Biomedical Engineering Reference
In-Depth Information
(Fig. 3a, b). The rate of fibril formation decreased with increasing EDC concentration, which
indicates EDC exhibited an inhibitory effect on collagen fibril formation. These inhibitory
effects were probably the results of the rapid reaction of EDC to monomeric collagens upon
mixing of the EDC containing buffer and acidic collagen solution, by which the ability to
form fibrils was reduced or lost through random and nonfibrous aggregation of monomeric
collagens. Further increased EDC concentration above 90 mM completely suppressed fibril
formation irrespective of NaCl concentrations. Based on these results, it appeared that a
buffer that would enable a faster fibril formation rate would be desirable. According to a
previous report, EDC is sufficiently stable and active under such conditions [35].
Fig. 4. Degree of crosslinking in the collagen. Values are mean ± SD (n=4).
The degree of crosslinking was determined as the decrease in the free amino group content
of the collagen molecules [19, 26]. The free amino group content was measured
spectrophotometrically after the reaction of the free amino groups with 2,4,6-trinitro-
benzensulfonic acid and was expressed as the decrease in the ratio of the free amino group
content of the crosslinked sample to that of the uncrosslinked sample. An increase in the
degree of crosslinking with increase in the EDC concentration was observed (Fig. 4). The
degree of crosslinking was slight lower at NaCl concentration of 100 mM compared to the
NaCl concentration of 50 mM. This may be attributed to lower ability of fibril formation
observed at NaCl concentration of 50 mM. Fig 2 shows that the plateau level in turbidity at
50 mM NaCl was lower than that at 100 mM NaCl. Lower fibril formation may result in an
increase of nonfibrous aggregates, which can lead to high degree of crosslinking due to
increased sites of crosslinking in monomeric collagens. The synergistic effects of
crosslinking and fibril formation were thought to be complete at EDC and NaCl
concentrations of 70 mM and 100 mM, respectively.
