Hamid Nurrohman

Apatite crystal protection against acid-attack beneath resin-dentin interface with four adhesives: TEM and crystallography evidence.

OBJECTIVES Interaction between specific functional groups and apatite crystals may contribute to adhesion. The present study investigated effects of four adhesives with different compositions on protection of crystals beneath the hybrid layer against acid-attack using transmission electron microscopy (TEM) and selected area electron diffraction (SAED). METHODS Human dentin was bonded with four adhesives; two with a carboxylic-based functional co-polymer (PAA): three-step etch-and-rinse Scotchbond Multi-Purpose (SMP, 3M ESPE) and one-step self-etch Adper Easy Bond (AEB, 3M ESPE), and two with a phosphate-based functional monomer (MDP): two-step etch-and-rinse Clearfil Photo Bond (CPB, Kuraray Medical) and two-step self-etch Clearfil SE Bond (CSE, Kuraray Medical). The specimens were the either left untreated (control) or subjected to acid-base challenge with demineralizing solution (pH 4.5) and 5% NaClO. All specimens were processed and observed by TEM. SAED was used to identify the presence or absence of apatite crystallites at the base or beneath hybrid layer before and after acid-base challenge. RESULTS An apatite-rich zone was observed beneath the partially demineralized hybrid layer of CSE. The zone was thinner in AEB, but a demineralization-susceptible area was found beneath it. The etch-and-rinse adhesives (SMP and CPB) demonstrated completely or predominantly demineralized hybrid layers, which were devoid of the acid-resistant apatite-rich zone. SIGNIFICANCE TEM/SAED evidence disclosed that the preserved dentin apatite crystals beneath the thin hybrid layer of the mild self-etch adhesives were protected against acid. Diffusion of reactive components beyond the hybrid layer, and their chemical bonding potential with the remaining crystals created the acid-base resistant zone.

Nanoleakage in Hybrid Layer and Acid–Base Resistant Zone at the Adhesive/Dentin Interface

The aim of interfacial nanoleakage evaluation is to gain a better understanding of degradation of the adhesive-dentin interface. The acid-base resistant zone (ABRZ) is recognized at the bonded interface under the hybrid layer (HL) in self-etch adhesive systems after an acid-base challenge. The purpose of this study was to evaluate nanoleakage in HL and ABRZ using three self-etch adhesives; Clearfil SE Bond (SEB), Clearfil SE One (SEO), and G-Bond Plus (GBP). One of the three adhesives was applied on the ground dentin surface and light cured. The specimens were longitudinally divided into two halves. One half remained as the control group. The others were immersed in ammoniacal silver nitrate solution, followed by photo developing solution under fluorescent light. Following this, the specimens were subjected to acid-base challenges with an artificial demineralization solution (pH4.5) and sodium hypochlorite, and prepared in accordance with common procedures for transmission electron microscopy (TEM) examination. The TEM images revealed silver depositions in HL and ABRZ due to nanoleakage in all the adhesives; however, the extent of nanoleakage was material dependent. Funnel-shaped erosion beneath the ABRZ was observed only in the all-in-one adhesive systems; SEO and GBP, but not in the two-step self-etch adhesive system; SEB.

Immobilization of phosphate monomers on collagen induces biomimetic mineralization.

BACKGROUND Immobilization of phosphoproteins on type-I collagen via covalent binding may induce extra- and intrafibrillar mineralization. OBJECTIVE This study tested the hypothesis that methacrylate phosphate esters immobilized on reconstituted type-I collagen can mimic the nucleating role of phosphoproteins. METHODS Three functional monomers (MDP, GPDM and Phenyl-P) that differed in chemical structure and steric hindrances around the phosphate moiety were evaluated. Reconstituted type-I collagen was either left untouched (control) or treated by 5% monomer/ethanol for 20 s. All samples were incubated in simulated dentinal fluid as mineralizing medium at 37°C for 7 or 14 days. The extra- and intrafibrillar mineralization were examined by SEM and TEM/SAED crystallography, respectively. RESULTS FT-IR spectroscopy showed that the phosphate groups were incorporated on reconstituted collagen, irrespective of their chemical structure. MDP immobilization induced dense growth of extrafibrillar mineral over time, while with GPDM- and Phenyl-P-immobilized collagen, mineralization was moderate and sparse, respectively. TEM/SAED evidence disclosed that intrafibrillar minerals exclusively occurred in MDP-immobilized collagen. CONCLUSIONS Immobilization of MDP, which had the lowest steric hindrance, could induce significant biomimetic extra- and intrafibrillar mineralization; resembling the lowest level of hierarchy organization of dentin.

Dentin bonding performance and ability of four MMA-based adhesive resins to prevent demineralization along the hybrid layer.

PURPOSE To evaluate the tensile bond durability and ability of four MMA-based adhesive resins to prevent demineralization along the hybrid layer when exposed to a demineralizing solution. MATERIALS AND METHODS A PMMA rod was bonded to human dentin using one of four MMA-based adhesive systems: Super-Bond C&B (SB), SBP-40TX (SBP, experimental), M-Bond (MB), and M-Bond II (MB II). Bonded specimens were sectioned into 0.9 mm x 0.9 mm beams and subjected to microtensile bond strength (µTBS) testing after water storage at 37°C for 24 h or 10,000 thermal cycles. Data were analyzed with two-way ANOVA and Bonferronis t-test. Fracture mode analysis of the bonding interface was performed using a scanning electron microscope (SEM) and statistically analysed using the chi-square test. To disclose the demineralization inhibition potential through formation of an acid-base resistant zone (ABRZ), the bonded interface was exposed to a demineralizing solution (pH 4.5) for 90 min, and then 5% NaOCl for 20 min. After argon-ion etching, the interfacial ultrastructure was observed using an SEM. RESULTS µTBS values without thermocycling were not significantly different (p > 0.05) among 4 adhesive resins. After thermocycling, a significant decrease in µTBS was found in MB and MB II (p < 0.001), whereas SB and SBP showed no significant change (p > 0.05). Failure modes were significantly different (p < 0.05). An ABRZ was seen in SBP, MB, and MB II, while this protective zone was absent in SB. CONCLUSION Dentin bonding performance after thermocycling was material dependent in MMA-based adhesive resins. The ABRZ formation was only observed in the self-etching systems.

The role of protease inhibitors on the remineralization of demineralized dentin using the PILP method

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.

Influence of fluoride on the mineralization of collagen via the polymer-induced liquid-precursor (PILP) process

OBJECTIVE The polymer-induced liquid-precursor (PILP) mineralization process has been shown to remineralize artificial dentin lesions to levels consistent with those of native dentin. However, nanoindentation revealed that the moduli of those remineralized lesions were only ∼50% that of native dentin. We hypothesize that this may be due to the PILP process having been previously optimized to obtain high amounts (∼70wt%) of intrafibrillar crystals, but without sufficient interfibrillar mineral, another significant component of dentin. METHODS Fluoride was added to the PILP-mineralization of collagen from rat tail tendon at varying concentrations to determine if a better balance of intra- versus inter-fibrillar mineralization could be obtained, as determined by electron microscopy. Nanoindentation was used to determine if fluoridated apatite could improve the mechanical properties of the composites. RESULTS Fluoride was successfully incorporated into the PILP-mineralization of rat tail tendon and resulted in collagen-mineral composite systems with the mineral phase of hydroxyapatite containing various levels of fluoridation. As the fluoride concentration increased, the crystals became larger and more rod-like, with an increasing tendency to form on the fibril surfaces rather than the interior. Nanomechanical testing of the mineralized tendons revealed that fluoride addition did not increase modulus over PILP mineralization alone. This likely resulted from the separated nature of collagen fibrils that comprise tendon, which does not provide lateral reinforcement and therefore may not be suited for the compressive loads of nanoindentation. SIGNIFICANCE This work contributes to the development of minimally invasive approaches to caries treatment by determining if collagen can be functionally mineralized.