Structural and biomechanical changes to dentin extracellular matrix following chemical removal of proteoglycans

Abstract

Proteoglycans are biomacromolecules with significant biomineralization and structural roles in the dentin extracellular matrix. This study comprehensively assessed the mechanical properties and morphology of the dentin extracellular matrix following chemical removal of proteoglycans to elucidate the structural roles of proteoglycans in dentin. Dentin extracellular matrix was prepared from extracted teeth after complete tissue demineralization. Chemical removal of proteoglycans was carried-out using guanidine hydrochloride for up to 10 days. The removal of proteoglycans was determined by dimethylmethylene blue colorimetric assay and histological staining analyses using transmission electron microscopy and optical microscopy. The modulus of elasticity of dentin matrix was determined by a 3-point bending test method. Partial removal of proteoglycans induced significant modifications to the dentin matrix, particularly to type I collagen. Removal of proteoglycans significantly decreased the modulus of elasticity of dentin extracellular matrix (p < 0.0001). In conclusion, the subtle disruption of proteoglycans induces pronounced changes to the collagen network packing and the bulk modulus of elasticity of dentin matrix.

Keywords: Collagen; Dentin; Histology; Modulus of elasticity; Proteoglycans.

Fig. 1

Results of the apparent modulus of elasticity (E) of dentin matrix incubated in GuHCl for 10 days. Symbol (δ) indicate where statistically significant differences were found between control and GuHCl groups at each incubation day (p < 0.05). Asterisk (*) indicates statistical differences between baseline measurements and incubation time for both groups (p < 0.05). There were no differences in E among incubation times for control and GuHCl groups (p > 0.05). Control group: incubation media; GuHCL: incubation with guanidine hydrochloride; D: incubation days.

Fig. 2

Representative light microscopy images of dentin section stained with Alcian blue (AB) to evaluate proteoglycans removal by GuHCl. Black arrows points to dentinal tubules. A, untreated group (control); B, 4 days of GuHCl incubation; C, 6 days of GuHCl incubation; D, 10 days of GuHCl incubation. Graph shows quantification of AB-staining in light microscopy images of AB-stained dentin sections after proteoglycans removal by GuHCl incubation for 4, 6 and 10 days. There were no statistical differences among groups (p > 0.05). D: Days. Scale bar = 50 μm.

Fig. 3

Representative transmission electron microscopy images of demineralized dentin matrix. A, control group specimens display dentin matrix morphology with abundance of proteoglycans (dark filaments), more prominent surrounding the dentin tubules (T); B and C, dentin matrices treated with GuHCl solution for 4 and 6 days respectively show similar dark filaments (proteoglycans) distribution as the control samples. Scale bar = 100 nm.

Fig. 4

Light microscopy representative images of demineralized dentin sections stained with picrosirius red. A-C, control group, without GuHCl; D-F, 6 days of GuHCl incubation, exhibiting loss on interfibrillar packing (increase interfibrilar spacing). Graph shows quantification of picrosirius red staining of collagen observed in light microscopy images of dentin sections after GuHCl or control incubation for 6 days (n= 10). (*) depicts statistical differences between groups (p > 0.05). Scale bar = 100 μm.