Abstract
Mineralized and sound dentin matrices contain inactive preforms of proteolytic enzymes that may be activated during the demineralization cycle. In this study, we tested the hypothesis that protease inhibitors (PI) preserve demineralized collagen fibrils and other constituents of the dentin matrix and thereby affect the potential for remineralization. Artificial carious lesions with lesion depths of 140 μm were created with acetate buffer (pH = 5.0, 66 hours), and remineralized using a polymer-induced-liquid-precursor (PILP) process (pH = 7.4, 14 days) containing poly(aspartic acid) (pAsp) as the process-directing agent. De- and remineralizing procedures were performed in the presence or absence of PI. Ultrastructure and mechanical recovery of demineralized dentin following PILP remineralization were examined and measured in water with atomic force microscopy (AFM) and nanoindentation. Nanomechanical properties of hydrated artificial lesions had a low elastic modulus (ER <0.4 GPa) extending about 100 μm into the lesion, followed by a sloped region of about 140 μm depth where values reached those of normal dentin (18.0–20.0 GPa). Mapping of mineral content by both micro-FTIR and micro x-ray computed tomography correlated well with modulus profiles obtained by nanoindentation. Tissue demineralized in the presence of PI exhibited higher elastic moduli (average 2.8 GPa) across the lesion and comprised a narrow zone in the outer lesion with strongly increased modulus (up to 8 GPa; p < 0.05), which might be related to the preservation of non-collagenous proteins that appear to induce calcium phosphate mineral formation even under demineralizing physical-chemical conditions. However, mechanical aspects of remineralization through the elastic modulus change, and the micromorphological aspects with SEM and TEM observation were almost identical with PILP treatments being conducted in the presence or absence of PI. Thus, the application of the protease inhibitors (PI) seemed to be less effective in promoting the remineralization of demineralized dentin.
Highlights
Extrafibrillar and more importantly intrafibrillar remineralization of collagen type I matrices has been suggested to be critical to restoration of the mechanical properties of dentin [1,2,3]
When protease inhibitors (PI) were present during the demineralization process (DEpi), shrinkage was considerably less (Fig 2B) and measured only 28.2 ± 3.2 μm
Remineralization succeeded in reinforcement of the dentin matrix and reduced the shrinkage to 4.2 ± 1.0, 4.1 ± 0.4 and 0.5 ± 0.7 μm for demineralization followed by remineralization (DE-REM), DE-REMpi and demineralization in the presence of PI (DEpi)-REMpi, respectively (Fig 2C–2E)
Summary
Extrafibrillar and more importantly intrafibrillar remineralization of collagen type I matrices has been suggested to be critical to restoration of the mechanical properties of dentin [1,2,3]. The PILP process consists of anionic polymer macromolecules poly(aspartic acid), a simple mimic for acidic or phosphorylated non-collagenous proteins, which stabilizes a supersaturated mineralization solution by interacting with calcium and phosphate ions and forming nanodroplets of about 15 to 30 nm in diameter [6, 7]. After PILP remineralization, there was complete recovery of mineral content throughout the lesion depth, while nanomechanical testing found the reduced elastic modulus (ER) recovered to 50–60% of normal dentin in the severely demineralized outer zone and full recovery of the inner zone. If process-directing agent like poly(aspartic acid) are unavailable, recovery of properties occurred only in the sloped inner zone of the lesion, while the outer fully demineralized zone did not show any enhancement at all as using calcium phosphate solutions alone was not able to induce intrafibrillar mineral in collagen [12]
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