Controlled Self-Assembly of Collagen Fibrils by an Automated Dialysis System

[+] Author and Article Information
Stefan Strasser, Albert Zink

Department für Geo- und Umweltwissenschaften, Ludwig-Maximilians-Universität, 80333 Munich, Germany

Wolfgang M. Heckl

Department für Geo- und Umweltwissenschaften, Ludwig-Maximilians-Universität, 80333 Munich, Germany and Deutsches Museum, Museumsinsel 1, 80538 Munich, Germany

Stefan Thalhammer1

Department für Geo- und Umweltwissenschaften, Ludwig-Maximilians-Universität, 80333 Munich, Germany and GSF-Forschungszentrum für Umwelt und Gesundheit GmbH, Ingolstädter Landstrasse 1, 85764 Oberschleissheim, Germanys.t@lmu.de


Corresponding author.

J Biomech Eng 128(5), 792-796 (Mar 12, 2006) (5 pages) doi:10.1115/1.2264392 History: Received August 25, 2005; Revised March 12, 2006

In vitro self-assembled collagen fibrils form a variety of different structures during dialysis. The self-assembly is dependent on several parameters, such as concentrations of collagen and α1-acid glycoprotein, temperature, dialysis time, and the acid concentration. For a detailed understanding of the assembly pathway and structural features like banding pattern or mechanical properties it is necessary to study single collagen fibrils. In this work we present a fully automated system to control the permeation of molecules through a membrane like a dialysis tubing. This allows us to ramp arbitrary diffusion rate profiles during the self-assembly process of macromolecules, such as collagen. The system combines a molecular sieving method with a computer assisted control system for measuring process variables. With the regulation of the diffusion rate it is possible to control and manipulate the collagen self-assembly process during the whole process time. Its performance is demonstrated by the preparation of various collagen type I fibrils and native collagen type II fibrils. The combination with the atomic force microscope (AFM) allows a high resolution characterization of the self-assembled fibrils. In principle, the represented system can be also applied for the production of other biomolecules, where a dialysis enhanced self-assembly process is used.

Copyright © 2006 by American Society of Mechanical Engineers
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Figure 3

AFM images of (A) a native collagen type I fibril. The banding is clearly visible and amounts to 67nm, the width to 300nm and the height to 20nm (scale bar 500nm, error signal, contact mode, decanol/end pH=6, cα1-acidglycoprotein=0.2mg∕ml/dialysis time: 17h). (B) Native and FLS collagen type I fibrils. The formation is changing along the axis of the fibril. This formation is typical for collagen clusters (scale bar 500nm, error signal, contact mode, decanol/end pH=6, cα1-acidglycoprotein=0.3mg∕ml/dialysis time: 17h). (C) An intermediate FLS fibril, assembled with increased α1-acid glycoprotein concentration (scale bar 500nm, error signal, contact mode, ambient conditions/end pH=4.5, cα1-acidglycoprotein=0.5mg∕ml/dialysis time: 10h). (D) Native collagen type II fibrils. The banding is clearly visible and amounts to 70nm. The diameter of collagen II (110nm in width and 13nm height) is much smaller than the diameter of collagen I (scale bar 500nm, topography signal, noncontact mode, ambient conditions/end pH=6, cα1-acidglycoprotein=0.2mg∕ml/dialysis time: 17h).

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Figure 2

Permeation time of the acetic acid. The bright (with) curve shows the pH change of the dialysis liquid during diffusion of acetic acid through the membrane of the dialysis tubing. The dark curve (without) shows the pH value of the dialysis liquid after addition of the same amount of acetic acid. ① marks the point in time when the acetic acid is added (pH value versus time (min)).

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Figure 1

(A) Sketch of the dialysis system, with the following workflow: The rinsing medium flows through the valve X1 into the beaker (via the inlet (air tight lid, with O-ring)). The outlet is fixed at a predetermined height, therefore a constant level of the dialysis liquid is guaranteed. The dialysis liquid in the beaker is stirred and the pH value is measured online. With a holding fixture the dialysis tubing is fastened in the medium. To achieve a volume compansation, mainly in the reservoir, the reservoir and the beaker are connected to a bag filled with nitrogen. The valve X1 is controlled by a USB interface card and diffusion rate in the system can be controlled online. (B) pH control curve during dialysis. Depicted are the setpoint value (set.value) and the actual pH value (act.value). The columns display the switching state of the valve within every 25min. One valve-open-cycle lasts 5s. The average variation can be calculated to ΔpH=0.033 and the maximal variation to ΔpH=0.09.



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