Dynamic shear-influenced collagen self-assembly

Abstract

The ability to influence the direction of polymerization of a self-assembling biomolecular system has the potential to generate materials with extremely high anisotropy. In biological systems where highly-oriented cellular populations give rise to aligned and often load-bearing tissue such organized molecular scaffolds could aid in the contact guidance of cells for engineered tissue constructs (e.g cornea and tendon). In this investigation we examine the detailed dynamics of pepsin-extracted type I bovine collagen assembly on a glass surface under the influence of flow between two plates. Differential Interference Contrast (DIC) imaging (60x-1.4NA) with focal plane stabilization was used to resolve and track the growth of collagen aggregates on borosilicate glass for 4 different shear rates (500, 80, 20, and 9 s -1 ). The detailed morphology of the collagen fibrils/aggregates was examined using Quick Freeze Deep Etch electron microscopy. Nucleation of fibrils on the glass was observed to occur rapidly (~2 min) followed by continued growth of the fibrils. The growth rates were dependent on flow in a complex manner with the highest rate of axial growth (0.1 microns/sec) occurring at a shear rate of 9 s -1 . The lowest growth rate occurred at the highest shear. Fibrils were observed to both branch and join during the experiments. The best alignment of fibrils was observed at intermediate shear rates of 20 and 80s -1 . However, the investigation revealed that fibril directional growth was not stable. At high shear rates, fibrils would often turn downstream forming what we term "hooks" which are likely the combined result of monomer interaction with the initial collagen layer or "mat" and the high shear rate. Further, QFDE examination of fibril morphology demonstrated that the assembled fibrillar structure did not possess native D-periodicity. Instead, fibrils comprised a collection of generally aligned, monomers which were self-assembled to form a fibril-like aggregate. In conclusion, though constant shear-rate clearly influences collagen fibrillar alignment, the formation of highlyorganized collagenous arrays of native-like D-banded fibrils remains a challenge. Modulation of shear in combination with surface energy patterning to produce a highly-aligned initial mat may provide significant improvement of both the fibril morphology and alignment.

Production of highly aligned collagen lamellae by combining shear force and thin film confinement

“…Previously, we have shown that hydrodynamic shear stress is capable of producing relatively aligned arrays of collagen fibrils in a microfluidic device [25]. In that study, “hooked” fibrils formed against the flow field due to surface interactions that competed with shear-induced collagen self-assembly.…”

“…thinnest chambers) the flow in this system was not strong enough to influence the organization of the individual collagen molecules but could influence the direction of larger aggregates (e.g. dimers and trimers) which spontaneously self-assemble under physiological conditions [25]. Thus the shear-flow technique is not suitable for assembling collagen layers with uniform fibril alignment mimetic of the cornea.…”

Isolation and Identification of Cryptic Bioactive Regions in Bovine Achilles Tendon Collagen

“…6, for all the test peptides, there was an almost hyperbolic increase in radical scavenging and chelating activity with increasing concentration. The peptides acted as potent antioxidative agents at lower concentrations but the activity asymptotically approached a maximum of 70 % at higher concentrations, probably due to the inherent recoiling nature of collagen type I peptides [43]. Upon comparison of the EC 50 values the scavenging activity was found to be in the given order, E1 followed by F3, E3 and F2.…”

Screening for novel cell adhesive regions in bovine Achilles tendon collagen peptides

“…However, at concentrations higher than 2 g·mL −1 , peptide binding decreased and cells were observed to adhere to collagen, an apparently unusual effect that arises due to the recoiling tendency of collagen-like peptides. At higher amounts, most collagen peptides recoil to each other forming transient helices (Saeidi et al 2009), and mostly remain unavailable for binding with cellular receptors. Free binding sites of the cellular receptors attach to collagen instead, increasing the observed cell count.…”