Biomedical Engineering Reference
In-Depth Information
been characterized [4,17,18,27]. The prevalence of studies utilizing collagen hydrogels originates from
the extremely important role and ubiquitous presence of collagen in connective tissues throughout the
body. Collagen is the most common protein in the body, comprising 6% of body weight and ~25-33%
of the total protein mass [28]. Self-assembled collagen hydrogels are not only useful
in vitro
models to
study cell-matrix interactions [29-32], cellular modulation of wound healing [8,33-41], fibrosis [42-
47], and microstructure-mechanics relations [21,22,48-52], but they also serve as starting scaffolds for
many tissue-engineering experiments and applications [42,43,45,53-55].
Cell-seeded collagen hydrogels are particularly amenable to analysis by three-dimensional LSM,
including SHG imaging, since the tissues may be imaged nondestructively at any time during
in vitro
culture [5,24,56-61]. Collagen self-assembly is controlled by polymerization conditions that influence
polymer aggregation, creating gels of varied microstructures and network properties [21,22,62-68]. The
cells seeded in or on such hydrogel scaffolds create tissue constructs that change dynamically due to
force interactions between cells and the surrounding scaffold, and by proteolysis and new matrix deposi-
tion. Dynamic remodeling of cell-seeded collagen gels may be tracked in four dimensions by assembling
z
-stacks of SHG image frames from tissues at different locations and culture time points. Other optical
signals, such as reflectance [69], optical coherence [70], one- and two-photon fluorescence (TPF) [56,71],
and coherent anti-Stokes Raman scattering (CARS) [72] may be imaged simultaneously or nearly on
a multimodal platform, increasing the structural, biochemical, and optical information derived from
the imaged tissue regions and allowing the study of interactions between the signal-producing species
within the tissue. SHG imaging of acellular and cell-seeded self-assembled gels is a powerful technique
to address the fundamental questions regarding tissue mechanics and cell behavior within a three-
dimensional matrix environment.
11.2 Background
11.2.1 collagen Structural Properties
Over 20 unique types of collagen have been described, including the fibril-forming collagens (types
I, II, III, V, and XI) [63,73]. Each of these fibril-forming collagens posses a similar noncentrosym-
metric structure that is required for both SHG and for self-assembly into supramolecular aggregates
and, finally, an entangled network. The three properties of SHG, self-assembly, and resistance to ten-
sion arise from the unique primary structure of fibril-forming collagens. This structure consists of
repeats of the triple amino acid sequence glycine-
X
-
Y
, where
X
and
Y
are most frequently proline and
hydroxyproline, respectively [73]. These repeats comprise ~10% of the collagen monomer sequence and
enable the formation of left-handed helices, termed alpha chains tightly coiled with about three amino
acids per turn. The three alpha chains interact with each other to form a right-handed, coiled-coil triple
helix. This triple helical procollagen molecule is capped by nonhelical propeptide regions that promote
solubility and whose presence and enzymatic removal are both necessary for fibrillogenesis to occur
[65]. Procollagen molecules are synthesized near the endoplasmic reticulum and must be packaged and
secreted by the Golgi apparatus before extracellular initiation of fibrillogenesis. Procollagen N- and
C-peptidase are extracellular enzymes that cleave the 15 and 10 nm-long N and C propeptides, respec-
tively, yielding a collagen monomer ~300 nm long and 1.5 nm wide [73]. Following procollagen cleav-
age, the collagen monomer contains only the helical region capped by nonhelical telopeptide regions
that consist of 10-25 amino acids, and are important in cross-linking and in directing monomer pack-
ing and fibrillogenesis. The interchain spacing within the triple helix is ~0.286 nm, close enough for
hydrogen bonding, hydrophobic interactions, and interchain cross-links to stabilize the monomer.
After the conversion of procollagen into collagen monomers, fibrillogenesis may occur. The process
is an entropy-driven self-assembly that may occur in a cell-free environment modulated by physical
variables (pH, temperature, ions) or in living tissue where cell-secreted molecules and enzymes may
modulate fibrillogenesis [73].
