|
We have developed a protocol employing dual-mode non-linear microscopy for the monitoring of the
biosynthesis of bacterial cellulose at a single-fiber level, with the fundamental aim to achieve a
product with material properties similar to those of human blood vessels. Grown in a tubular geometry
it could then be used as a natural and biocompatible source of replacement tissue in conjunction with
cardiovascular surgery. The bacteria (Acetobacter xylinum) were selectively visualized based on the
CH2 vibration of its organic macromolecular contents by the Coherent Anti-Stokes Raman Scattering
(CARS) process and, simultaneously, the non-centrosymmetrically ordered, birefringent cellulose
fibers were depicted by the Second Harmonic Generation (SHG) process. This dual-channel detection
approach allows the monitoring of cellulose-fiber formation in vivo and to determine the influence of
e.g. different growth conditions on fiber thickness and orientation, their assembling into higher-order
structures and overall network density. The bacterial and fiber distributions were monitored in a
simple microscope cultivation chamber, as well as in samples harvested during the actual fermentation
process of tubular cellulose grafts. The CARS and SHG
co-localization images reveal that highest
bacterial population densities can be observed in the surface regions of the cellulose tissue, where the
primary growth presumably takes place. The cellulose network morphology was also compared with
that of human arteries and veins, from which we conclude that the cellulose matrix is comparatively
homogeneous in contrast to the wavy band-like supra-formations of collagen in the native tissue. This
prompts for sophisticated fermentation methods by which tunnels and pores of appropriate sizes and
shapes can be introduced in the cellulose network in a controllable way. With this protocol we hope to
contribute to the fundamental knowledge required for optimal production of bioengineered cellulose
tissues, eventually being available for clinical use.
|
