Biomedical Engineering Reference
In-Depth Information
swelling, and new processes have been described which attempt to better
preserve the tissue architecture prior to fixation (Cunanan et al., 2009).
5.7.4 Crosslinking
Crosslinking of the tissue can be done either with or without an applied force, as
discussed previously (Lee et al., 1989a,b). Determining the completion of
crosslinking is an important parameter in controlling the process. While many
believe shrinkage temperature to be a measure of crosslinking, it is not in a pure
sense of the term (Lee et al., 1995). Shrinkage temperature is defined as the
temperature at which the tissue shrinks by 1%. While it is true that the shrinkage
temperature of crosslinked tissue is greater than the shrinkage temperature of
fresh tissue (Cunanan et al., 2001), this merely demonstrates that the tissue has a
greater resistance to thermal denaturation after crosslinking. A better measure of
the degree of crosslinking would be to measure the consumption of the reactants
involved in the crosslinking reaction. Since it is difficult to accurately measure
minute changes in glutaraldehyde during the reaction (because it is present in
great excess), it is easier to measure the component to which the glutaraldehyde
reacts, that is, the amino acid lysine.
Lysine can be easily measured using amino acid analysis, of which many
commercial systems are available. Because of sampling difficulties, it is best to
normalize the amount of lysine to another amino acid present in collagen which
is not consumed in the crosslinking reaction. Glycine is a particularly abundant
amino acid in collagen which cannot participate in the cross-linking reaction,
due to its non-reactive side chain. This then leads to the development of a Lys/
Gly ratio (L/GR) and a percentage crosslinked, as defined below:
Lys/Gly ratio
residues of lysine in the sample (per 1000 amino acids)
residues of glycine in the sample (per 1000 amino acids)
5:1
% crosslinked
L/GR
initial
ÿL/GR
final
L/GR
initial
5:2
As lysine is consumed in the crosslinking reaction, amino acid analysis will
reveal a decline in the lysine content with time. At the point where the reaction
has reached a steady state, the percentage crosslinking will remain unchanged
with further reaction time. This is the point of complete fixation.
An important point worth mentioning is that the lysine content will never be
zero in an intact crosslinked tissue. This is because some of the lysine side
chains will be protected by other molecules in the tissue and thus are sterically
hindered from reacting with the glutaraldehyde. Table 5.3 contains some amino
acid data published by Schoen et al. (1986). In this study he characterizes the
kinetics of glutaraldehyde incorporation into the tissue using tritium-labeled
glutaraldehyde. This is an elegant way to measure crosslinking; however, if
