Biomedical Engineering Reference
In-Depth Information
The Matrigel treatment was found to promote outgrowth of cells as well as cell survival. The collagen
structure was monitored by SHG microscopy and it was observed that increasing amounts of added
Matrigel resulted in a less homogenous matrix.
Dumas et al. have presented combined two-photon fluorescence and SHG microscopy with detec-
tion utilizing time-correlated single-photon counting to distinguish between the signals. The technique
was applied to investigate mesenchymal human stem cells seeded on collagen sponge scaffolds (Dumas
et al. 2010). The advance of SHG microscopy as a tool in tissue engineering also requires methods for
image analysis. One example of this is an algorithm for analysis of fiber content and orientation in thick
collagen gels developed by Bayan et al. and applied to study the influence of fibroblast seeding on the
scaffold (Bayan et al. 2009).
18.2.2 extracellular Matrix collagen
In addition to being employed as a scaffold material, collagen is a major component of the ECM syn-
thesized by many types of cells. ECM collagen production has been traced using SHG microscopy in a
number of investigations, for example, in studies of chondrocytes differentiated from stem cells (Chen
et al. 2008, 2010) or in explanted cartilage tissue (Werkmeister et al. 2010). A delayed collagen produc-
tion (Chen et al. 2008) as well as a more aligned fiber arrangement could be observed for samples under
mechanical stress compared to unstressed samples (Chen et al. 2008, Werkmeister et al. 2010). Studies
of ECM collagen have also been carried out on mesenchymal stem cells differentiated into osteogenic
cells (Pallotta et al. 2009, Rice et al. 2010). In a study of megakaryocytes, Pallotta et al. compared the
production of ECM collagen at oxygen levels of 5% and 20% for isolated osteoblasts as well as for co-
cultures with hematopoietic stem cells (Pallotta et al. 2009). In agreement with the trend observed for
collagen levels monitored via hydroxyproline detection, SHG data showed an earlier development of
ECM collagen from osteoblasts in co-culture at the lower oxygen condition. Results from a similar study
carried out by Rice et al., comparing osteoblast collagen production at low (5%) and high (20%) levels
of oxygen access, also showed an earlier and in total higher production of collagen for cells grown at
low oxygen supply. Another example of ECM collagen produced by osteoblasts and monitored by SHG
microscopy is presented in Section 18.3.2.2 (Figure 18.9). The technique has also been employed in car-
diovascular tissue engineering where König and coworkers studied the influence of pressure on collagen
synthesis in engineered heart-valve leaflets (König 2008). Images of tissue exposed to pressures above
physiological levels showed signs of disturbed matrix formation and cell growth compared to tissue at
normal pressure level. The same author has also presented results from SHG applied to investigate col-
lagen generation by differentiated human stem cells (König 2008). In addition to investigations,
in vitro
SHG microscopy has also been employed for imaging ECM collagen in native tissue such as heart valve
leaflets, cornea, and cartilage (Schenke-Layland 2008).
18.3 Biosynthesized cellulose
Cellulose is the most abundant naturally occurring polymer and a major component of biomass from
plants. It can also be produced by bacteria and algae, resulting in a matrix of crosslinked fibers (Ross
et al. 1991). The cellulose polymer consists of a linear arrangement of glucose molecules, where parallel
polymer chains attach with hydrogen bonds and form the crystalline cellulose structure. The molecular
structure of cellulose and a schematic of the synthesis carried out by bacteria of strain
Gluconacetobacter
xylinus
are shown in Figure 18.4.
In the cellulose synthesis, glucan chains, extruded from cellulose synthase complexes located on the
bacterium surface, are initially assembled into a sub-elementary fibril, then further on into a crystalline
microfibril and finally a fiber ribbon assembly, which is a few hundred nanometers in diameter (Brown
et al. 1976, Ross et al. 1991). The ordered crystalline arrangement makes the material non-centrosymmetric
and it has also proven to be SHG-active (Brown et al. 2003). The first visualizations of microbial cellulose
