Biomedical Engineering Reference
In-Depth Information
achieved within hours by exposure to temperatures in
excess of 105 C under atmospheric pressure. The possi-
bility that cross-linking achieved under these conditions is
caused by a pyrolytic reaction has been ruled out. Fur-
thermore, chromatographic data have shown that the
amino acid composition of collagen remains intact after
exposure to 105 C for several days. In fact, it has been
observed that gelatin can be cross-linked by exposure to
temperatures as low as 25 C provided that a sufficiently
high vacuum is present to achieve the drastic moisture
removal that drives the cross-linking reaction.
Exposure of highly hydrated collagen to temperatures
in excess of ca. 37 C is known to cause reversible melting
of the triple helical structure, as described earlier. The
melting point of the triple helix increases with the
collagen-diluent ratio from 37 C, the helix-coil transi-
tion of the infinitely dilute solution, to about 120 C for
collagen swollen with as little as 20 wt.% diluent and up
to 210 C, the approximate melting point of anhydrous
collagen. Accordingly, it is possible to cross-link collagen
using the drastic dehydration procedure described above
without loss of the triple helical structure. It is simply
sufficient to adjust the moisture content of collagen to
a low enough level prior to exposure to the high tem-
perature levels required for rapid dehydration.
Dialdehydes have been long known in the leather in-
dustry as effective tanning agents and in histological
laboratories as useful fixatives. Both of these applications
are based on the reaction between the dialdehyde and the
3-amino group of lysyl residues in the protein, which
induces formation of interchain cross-links. Glutaralde-
hyde cross-linking is a relatively widely used procedure in
the preparation of implantable biomaterials. Free glu-
taraldehyde is a toxic substance for cells; it cross-links
vital cell proteins. However, clinical studies and exten-
sive clinical use of implants have shown that the toxicity
of glutaraldehyde becomes effectively negligible after the
unreacted glutaraldehyde has been carefully rinsed out
following reaction with an implant, e.g., one based on
collagen. The nature of the cross-link formed has been
the subject of controversy, primarily due to the complex,
apparently polymeric, character of this reagent. Consid-
erable evidence supports a proposed anabilysine struc-
ture, which is derived from two lysine side chains and
two molecules of glutaraldehyde.
Evidence for other mechanisms has been presented.
By comparison with other aldehydes, glutaraldehyde has
shown itself to be a particularly effective cross-linking
agent, as judged, for example, by its ability to increase
the crosslink density to very high levels. Values of the
average molecule weight between cross-links ( M c ) pro-
vide the experimenter with a series of collagens in which
the enzymatic degradation rate can be studied over
a wide range, thereby affording implants that effectively
disappear from tissue between a few days and several
months following implantation. The mechanism of the
reaction between glutaraldehyde and collagen at neutral
pH is understood in part; however, the reaction in acidic
media has not been studied extensively. Evidence that
covalent cross-linking is involved comes from measure-
ments of the equilibrium tensile modulus of films that
have been treated to induce cross-linking and have sub-
sequently been gelatinized by treatment in 1 M NaCl at
70 C. Under such conditions, only gelatin films that have
been converted into a three-dimensional network by
cross-linking support an equilibrium tensile force; by
contrast, un-cross-linked specimens dissolve readily in
the hot medium.
Several other methods for cross-linking collagen have
been studied, including hexamethylene diisocynate, acyl
azide, and a carbodiimide, 1-ethyl-3-(3-dimethlyamino-
propyl) carbodiimide (EDAC).
The immunogenicity of the collagen used in implants
is not insignificant and has been studied assiduously using
laboratory preparations. However, the clinical signifi-
cance of such immunogenicity has been shown to be very
low and is often considered to be negligible. The validity
of this simple approach to using collagen as a biomaterial
was long ago recognized by manufacturers of collagen-
based sutures. The apparent reason for the low antige-
nicity of type I collagen mostly stems from the small
species difference among type I collagens (e.g., cow
versus human). Such similarity is, in turn, probably un-
derstandable in terms of the inability of the triple helical
configuration to incorporate the substantial amino acid
substitutions that characterize species differences with
other proteins. The relative constancy of the structure of
the triple helix among the various species is, in fact, the
reason why collagen is sometimes referred to as a ''suc-
cessful'' protein in terms of its evolution or, rather, the
relative lack of it.
In order to reduce the immunogenicity of collagen it is
useful to consider the location of its antigenic de-
terminants, i.e., the specific chemical groups that are
recognized as foreign by the immunological system of the
host animal. The configurational (or conformational)
determinants of collagen depend on the presence of the
intact triple helix and, consequently, are abolished when
collagen is denatured into gelatin; the latter event (see
earlier discussion) occurs spontaneously after the triple
helix is cleaved by a collagenase. Gelatinization exposes
effectively the sequential determinants of collagen over
the short period during which gelatin retains its macro-
molecular character, before it is cleared away following
attack by one of several nonspecific proteases. Control of
the stability of the triple helix during processing of col-
lagen, therefore, partially prevents the display of the
sequential determinants.
Sequential determinants also exist in the nonhelical
end (telopeptide region) of the collagen molecule, and
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