameter compared to the arms. There was also a slight shift in average orientation in different regions of the cruciform.

Theory of the short time mechanical relaxation in articular cartilage.

Articular cartilage is comprised of macromolecules, proteoglycans, with (charged) chondroitin sulfate side-chains attached to them. The proteoglycans are attached to longer hyaluronic acid chains, trapped within a network of type II collagen fibrils. As a consequence of their relatively long persistence lengths, the number of persistence lengths along the chondroitin sulfate and proteoglycan chains is relatively small, and consequently, the retraction times for these side chains are also quite short. We argue that, as a consequence of this, they will not significantly inhibit the reptation of the hyaluronic acid chains. Scaling arguments applied to this model allow us to show that the shortest of the mechanical relaxation times of cartilage, that have been determined by Fyhrie and Barone to be due to reptation of the hyaluronic acid polymers, should have a dependence on the load, i.e., force per unit interface area P, carried by the cartilage, proportional to P(3/2).

Human primary corneal fibroblasts synthesize and deposit proteoglycans in long-term 3-D cultures.

Our goal was to develop a 3-D multi-cellular construct using primary human corneal fibroblasts cultured on a disorganized collagen substrate in a scaffold-free environment and to use it to determine the regulation of proteoglycans over an extended period of time (11 weeks). Electron micrographs revealed multi-layered constructs with cells present in between alternating parallel and perpendicular arrays of fibrils. Type I collagen increased 2-4-fold. Stromal proteoglycans including lumican, syndecan4, decorin, biglycan, mimecan, and perlecan were expressed. The presence of glycosaminoglycan chains was demonstrated for a subset of the core proteins (lumican, biglycan, and decorin) using lyase digestion. Cuprolinic blue-stained cultures showed that sulfated proteoglycans were present throughout the construct and most prominent in its mid-region. The size of the Cuprolinic-positive filaments resembled those previously reported in a human corneal stroma. Under the current culture conditions, the cells mimic a development or nonfibrotic repair phenotype.

Mathematical modelling of corneal swelling.

This paper presents a differential model of the corneal transport system capable of modelling thickness changes in response to osmotic perturbations applied to either limiting membrane. The work is directed towards understanding corneal behaviour in vivo. The model considers the coupled viscous flows within the corneal stroma and across the epithelial and endothelial membranes. The flows within the stroma are established based on transport theory in porous media, while the flows across the membranes are described using the phenomenological equations of irreversible thermodynamics. The ability of the numerical model to reproduce corneal thickness changes in response to endothelial perturbations was tested against available experimental data. The sensitivity of the model to changes in stromal and membrane transport coefficients was examined.

Evidence for a central role for electro-osmosis in fluid transport by corneal endothelium.

The mechanism of transepithelial fluid transport remains unclear. The prevailing explanation is that transport of electrolytes across cell membranes results in local concentration gradients and transcellular osmosis. However, when transporting fluid, the corneal endothelium spontaneously generates a locally circulating current of approximately 25 microA cm(-2), and we report here that electrical currents (0 to +/-15 microA cm(-2)) imposed across this layer induce fluid movements linear with the currents. As the imposed currents must be approximately 98% paracellular, the direction of induced fluid movements and the rapidity with which they follow current imposition (rise time < or =3 sec) is consistent with electro-osmosis driven by sodium movement across the paracellular pathway. The value of the coupling coefficient between current and fluid movements found here (2.37 +/- 0.11 microm cm(2) hr(-1) microA (-1), suggests that: 1) the local endothelial current accounts for spontaneous transendothelial fluid transport; 2) the fluid transported becomes isotonically equilibrated. Ca(++)-free solutions or endothelial damage eliminate the coupling, pointing to the cells and particularly their intercellular junctions as a main site of electro-osmosis. The polycation polylysine, which is expected to affect surface charges, reverses the direction of current-induced fluid movements. Fluid transport is proportional to the electrical resistance of the ambient medium. Taken together, the results suggest that electro-osmosis through the intercellular junctions is the primary process in a sequence of events that results in fluid transport across this preparation.

Anomalous acute inflammatory response in rabbit corneal stroma.

PURPOSE: To investigate the nature and cause of an acute, anomalous stromal edema after epithelial debridement in the rabbit cornea. METHODS: Series I: Adult New Zealand White rabbit corneas were mounted in perfusion chambers. The endothelium was bathed with Ringer's fluid, and the outer surface was covered with silicone oil. The epithelium of one eye was débrided with a scalpel before mounting, and the cornea of the fellow eye was débrided with a rotating brush after stabilization in the perfusion chamber. Using specular microscope tracking software, it was possible to measure total swelling and local swelling within the cornea. Series II: Diclofenac sodium ophthalmic solution 0.1% or a placebo was applied topically, 1 drop per 45 minutes for 3 hours before animals were euthanatized. RESULTS: Series I: Corneas with their epithelium scraped with a scalpel before mounting were 37.5 +/- 17.5 microm (n = 6; P < 0.001) thicker in vitro than the stromas of perfused, intact fellow corneas. Epithelial débridement with a rotating brush after mounting resulted in an immediate (within 8 minutes) stromal swelling that plateaued in 1 hour at 31.0 +/- 5.3 microm (n = 6; P < 0.001). Curiously, in six of six corneas, the anterior stroma swelled more than the posterior stroma. In four of six corneas, the posterior stroma thinned. Analysis showed this pattern to be consistent with a sudden increase in anterior swelling pressure or osmotic pressure and to be inconsistent with a change in endothelial transport properties. Series II: Placebo-treated corneas swelled 30.6 +/- 7.7 microm (n = 5) 1 hour after débridement, whereas corneas pretreated with diclofenac sodium swelled only 19.2 +/- 3.1 microm (n = 6; P < 0.008). CONCLUSIONS: The anterior stromal swelling occurs rapidly and near the site of epithelial injury suggesting messenger and/or enzymatic involvement with an effect parallel to apoptosis. Reduction of the swelling response with nonsteroidal anti-inflammatory drugs (NSAIDs) implicates the cyclooxygenase pathway. The swelling is similar to the unexplained acute edema that occurs during inflammation in the rat paw edema model, and may represent a general mechanism for mobilization of inflammatory cells.

Steady flow in models of abdominal aortic aneurysms. Part I: Investigation of the velocity patterns.

As an investigation into the mechanical factors that lead to rupture of abdominal aortic aneurysms, both color Doppler flow imaging and laser Doppler velocimetry measurements of steady flow through a series of aneurysm models are presented. The flow pattern in each model consisted of a core of relatively fast-moving fluid traveling through the center of the dilation, surrounded by an outer annulus of slowly recirculating fluid. At flow rates below a Reynolds number of 1750 +/- 150, the flow was smooth, steady, and laminar. At higher flow rates (Reynolds number above 2250 +/- 250), the flow was always irregular and turbulent. Between these fully laminar and fully turbulent regimes the flow was intermittently turbulent. Larger models showed a tendency to become turbulent at lower Reynolds numbers than smaller models. In addition, turbulence was amplified in the distal half of the model dilation, with the largest models producing velocity fluctuations as great as 35% of the time-average centerline velocities. These data suggest that larger aneurysms in vivo may be subject to more frequent and intense turbulence than smaller aneurysms. Concomitantly, increased turbulence may contribute significantly to risk of rupture, as is discussed in Part II.

Experimental investigation of steady flow in rigid models of abdominal aortic aneurysms.

Abdominal aortic aneurysms occur in as much as 2-3% of the population, and their rupture produces a mortality rate of 78-94% (1), causing 15,000 deaths per year in the U.S. alone. As an investigation into the mechanical factors that lead to aneurysm rupture, flow field measurements are presented for steady flow through a range of aneurysm sizes and Reynolds numbers. Seven rigid symmetric models of aneurysms were constructed with uniform lengths of 4d and diameters that ranged from 1.4 to 3.3d, where d is the inner diameter of the undilated entrance tube. Color Doppler flow imaging was used to visualize the flow fields, while laser Doppler velocimetry was used to quantify the flow field velocities and to determine critical Reynolds numbers for the onset of, and complete transition to, turbulent flow. Estimates of mean and peak wall shear stresses were derived from velocity measurements. Flow in these models varied from fully laminar to fully turbulent over the range of Reynolds numbers corresponding to in vivo flows. There was a large range over which the flow was intermittently turbulent. High wall shear occurred in the models when the flow was turbulent, suggesting that turbulence in in vivo aneurysms may contribute significantly to their risk of rupture.