Characterization of biomodified dentin matrices for potential preventive and reparative therapies

Abstract

Biomodification of existing hard tissue structures, specifically tooth dentin, is an innovative approach proposed to improve the biomechanical and biochemical properties of tissue for potential preventive or reparative therapies. The objectives of the study were to systematically characterize dentin matrices biomodified by proanthocyanidin-rich grape seed extract (GSE) and glutaraldehyde (GD). Changes to the biochemistry and biomechanical properties were assessed by several assays to investigate the degree of interaction, biodegradation rates, proteoglycan interaction, and effect of collagen fibril orientation and environmental conditions on the tensile properties. The highest degree of agent-dentin interaction was observed with GSE, which exhibited the highest denaturation temperature, regardless of the agent concentration. Biodegradation rates decreased remarkably following biomodification of dentin matrices after 24h collagenase digestion. A significant decrease in the proteoglycan content of GSE-treated samples was observed using a micro-assay for glycosaminoglycans and histological electron microscopy, while no changes were observed for GD and the control. The tensile strength properties of GD-biomodified dentin matrices were affected by dentin tubule orientation, most likely due to the orientation of the collagen fibrils. Higher and/or increased stability of the tensile properties of GD- and GSE-treated samples were observed following exposure to collagenase and 8 months water storage. Biomodification of dentin matrices using chemical agents not only affects the collagen biochemistry, but also involves interaction with proteoglycans. Tissue biomodifiers interact differently with dentin matrices and may provide the tissue with enhanced preventive and restorative/reparative abilities.

Figure 1

Illustration of sample preparation: A –Dentin section obtained from sectioning crowns at either 0.5 or 0.2 thickness; B – Dentin sample obtained for temperature denaturation analysis (dimensions 0.5 × 2.0 × 2.0 mm); and C – Sample for ultimate tensile strength testing prepared (dimensions 0.5 × 0.5 mm neck) according to the tubule orientation (parallel or perpendicular).

Figure 2

High resolution field emission scanning electron micrograph (left image) of demineralized dentin showing the orientation of collagen fibrils from inter-tubular dentin surrounding the dentinal tubules and the tensile force orientation for the ultimate tensile strength evaluation. Schematic picture on the right shows the presence and location of intra-, inter-molecular and inter-microfibrillar cross-links.

Figure 3

Representative standard DSC graphs (Temperature °C × heat flow mW) of specimens biomodified with 6.5% GSE and untreated (control). Denaturation temperature (T_d), is detected by abrupt changes in the heat flow. Note the multiple peaks for GSE; the most defined peak (asterisk) was selected to characterize the tissue.

Figure 4

Representative transmission electron images of demineralized dentin matrix: A – dentin matrices biomodified with 5% GD depicted dark filaments (arrows) indicative of proteoglycans throughout the sample; B – dentin matrices biomodified with 6.5% GSE showed a remarkable decrease in the presence of filaments, no filaments were observed in most sections; C – control samples shows similar morphology as GD groups, where proteoglycans (arrows) are abundant throughout the sample. (scale bar = 200µm)