Chemical Markers of Human Tendon Health Identified Using Raman Spectroscopy: Potential for In Vivo Assessment

Abstract

The purpose of this study is to determine whether age-related changes to tendon matrix molecules can be detected using Raman spectroscopy. Raman spectra were collected from human Achilles (n = 8) and tibialis anterior (n = 8) tendon tissue excised from young (17 ± 3 years) and old (72 ± 7 years) age groups. Normalised Raman spectra underwent principal component analysis (PCA), to objectively identify differences between age groups and tendon types. Certain Raman band intensities were correlated with levels of advanced glycation end-product (AGE) collagen crosslinks, quantified using conventional destructive biochemistry techniques. Achilles and tibialis anterior tendons in the old age group demonstrated significantly higher overall Raman intensities and fluorescence levels compared to young tendons. PCA was able to distinguish young and old age groups and different tendon types. Raman intensities differed significantly for several bands, including those previously associated with AGE crosslinks, where a significant positive correlation with biochemical measures was demonstrated. Differences in Raman spectra between old and young tendon tissue and correlation with AGE crosslinks provides the basis for quantifying age-related chemical modifications to tendon matrix molecules in intact tissue. Our results suggest that Raman spectroscopy may provide a powerful tool to assess tendon health and vitality in the future.

Keywords: Achilles tendon; Raman spectroscopy; advanced glycation end-product; collagen crosslinks; extracellular matrix.

Figure 1

Averaged (A) and normalised (B) raw CCD counts (n = 160, 40 per group) between young and old Achilles tendons and tibialis anterior tendons. Inset: representative figure of different background shapes between young (black) and old (red) tendons.

Figure 2

Principle component analysis of pooled Raman spectra (n = 160).

Figure 3

Averaged Raman spectra of young (n = 80, black lines) and old (n = 80, red lines) tendons (upper row) and the PC2 loading (lower row).

Figure 4

Group comparison between different Raman band intensities. Young group: n = 8 specimens, 10 spectra per specimen; Old group: n = 8 specimens, 10 spectra per specimen; *: p < 0.05.

Figure 5

Averaged Raman spectra of Achilles (n = 80, black lines, upper row) and tibialis anterior tendons (n = 80, red lines) and the PC1 loading (lower row).