Speaker
Description
Crosslinking of collagen is a critical mechanism in bioengineering and human health. Crosslinks are used to strengthen in-vitro assembled collagen materials, yet excessive crosslinking in-vivo, due to factors such as aging and high blood sugar, can lead to functional loss in human tissues. There is a lack of understanding regarding the effects of crosslinking on collagen molecular structure. The triple helix structure of collagen has traditionally only been accessible using X-ray scattering techniques, measured over macroscopic regions. Second harmonic generation (SHG) microscopy is uniquely positioned to measure the triple-helix structure of collagen molecules with sub-micron resolution.
In this work, we used a laser-scanning polarization-in polarization-out second harmonic generation (PIPO-SHG) microscope to investigate changes in the collagen triple-helix structure under different crosslinking conditions. Collagen fibrils were assembled in-vitro from type I bovine telocollagen, then incubated in known crosslinkers glutaraldehyde, methylglyoxal, or ribose.
By fitting to SHG intensity images at different polarizations, we measured a decrease in molecular tilt angle from 44.9 ± 0.3° to 39.7 ± 0.6° after glutaraldehyde treatment. Similar trends were observed for methylglyoxal and ribose. This suggested a stretching of the triple helix due to intermolecular strain. However, the glutaraldehyde-treated fibrils had an angle very close to the SHG “magic angle” of 39.2°, observed when there are significant levels of molecular disorder (Simpson, 1999). To distinguish between the two interpretations, we imaged crosslinked fibrils with atomic force microscopy (AFM), which revealed that the fibril D-band repeat remained unchanged. This indicates that the decrease in molecular tilt angle after collagen crosslinking is due to molecular disorder, rather than a strain-induced shift in the triple helix structure. Our work offers an empirical measurement of the molecular structure of collagen crosslinking, with implications for in-vitro collagen material design and quality control as well as aging and disease mechanisms in in-vivo collagen tissues.
| Keyword-1 | Collagen |
|---|---|
| Keyword-2 | Second harmonic generation |
| Keyword-3 | Crosslinking |