Loss of Stiffness of Demineralized Dentin Associated with Collagen Solubilization

Dentin collagen is the major organic structure of the hybrid layer and its stability may have a great impact on longevity of restorations. Objectives: To evaluate the effect of chlorhexidine (CHX) on the mechanical and biochemical integrity of dentin matrix.

Methods: Sixteen extracted non-carious human molars were sectioned into dentin discs that were further trimmed to generate 1-mm² dentin beams. Specimens were demineralized in 10% phosphoric acid and had their baseline stiffness measured by non-destructive three-point bending test. Specimens were preincubated (30 min) in one of the solutions:  4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES-Control); 2 mM amino-phenylmercuric-acetate (APMA); 0.2% CHX or 2% CHX. All specimens were individually incubated at 37^oC with 1 mL of CaCl₂/ZnCl₂-containing artificial saliva (AS) (pH 7.4) for 4 weeks under constant shaking. Stiffness was re-measured and the supernatant solution was hydrolyzed to reduce any solubilized collagen to amino acids. The concentration of hydroxyproline was spectrophotometrically assayed to estimate the percentage of solubilized collagen.

Results:

		Stiffness of Demineralized Dentin (MPa)		Solubilized Collagen (%)
Preincubation	Incubation	Baseline	after 4 week-incubation	after 4 week-incubation
Control-HEPES	AS	4.64±0.76a	3.01±0.62b	10.8±1.32c
APMA		4.91±1.00a	2.96±0.96b	12.73±0.70c
0.2% CHX		4.75±0.98a	4.87±0.68a	5.08±1.65d
2% CHX		4.34±0.45a	4.18±0.46a	0.46±0.02e

Values are means ± S.D (n=8).  Different superscript letters indicate statistical differences (p<0.05). (two-way-repeated-measures-ANOVA/post-hoc Holm-Sidak).

Conclusion: After 4 weeks, significant loss of stiffness occurred for demineralized dentin specimens that were preincubated in HEPES (Control) and APMA. Conversely, stiffness was preserved for specimens preincubated in CHX. Collagen was found to be slightly more solubilized in APMA than in Control. The amount of solubilized collagen in CHX-pretreated specimens was significantly lower, especially for the most concentrated solution (2% CHX). This method can be used to screen potential inhibitors of collagenase activity of dentin. Supported, in part, by grants R01DE015306 from the NIDCR (D.P.-P.I.) and 473164/2007-8 from the CNPq (M.C.-P.I).