On the bulk biomechanical behavior of densely cross-linked dentin matrix: The role of induced-glycation, regional dentin sites and chemical inhibitor

Abstract

Collagen glycation takes place under physiological conditions during chronological aging, leading to the formation of advanced glycation end-products (AGEs). AGEs accumulation induces non-enzymatic collagen cross-links increasing tissue stiffness and impairing function. Here, we focused on determining the cumulative effect of induced glycation on the mechanical behavior of highly collagen cross-linked dentin matrices and assess the topical inhibition potential of aminoguanidine. Bulk mechanical characterization suggests that early glycation cross-links significantly increase the tensile strength and stiffness of the dentin matrix and promote a brittle failure response. Histologically, glycation yielded a more mature type I collagen in a densely packed collagen matrix. The time-dependent effect of glycation indicates cumulative damage of dentin matrices that is partially inhibited by aminoguanidine. The regional dentin sites were differently affected by induced-glycation, revealing the crown dentin to be mechanically more affected by the glycation protocol. These findings in human dentin set the foundation for the proposed in vitro ribose-induced glycation model, which produces an early matrix stiffening mechanism by reducing tissue viscoelasticity and can be partially inhibited by topical aminoguanidine.

Keywords: Advanced glycation end-products; Aminoguanidine; Dentin; Glycation; Ribose; Tensile strength.

Fig. 1.

Schematic of specimen preparation for tensile strength test. (A) Crown and root dentin were sectioned axially into 0.5 mm-thick slabs. Sections were trimmed into an hour-glass shape to have the tubule orientation perpendicular to the tensile crosshead direction. (B) Demineralized hour-glass shaped specimen before and after tensile fracture.

Fig. 2.

Ultimate tensile strength. (A) Results of UTS (mean and standard deviation) of the dentin matrix for all variables (pooled means): condition, cumulative time and dentin site. Interactions among study factors were only significant for dentin site vs. time (p = 0.007). Statistical differences were observed within all study factors (p = 0.001). Different symbols represent statistically significant differences (p < 0.05) among groups in each graph. (B) Graphs describe UTS results from crown and root dentin ECM as a function of time. Different symbols represent statistically significant differences (p < 0.05) from all conditions among time points. Gly: ribose-induced glycation, AMG: aminoguanidine-mediated inhibition of glycation.

Fig. 3.

Energy to fracture. (A) Results of the energy to fracture of the dentin matrix presented by studied variables (pooled means). Interactions among factors were only significant for dentin site vs. time (p = 0.004). Statistical differences were observed for study factors: dentin site (p < 0.001) and cumulative time (p < 0.001), but not condition (p = 0.443). Different symbols represent statistically significant differences (p < 0.05) among groups in each graph. (B) Graphs describe energy to fracture results of crown and root dentin ECM as a function of time. Different symbols represent statistically significant differences (p < 0.05) from all conditions among time points. Gly: ribose-induced glycation, AMG: aminoguanidine-mediated inhibition of glycation.

Fig. 4.

Apparent modulus of elasticity. (A) Results of the apparent modulus of elasticity of the dentin matrix presented by study factors (pooled means). No interactions were found between studied factors (p = 0.326). Differences were observed within condition (p < 0.001) and cumulative time (p = 0.016). Different symbols represent statistically significant differences (p < 0.05) among groups in each graph. (B) Graphs describe the apparent modulus of elasticity of crown and root dentin ECM as a function of their experimental conditions. Different symbols represent statistically significant differences (p < 0.05) among all conditions. Gly: ribose-induced glycation, AMG: aminoguanidine-mediated inhibition of glycation.

Fig. 5.

Representative tensile stress-strain curves from experimental groups (plots used force and known cross-section area to calculate the stress over the displacement deformation of the specimen) after 7 (A), 14 (B) and 21 (C) days from crown and root dentin. Different from control group, the stress-strain slope of the ribose-induced glycation group showed little to no plastic deformation upon fracture, revealing a brittle failure with more pronounced changes after 21 days. After 14 days, curves from root dentin ECM showed a characteristic initial plateau before a rise in the stress until fracture, likely due to the alignment of the fibrils towards the force direction. Gly: ribose-induced glycation, AMG: aminoguanidine-mediated inhibition of glycation.

Fig. 6.

Representative images of the picrosirius red staining of crown dentin extracellular matrix from control (A), Gly (B) and AMG (C) groups at different time points. Dentin matrix from Gly group showed more densely packed mature collagen as it increased 1.5 to 2 times the red birefringence when compared to control. Scale bar corresponds to 50 μm. Gly: ribose-induced glycation, AMG: aminoguanidine-mediated inhibition of glycation.

Fig. 7.

Results of percentage of mature collagen detected under red birefringence polarized microscopy of crown dentin sections stained with picrosirius red under experimental conditions and different time points. No interaction was found between conditions and cumulative times (p = 0.359). Statistical differences were observed within conditions (p < 0.001). Bar represents lack of statistical significant differences (p > 0.05). Gly: ribose-induced glycation, AMG: aminoguanidine-mediated inhibition of glycation.

Fig. 8.

Raman spectroscopy analysis of the dentin matrix after 21-day incubation. Ratios were calculated with the integrated peak of designated Raman bands. Graphs exhibit ratios (mean and standard error) of collagen maturity (A), post-translational modifications of the extracellular matrix (B), and pentosidine content (C). The symbol (*) depicts statistically significant differences when compared to other groups (p < 0.05). Gly: ribose-induced glycation, AMG: aminoguanidine-mediated inhibition of glycation.