ATR-FTIR Characterization of Dentin Surface Exposed to Collagen Cross-linking Agents

Objectives: To determine the degree of non-enzymatic collagen cross-linking of human crown dentin treated with various cross-linking agents used at different exposure times.

Methods: Dentin surfaces were analyzed using attenuated total reflectance fourier transform-infrared spectroscopy (ATR-FTIR). Spectra were recorded using a FTLA2000 Series spectrometer (ABB Bomem Inc.) in the range of 400-4000 cm^-1 using single-beam with 4 cm^-1 resolution and averaging 256 scans. Spectra from the same sections (N=5) were obtained at different timepoints: undemineralized, demineralized surface (20-sec 35% phosphoric acid), cumulative exposure to cross-linking agents: 5% glutaraldehyde, carbodiimide/N-Hydroxysuccinimide (5.75%/1.38% w:v in water), and grape seed extract (6.5 % and 30% GSE w:v in water) for 30-sec, 60-sec, 2-min, 5-min and 10-min. The data was statistically analyzed by Kruskal-Wallis test, followed by Mann-Whitney analysis for independent samples (cross-linking agents), and Wilcoxon signed-rank test for dependent values (different exposure times); 95% confidence level.

Results: The IR absorption peaks assigned to the dentin collagen; amide I and II band complexes observed between 1700-1600 cm^-1, 1575-1520 cm^-1 respectively and peaks assigned to the phosphate group of hydroxyapatite observed between 1105-1026 cm^-1 were consistent with previous publications. After 5-min exposure to the cross-linking agents, 30% GSE group showed the highest intensity (p=0.016) for the peak observed approximately at 1653 cm^-1, an indicator of intermolecular hydrogen bonded carbonyls. Also, the intensity of the peak indicating trivalent cross-linking in collagen (approximately at 1457 cm^-1) was used to make direct comparisons of the efficacy of cross-linking agents, the average intensity of this peak was highest at 30% GSE group, compared to the other cross-linking agents.

Conclusions: Exposing dentin to cross-linking agents alters its chemical composition by increasing intra and interfibrillar cross-linking of type-I collagen. Grape seed extract induced the highest extend of hydrogen bonding and trivalent interfibrillar cross-linking of dentin collagen in comparison to other cross-linking agents.