Dentin collagen denaturation status assessed by collagen hybridizing peptide and its effect on bio-stabilization of proanthocyanidins

Abstract

Objective: To assess dentin collagen denaturation from phosphoric acid and enzyme treatments using collagen hybridizing peptide (CHP) and to investigate the effect of collagen denaturation on bio-stabilization promoted by proanthocyanidins (PA).

Methods: Human molars were sectioned into 7-µm-thick dentin films, demineralized, and assigned to six groups: control with/without PA modification, H₃PO₄-treated collagen with/without PA modification, enzyme-treated collagen with/without PA modification. PA modification involved immersing collagen films in 0.65% PA for 30 s. H₃PO₄ and enzyme treatments were used to experimentally induce collagen denaturation, which was quantitated by fluorescence intensity (FI) from the fluorescently-conjugated-CHP (F-CHP) staining (n = 4). FTIR was used to characterize collagen structures. All groups were subject to collagenase digestion to test the bio-stabilization effect of PA on denatured collagen using weight loss analysis and hydroxyproline assay (n = 6). Data were analyzed using two-factor ANOVA and Games-Howell post hoc tests (α = 0.05).

Results: FTIR showed collagen secondary structural changes after denaturation treatments and confirmed the incorporation and cross-linking of PA in control and treated collagen. F-CHP staining indicated high-degree, medium-degree, and low-degree collagen denaturation from H₃PO₄-treatment (FI = 83.22), enzyme-treatment (FI = 36.54), and control (FI = 6.01) respectively. PA modification significantly reduced the weight loss and hydroxyproline release of all groups after digestion (p < 0.0001), with the results correlated with FI values at r = 0.96-0.98.

Significance: A molecular method CHP is introduced as a sensitive technique to quantitate dentin collagen denaturation for the first time. PA modification is shown to effectively stabilize denatured collagen against collagenase digestion, with the stabilization effect negatively associated with the collagen denaturation degree.

Keywords: Collagen bio-stabilization; Collagen hybridizing peptide; Cross-linking; Dentin collagen denaturation; FTIR; Hydroxyproline assay; Proanthocyanidins; Weight loss.