Directed Assembly of Elastic Fibers via Coacervate Droplet Deposition on Electrospun Templates.

Elastic fibers provide critical elasticity to the arteries, lungs, and other organs. Elastic fiber assembly is a process where soluble tropoelastin is coacervated into liquid droplets, cross-linked, and deposited onto and into microfibrils. While much progress has been made in understanding the biology of this process, questions remain regarding the timing of interactions during assembly. Furthermore, it is unclear to what extent fibrous templates are needed to guide coacervate droplets into the correct architecture. The organization and shaping of coacervate droplets onto a fiber template have never been previously modeled or employed as a strategy for shaping elastin fiber materials. Using an in vitro system consisting of elastin-like polypeptides (ELPs), genipin cross-linker, electrospun polylactic-co-glycolic acid (PLGA) fibers, and tannic acid surface coatings for fibers, we explored ELP coacervation, cross-linking, and deposition onto fiber templates. We demonstrate that integration of coacervate droplets into a fibrous template is primarily influenced by two factors: (1) the balance of coacervation and cross-linking and (2) the surface energy of the fiber templates. The success of this integration affects the mechanical properties of the final fiber network. Our resulting membrane materials exhibit highly tunable morphologies and a range of elastic moduli (0.8-1.6 MPa) comparable to native elastic fibers.

Globule and fiber formation with elastin-like polypeptides: a balance of coacervation and crosslinking.

Elastic fiber assembly is a complex process that requires the coacervation and cross-linking of the protein building block tropoelastin. To date, the order, timing, and interplay of coacervation and crosslinking is not completely understood, despite a great number of advances into understanding the molecular structure and functions of the many proteins involved in elastic fiber assembly. With a simple in vitro model using elastin-like polypeptides and the natural chemical crosslinker genipin, we demonstrate the strong influence of the timing and kinetics of crosslinking reaction on the coacervation, crosslinking extent, and resulting morphology of elastin. We also outline a method for analyzing elastin droplet network formation as a heuristic for measuring the propensity for elastic fiber formation. From this we show that adding crosslinker during peak coacervation dramatically increases the propensity for droplet network formation.

Initiation of Medial Calcification: Revisiting Calcium Ion Binding to Elastin.

Pathological calcification of elastin, a key connective tissue protein in the medial layers of blood vessels, starts with the binding of calcium ions. This Mini-Review focuses on understanding how calcium ions interact with elastin to initiate calcification at a molecular level, and emphasizes water's critical role in mediating this interaction. In the past decade, great strides have been made in understanding and modeling ion-specific hydration and its effects on biomolecule interactions. However, these advances have been largely absent from our understanding of elastin calcification. Historically, charge-neutral backbone carbonyls and negatively charged carboxyl groups have been proposed as elastin's calcium binding sites. Recently, tropoelastin's only four carboxyl groups have been identified as binding sites from classical molecular dynamics (MD). While carboxyl groups have a much higher affinity for binding calcium ions than backbone carbonyls, conflicting evidence persists for both functional group's importance in elastin calcification. This can be attributed to the fact that divalent ions strongly polarize water, leading to a hydration shell that shields electrostatic forces. The hydration shell surrounding both a calcium ion and either of the proposed binding sites must be displaced to enable binding. Providing our own extended X-ray absorption fine structure (EXAFS) data and complementary simulations, we discuss the potential structures of calcium binding in elastin and review prior knowledge regarding the relative importance of the two proposed binding sites.