Electrophoresis-Aided Biomimetic Mineralization System Using Graphene Oxide for Regeneration of Hydroxyapatite on Dentin

Graphene oxide (GO) is an emerging luminescent carbon nanomaterial with the ability to foster hydroxyapatite (HA). A specially designed electrophoresis system can be used to accelerate the mineralization process. The aim of this study was to promote HA crystal growth on demineralized dentin using a GO incorporated electrophoresis system. GO was successfully synthesized by carbonization of citric acid and its presence was confirmed by Fourier transform infrared and UV-visible spectrophotometry evaluation. Dentin slices were placed in demineralized solution and divided into control (without the electrophoresis system) and experimental group. Demineralized dentin slices in the experimental group were remineralized using the electrophoresis system for 8 h/1.0 mA, with one subgroup treated without GO and the other with GO. Energy dispersive spectroscopy evaluation showed that the calcium/phosphate ratio of the crystal formed in control and experimental group with addition of GO was close to natural hydroxyapatite. However, scanning electron microscopy evaluation showed that the exposed dentinal tubules were occluded with rod-like crystals, which is similar to native enamel morphology, in the experimental group with addition of GO compared to the flake-like crystal in the control group. Mechanical evaluation revealed that the nanohardness and modulus of remineralized dentin were significantly higher in the experimental group. In conclusion, GO is a promising material to remineralize dentin and the introduction of an electrophoresis system can accelerate its process.

Chemical Properties of Human Dentin Blocks and Vertical Augmentation by Ultrasonically Demineralized Dentin Matrix Blocks on Scratched Skull without Periosteum of Adult-Aged Rats

Vertical augmentation is one of the most challenging techniques in bone engineering. Several parameters, such mechano-chemical characteristics, are important to optimize vertical bone regeneration using biomaterials. The aims of this study were to chemically characterize human dentin blocks (calcified demineralized dentin matrix: CDM, partially demineralized dentin matrix: PDDM and completely demineralized dentin matrix: CDDM) (2 × 2 × 1 mm3) chemically and evaluate the behavior of PDDM blocks on non-scratched or scratched skulls without periosteum of adult rats (10–12 months old, female) as a vertical augmentation model. The dissolved efficiency of CDM showed 32.3% after ultrasonic demineralization in 1.0 L of 2% HNO3 for 30 min. The 30 min-demineralized dentin was named PDDM. The SEM images of PDDM showed the opening of dentinal tubes, nano-microcracks and the smooth surface. In the collagenase digestion test, the weight-decreasing rates of CDM, PDDM and CDDM were 9.2%, 25.5% and 78.3% at 12 weeks, respectively. CDM inhibited the collagenase digestion, compared with PDDM and CDDM. In the PDDM onlay graft on an ultrasonically scratched skull, the bone marrow-space opening from original bone was found in the bony bridge formation between the human PDDM block and dense skull of adult senior rats at 4 and 8 weeks. On the other hand, in the cases of the marrow-space closing in both non-scratched skulls and scratched skulls, the bony bridge was not formed. The results indicated that the ultrasonic scratching into the compact parietal bone might contribute greatly to the marrow-space opening from skull and the supply of marrow cells, and then bony bridge formation could occur in the vertical augmentation model without a periosteum.

Hydroxypropylmethylcellulose as a film and hydrogel carrier for ACP nanoprecursors to deliver biomimetic mineralization

Demineralization of hard tooth tissues leads to dental caries, which cause health problems and economic burdens throughout the world. A biomimetic mineralization strategy is expected to reverse early dental caries. Commercially available anti-carious mineralizing products lead to inconclusive clinical results because they cannot continuously replenish the required calcium and phosphate resources. Herein, we prepared a mineralizing film consisting of hydroxypropylmethylcellulose (HPMC) and polyaspartic acid-stabilized amorphous calcium phosphate (PAsp-ACP) nanoparticles. HPMC which contains multiple hydroxyl groups is a film-forming material that can be desiccated to form a dry film. In a moist environment, this film gradually changes into a gel. HPMC was used as the carrier of PAsp-ACP nanoparticles to deliver biomimetic mineralization. Our results indicated that the hydroxyl and methoxyl groups of HPMC could assist the stability of PAsp-ACP nanoparticles and maintain their biomimetic mineralization activity. The results further demonstrated that the bioinspired mineralizing film induced the early mineralization of demineralized dentin after 24 h with increasing mineralization of the whole demineralized dentin (3–4 µm) after 72–96 h. Furthermore, these results were achieved without any cytotoxicity or mucosa irritation. Therefore, this mineralizing film shows promise for use in preventive dentistry due to its efficient mineralization capability. Graphical Abstract

Evaluation of surface roughness and Vickers microhardness of various nano-herbal extracts on demineralized dentin and their bactericidal efficacy with 970-nm wavelength diode laser irradiation

Objective Was to evaluate effect of clove, turmeric and garlic nano-herbal extracts on surface roughness and microhardness of demineralized dentin, and their bactericidal effect on Streptococcus mutans and Streptococcus sobrinus with or without diode laser irradiation. Methods Three 5% nano-formulas were prepared and characterized using transmission electron microscope. MI paste Plus™ was used as control. A total of 100 specimens of demineralized dentin were prepared and treated with 3 W-power diode laser; then, the different tested materials for 10-min before the surface roughness and Vickers microhardness tests were conducted. Eighty coronal cavities were prepared (1-mm diameter × 2-mm depth). Cavities were inoculated with the tested materials with S. mutans or S. sobrinus bacteria, with or without diode laser irradiation for 20-s. Colony-forming unit method was used for counting the viable bacteria. Data were explored for normality using Kolmogorov–Smirnov and Shapiro–Wilk tests and showed parametric distribution for the surface roughness and microhardness tests, and non-parametric distribution for the bactericidal activity test. Results The herbal formulas had a significant surface roughness and microhardness mean values. It showed a significant antimicrobial effect on the tested bacteria. When they were combined with diode laser, they showed a significantly higher antimicrobial effect. Conclusions The tested herbal formulas represent potent topical remineralizing and antibacterial agents especially when they are used in conjunction with diode laser irradiation.

Effect of Novel Bioactive Glass-Containing Dentin Adhesive on the Permeability of Demineralized Dentin

This study aimed to evaluate the effect of a novel bioactive glass (BAG)-containing dentin adhesive on the permeability of demineralized dentin. Bioactive glass (85% SiO2, 15% CaO) was fabricated using the sol-gel process, and two experimental dentin adhesives were prepared with 3 wt% silica (silica-containing dentin adhesive; SCA) or BAG (BAG-containing dentin adhesive; BCA). Micro-tensile bond strength (μTBS) test, fracture mode analysis, field-emission scanning electron microscopy (FE-SEM) analysis of adhesive and demineralized dentin, real-time dentinal fluid flow (DFF) rate measurement, and Raman confocal microscopy were performed to compare SCA and BCA. There was no difference in μTBS between the SCA and BCA (p > 0.05). Multiple precipitates were evident on the surface of the BCA, and partial occlusion of dentinal tubules was observed in FE-SEM of BCA-approximated dentin. The DFF rate was reduced by 50.10% after BCA approximation and increased by 6.54% after SCA approximation. Raman confocal spectroscopy revealed an increased intensity of the hydroxyapatite (HA) peak on the dentin surface after BCA application. The novel BAG-containing dentin adhesive showed the potential of both reducing dentin permeability and dentin remineralization.

Bone regeneration of demineralized dentin matrix with platelet-rich fibrin and recombinant human bone morphogenetic protein-2 on the bone defects in rabbit calvaria

Background This study was to evaluate the bone formation ability of demineralized dentin matrix (DDM) combined with platelet-rich fibrinogen (PRF) and DDM combined with recombinant human bone morphogenetic protein-2 (rhBMP-2) to improve the osteoinductive ability of DDM. Methods After four bone defects with a diameter of 8mm were created in the calvarium of each rabbit, DDM was grafted into the first defect (experimental groups 1), a combination of DDM and PRF was grafted into the second defect (experimental groups 2), and DDM with absorbed rhBMP-2 was grafted into the third defect (experimental groups 3). The fourth defect was used as the control group. Twelve healthy male rabbits (New Zealand, white rabbits) weighing around 3.0–4.0 kg were used. Among 12 rabbits, 3 rabbits were sacrificed immediately after surgery and at 2, 4, and 8 weeks after surgery, respectively. Histopathologic analysis and histomorphometric analysis were conducted to evaluate bone formation in each group. Results The PRF/DDM group did not show a significantly higher degree of new bone formation in calvarial bone defects than the DDM group at 2, 4, and 8 weeks postoperatively in histopathological findings and histomorphometric results. On the other side, the rhBMP-2/DDM group showed higher degrees of new bone formation and calcification, and the lamellae of bone matrix, which are observed in mature bone tissue, were more distinctly visible in the rhBMP-2/DDM group. Moreover, the rhBMP-2/DDM group showed a significantly higher amount of new bone formation, compared to the DDM group at 4 and 8 weeks postoperatively (P<0.05) in histomorphometric results. Conclusion The DDM has great potential as a carrier for the maintenance and sustained release of rhBMP-2, which has been recently receiving wide attention as a type of signaling molecules to promote bone formation.

A Novel Mineralizing film Induces Biomimetic Mineralization

Demineralization of tooth hard tissues leads to dental caries, which causes health problem and economic burden throughout the world. Biomimetic mineralization strategy is expected to reverse initial dental caries. Commercially available mineralizing anti-carious products lead to indefinite clinical results because they could not continuously replenish calcium and phosphate resources. Herein, we prepared a novel mineralizing film (NMF) consisting of hydroxypropylmethylcellulose (HPMC) and polyaspartic acid-stabilized amorphous calcium phosphate (PAsp-ACP) nanoparticles. HPMC containing multiple hydroxyls is a gel-forming material that could be either desiccated to be a dry film or gradually changed into gel in moist milieu. The HPMC was, for the first time, used as a carrier of PAsp-ACP nanoparticles for delivering biomimetic mineralization in moist milieu. Our results indicated the hydroxyls and methoxyls of HPMC could assist the stability of PAsp-ACP nanoparticles and maintain their bioactivity of mineralization. The results further demonstrated that the NMF bioinspired the early mineralization of demineralized dentin at 24 hrs and the heavy mineralization of the whole demineralized dentin (3-4 µm) at 72-96 hrs without any cytotoxicity and mucosa irritation. Therefore, the NMF might be promising mineralizing strategy for preventive dentistry with efficient mineralization capability.

The Amelogenin-Derived Peptide TVH-19 Promotes Dentinal Tubule Occlusion and Mineralization

In this study, the amelogenin-derived peptide, TVH-19, which has been confirmed to promote mineralization, was evaluated to derive its potential to induce dentinal tubule occlusion. The binding capability of fluorescein isothiocyanate (FITC)-labeled TVH-19 to the demineralized dentin surface was analyzed by confocal laser scanning microscopy (CLSM). Additionally, the sealing function of the peptide was studied through the remineralization of demineralized dentin in vitro. The adsorption results showed that TVH-19 could bind to the hydroxyapatite and demineralized dentin surfaces, especially to periodontal dentin. Scanning electron microscopy analysis further revealed that TVH-19 created mineral precipitates. The plugging rate in the TVH-19 group was higher than that in the PBS group. Moreover, energy-dispersive X-ray spectroscopy (EDX) results indicated that the calcium/phosphorus (Ca/P) ratio of the new minerals induced by TVH-19 was close to that of the hydroxyapatite. Attenuated total internal reflection-Fourier transform infrared (ATR-FTIR) spectrometry and X-ray diffraction (XRD) results indicated that the hydroxyapatite crystals formed via remineralization elongated the axial growth and closely resembled the natural dentin components. These findings indicate that TVH-19 can effectively promote dentin sealing by binding to the periodontal dentin, promoting mineral deposition, and reducing the space between the dentin tubules.

Cranberry Juice Extract Rapidly Protects Demineralized Dentin against Digestion and Inhibits Its Gelatinolytic Activity

Improving the longevity of composite restorations has proven to be difficult when they are bonded to dentin. Dentin demineralization leaves collagen fibrils susceptible to enzymatic digestion, which causes breakdown of the resin–dentin interface. Therefore, measures for counteracting the enzymatic environment by enhancing dentin collagen’s resistance to degradation have the potential to improve the durability of dental composite restorations. This study aimed to evaluate the effects of polyphenol-rich extracts and a chemical cross-linker on the cross-linking interaction, resistance to digestion, and endogenous matrix metalloproteinase (MMP) activities of dentin collagen under clinically relevant conditions. Ten-µm-thick films were cut from dentin slabs of non-carious extracted human third molars. Following demineralization, polyphenol-rich extracts—including grape seed (GSE), green tea (GTE), and cranberry juice (CJE)—or chemical cross-linker carbodiimide with n-hydroxysuccinimide (EDC/NHS) were applied to the demineralized dentin surfaces for 30 s. The collagen cross-linking, bio-stabilization, and gelatinolytic activities of MMPs 2 and 9 were studied by using Fourier-transform infrared spectroscopy, weight loss, hydroxyproline release, scanning/transmission electron microscopy, and in situ zymography. All treatments significantly increased resistance to collagenase degradation and reduced the gelatinolytic MMP activity of dentin collagen compared to the untreated control. The CJE- and GSE-treated groups were more resistant to digestion than the GTE- or EDC/NHS-treated ones (p < 0.05), which was consistent with the cross-linking interaction found with FTIR and the in situ performance on the acid-etched dentin surface found with SEM/TEM. The collagen films treated with CJE showed the lowest MMP activity, followed by GSE, GTE, and, finally, EDC/NHS. The CJE-treated dentin collagen rapidly increased its resistance to digestion and MMP inhibition. An application of CJE as short as 30 s may be a clinically feasible approach to improving the longevity of dentin bonding in composite restorations.