Biomedical Engineering Reference
In-Depth Information
11.2.2 collagen Gel Self-Assembly
Collagen for the
in vitro
construction of collagen gels is derived by soaking collagen-rich tissues (such as
rat-tail tendons or calf skin) in acetic acid for several days, followed by dialysis to concentrate the solubi-
lized, triple-helical collagen monomers [74]. The resulting soluble collagen contains few intramolecular
cross-links, though aggregates of 5-17 monomers may exist in soluble form [63,73]. Raising the tem-
perature, pH, and ionic strength to physiological levels typically initiates self-assembly, in which collagen
fibrils form via lateral and linear fusion of monomer aggregates. Fibril length, diameter, and aggregation
into fibers are affected by polymerization, pH, temperature, and ionic strength [21,22,62-68,75]. Self-
assembly results in the formation of a collagen gel, that is, an entangled network of highly hydrated
collagen fibrils surrounding fluid-filled pores. The gelation process proceeds with an initial lag phase,
during which the monomer aggregates initiate fusion, followed by a rapid growth phase, and eventual
plateau. The mechanical and optical properties of the gel change with gelation time in a similar, sigmoi-
dal shape. For example, gel turbidity (a measure of light scattering) increases as
x
= −
1
exp(
−
Z
n
n
),
(11.1)
where
x
is the mass fraction of precipitated collagen (linearly related to turbidity),
Z
is a rate constant,
t
is the gelation time, and
n
is a constant related to collagen fiber nucleation [65]. Bulk shear modulus
of the gel also increases rapidly after a lag and prior to a plateau value [73]. The collagen self-assembly
process is hypothesized to occur through simultaneous nucleation and linear growth of fibrils [73].
In this model of collagen fibrillogenesis, collagen monomers exist in equilibrium with small, soluble
aggregates of 5-17 monomers, termed microfibrils. The existence of these purported microfibrils is
supported by x-ray diffraction and electron microscopy measurements of collagen fibrils, which sug-
gest that fibrils exist with quantized diameters, of integer multiples of ~4 nm. Self-assembly proceeds
by both lateral and axial accretion of these microfibrillar subunits [76]. Increasing ion concentra-
tion or decreasing temperature or pH tends to favor the lateral aggregation of microfibrils, leading to
increased fibril diameters [73,77]. The physical parameters that increase fibril diameter tend to delay
fibrillogenesis and prolong the lag phase, during which nucleation is the predominant process [63].
Other extracellular matrix constituents present during collagen self-assembly may also modulate the
collagen fibril and network structure. Proteoglycan and glycosaminoglycan binding to collagen may
either delay or accelerate fibrillogenesis, thus affecting the collagen fibril diameter. Hyaluronic acid
and decorin tend to decrease the diameter of fibrils formed in their presence [23], whereas dermatan
sulfate binding favors the formation of thicker fibers [78]. The physical and chemical parameters can
alter
in vitro
fibrillogenesis that affects collagen fiber dimensions and, given a limited concentration of
collagen monomers, can vary the fiber number density and therefore the pore size of the formed col-
lagen network, thus impacting network mechanics. The final result of collagen self-assembly and fibril-
logenesis is an ordered and hierarchical array of collagen monomers, forming an entangled biopolymer
network of fibrils and fibril bundles.
The fibril level of organization consists of arrays of monomers that are ordered in an axially stag-
gered pattern in which molecules are stacked and staggered by one-quarter of the molecular length,
or about 68 nm. This staggered array allows for interchain cross-link formation, and results in native
fibrils 20-500 nm wide and up to 1 cm long [79] displaying
D
-banding: 68 nm wide bands that stripe
the collagen fibril, apparent in metal-stained fibrils and resulting from the quarter-staggered array of
monomers [80]. Bundles of fibrils form collagen fibers, typically 1-20 μm wide; bundles of fibers form
thicker fascicles that form bundles within tendons [81]. Between the monomer and the assembled col-
lagen unit, physical and biological factors alter the assembly process. Importantly, collagen structure
and cross-link content affect the mechanical properties on the level of single-collagen fibers, collagen
networks, and bulk tissue. Knowledge of the mechanisms of collagen fibrillogenesis allows control over
the formation of collagen-containing tissues
in vitro
and
in vivo
.
