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N. Saeidi, J.W. Ruberti; Kinetics of Tethered Collagen Assembly in Shearing Flow Assessed by Dynamic Optical Imaging . Invest. Ophthalmol. Vis. Sci. 2006;47(13):3949.
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© ARVO (1962-2015); The Authors (2016-present)
The corneal stroma comprises a nematic stack of highly monodisperse, uniformly–spaced, oriented, collagen fibrils arranged in thin lamellae which alternate in direction. Corneal transparency and mechanical strength are derived from this remarkable architecture. To duplicate this structure de novo for the purpose of corneal tissue engineering requires exquisite control over the self–assembly of collagen. Our laboratory has demonstrated the ability to shear–align collagen during fibrillogenesis but has lacked the ability observe the kinetics of the process directly. This investigation demonstrates dynamic optical observation of the kinetics of collagen self–assembly under the influence of shear flow.
Cold, neutralized, acid–soluble bovine collagen monomers (3.0 mg/mL, Cohesion, Palo Alto, CA) in PBS were injected through an in–line solution heater (SC–20, Hamden, CT) into an environmentally–controlled microfluidics chamber (FCS2 Chamber, Bioptechs Butler, PA) mounted on the stage of an inverted microscope (Nikon TE2000–U, Melville, NY). The solution was warmed to 37°C prior to entry into the chamber and maintained at that temperature throughout the experiment. Subsequent to the initial injection, nucleation of collagen fibrils on the surface of the glass bottom of the chamber was followed dynamically (images every 20 sec) with Differential Interference Contrast imaging (DIC – 60x objective; 1.4 NA; 1.5x). After four minutes, flow was either restarted (experimental – shear rate of 264 sec–1) or the solution was left static in the chamber (control).
The dynamic DIC images capture the nucleation and axial growth of collagen fibrils under both static and shearing flow conditions. Observable nucleation begins at approximately 150 seconds. The axial growth rate of the collagen was found to be 8.76 µm/min ± 3.2.
DIC imaging in combination with a temperature–controlled microfluidics chamber is an effective system which can be used to observe the kinetics of collagen self–assembly. It has already demonstrated the time–to–nucleation on glass which will inform our higher shear rate collagen alignment work for artificial cornea. It has also shown us the rate of axial growth of collagen fibrils which might be expected in a shear flow. Future experiments will include the effect of various parameter changes including the addition of fibrillogenesis mediators (i.e. proteoglycans) on the polymerization rates and pattern of self–assembly of collagen monomers in vitro.
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