Nanoparticle-Based Targeting and Detection of Microcavities.

Although dental caries is the most prevalent oral disease worldwide, currently, many dentists continue to use the traditional mirror and probe (dental explorer) method of caries diagnosis. This method of caries detection has the drawback that it is often difficult to distinguish between active and inactive carious lesions. In this work, novel bio-based nanoparticles are developed to specifically detect active caries in vitro. The nanoparticles are made from a cationic fluorescein-labeled food-grade starch in order to fluoresce when illuminated by a standard dental curing light, and to degrade in the oral cavity into nontoxic compounds after detecting the active carious lesion. When exposed to extracted human teeth, cationic fluorescent (+5.8 ± 1.2 mV) nanoparticles (size 101 ± 56 nm) selectively illuminate active caries, but not the healthy tooth surface. Two-photon microscopy confirms the selective binding and accumulation of cationic fluorescent nanoparticles into microscopic carious pores in enamel. These novel nanoparticles provide a unique method to assist in the early diagnosis of active carious lesions with the potential to directly impact dental treatment.

Nano/micro fluorhydroxyapatite crystal pastes in the treatment of dentin hypersensitivity: an in vitro study.

OBJECTIVES: Dentin hypersensitivity (DH) is a prevalent problem. This study aimed to formulate a paste using fluorhydroxyapatite (FA) crystals dispersed in different carriers to treat DH. The ability to occlude patent dentinal tubules and to release ions was investigated. MATERIALS AND METHODS: Twenty percent FA/sodium alginate, 40% FA/poly(hydroxyethyl methacylate(HEMA)), and 40% FA/poly(DMA-co-MEA) were applied to etched dentin samples and examined with scanning electron microscopy (SEM) to determine the degree of tubule occlusion. Fluoride electrode was used to measure F release and spectroscopy to evaluate Ca and PO4 release. The cytotoxicity of the synthesized poly(DMA-co-MEA) gel was tested. Kruskall-Wallis test was used to test the differences in ion release between the groups. RESULTS: FA/poly(DMA-co-MEA) paste obstructed up to 80% of the dentinal tubules, while the coverage was up to 70% for FA/poly(HEMA) and less than 50% for FA/sodium alginate. Fluoride and Ca release was the highest for FA/P(HEMA), 7.2 ± 0.7 and 139.8 ± 32.5 ppm, respectively. The highest concentration of PO4 was 46.2 ± 16.4 ppm for FA/Sodium alginate. No statistical significance was found. CONCLUSIONS: FA/Poly(DMA-co-MEA) and FA/poly(HEMA) pastes may offer immediate short-term relief of DH because of their ability to occlude the tubules and adhere to wet dentin surfaces. The release of the F, Ca, and PO4 ions may offer long-term relief by forming a mineral barrier both within the dentinal tubules and on the dentin surface. CLINICAL SIGNIFICANCE: The tested materials may offer a long-term treatment for DH.

Enamel crystals of mice susceptible or resistant to dental fluorosis: an AFM study.

OBJECTIVE: This study aimed to assess the overall apatite crystals profile in the enamel matrix of mice susceptible (A/J strain) or resistant (129P3/J strain) to dental fluorosis through analyses by atomic force microscopy (AFM). MATERIAL AND METHODS: Samples from the enamel matrix in the early stages of secretion and maturation were obtained from the incisors of mice from both strains. All detectable traces of matrix protein were removed from the samples by a sequential extraction procedure. The purified crystals (n=13 per strain) were analyzed qualitatively in the AFM. Surface roughness profile (Ra) was measured. RESULTS: The mean (±SD) Ra of the crystals of A/J strain (0.58±0.15 nm) was lower than the one found for the 129P3/J strain (0.66±0.21 nm) but the difference did not reach statistical significance (t=1.187, p=0.247). Crystals of the 129P3/J strain (70.42±6.79 nm) were found to be significantly narrower (t=4.013, p=0.0013) than the same parameter measured for the A/J strain (90.42±15.86 nm). CONCLUSION: enamel crystals of the 129P3/J strain are narrower, which is indicative of slower crystal growth and could interfere in the occurrence of dental fluorosis.

Enamel crystals of mice susceptible or resistant to dental fluorosis: an AFM study

Objective: This study aimed to assess the overall apatite crystals profile in the enamel matrix of mice susceptible (A/J strain) or resistant (129P3/J strain) to dental fluorosis through analyses by atomic force microscopy (AFM). Material and Methods: Samples from the enamel matrix in the early stages of secretion and maturation were obtained from the incisors of mice from both strains. All detectable traces of matrix protein were removed from the samples by a sequential extraction procedure. The purified crystals (n=13 per strain) were analyzed qualitatively in the AFM. Surface roughness profile (Ra) was measured. Results: The mean (±SD) Ra of the crystals of A/J strain (0.58±0.15 nm) was lower than the one found for the 129P3/J strain (0.66±0.21 nm) but the difference did not reach statistical significance (t=1.187, p=0.247). Crystals of the 129P3/J strain (70.42±6.79 nm) were found to be significantly narrower (t=4.013, p=0.0013) than the same parameter measured for the A/J strain (90.42±15.86 nm). Conclusion: enamel crystals of the 129P3/J strain are narrower, which is indicative of slower crystal growth and could interfere in the occurrence of dental fluorosis. .

Fluorapatite enhances mineralization of mesenchymal/endothelial cocultures.

In addition to the widely used mesenchymal stem cells (MSCs), endothelial cells appear to be a favorable cell source for hard tissue regeneration. Previously, fluorapatite was shown to stimulate and enhance mineralization of MSCs. This study aims to investigate the growth of endothelial cells on synthesized ordered fluorapatite surfaces and their effect on the mineralization of adipose-derived stem cells (ASCs) through coculture. Endothelial cells were grown on fluorapatite surfaces and characterized by cell counting, flow cytometry, scanning electron microscopy, and enzyme-linked immunosorbent assay (ELISA). Cells were then cocultured with ASCs and stained for alkaline phosphatase and mineral formation. Fibroblast growth factor (FGF) pathway perturbation and basic FGF (bFGF) treatment of the ASCs were also conducted to observe their effects on differentiation and mineralization of these cells. Fluorapatite surfaces showed good biocompatibility in supporting endothelial cells. Without a mineralization supplement, coculture with endothelial cells induced osteogenic differentiation of ASCs, which was further enhanced by the fluorapatite surfaces. This suggested a combined stimulating effect of endothelial cells and fluorapatite surfaces on the enhanced mineralization of ASCs. Greater amounts of bFGF release by endothelial cells alone or cocultures with ASCs stimulated by fluorapatite surfaces, together with FGF pathway perturbation and bFGF treatment results, suggested that the FGF signaling pathway may function in this process.

In vitro differentiation and mineralization of dental pulp stem cells on enamel-like fluorapatite surfaces.

Our previous studies have shown good biocompatibility of fluorapatite (FA) crystal surfaces in providing a favorable environment for functional cell-matrix interactions of human dental pulp stem cells (DPSCs) and also in supporting their long-term growth. The aim of the current study was to further investigate whether this enamel-like surface can support the differentiation and mineralization of DPSCs, and, therefore, act as a potential model for studying the enamel/dentin interface and, perhaps, dentine/pulp regeneration in tooth tissue engineering. The human pathway-focused osteogenesis polymerase chain reaction (PCR) array demonstrated that the expression of osteogenesis-related genes of human DPSCs was increased on FA surfaces compared with that on etched stainless steel (SSE). Consistent with the PCR array, FA promoted mineralization compared with the SSE surface with or without the addition of a mineralization promoting supplement (MS). This was confirmed by alkaline phosphatase (ALP) staining, Alizarin red staining, and tetracycline staining for mineral formation. In conclusion, FA crystal surfaces, especially ordered (OR) FA surfaces, which mimicked the physical architecture of enamel, provided a favorable extracellular matrix microenvironment for the cells. This resulted in the differentiation of human DPSCs and mineralized tissue formation, and, thus, demonstrated that it may be a promising biomimetic model for dentin-pulp tissue engineering.

The stimulation of adipose-derived stem cell differentiation and mineralization by ordered rod-like fluorapatite coatings.

In this study, the effect of ordered rod-like FA coatings of metal discs on adipose-derived stem cell (ASC)'s growth, differentiation and mineralization was studied in vitro; and their mineral inductive effects in vivo. After 3 and 7 days, the cell number on the metal surfaces was significantly higher than those on the ordered and disordered FA surfaces. However, after 4 weeks much greater amounts of mineral formation was induced on the two FA surfaces with and even without osteogenesis induction. The osteogenic profiles showed the up regulation of a set of pro-osteogenic transcripts and bone mineralization phenotypic markers when the ASCs were grown on FA surfaces compared to metal surfaces at 7 and 21 days. In addition to BMP and TGFß signaling pathways, EGF and FGF pathways also appeared to be involved in ASC differentiation and mineralization. In vivo studies showed accelerated and enhanced mineralized tissue formation integrated within ordered FA coatings. After 5 weeks, over 80% of the ordered FA coating was integrated with a mineralized tissue layer covering the implants. Both the intrinsic properties of the FA crystals and the topography of the FA coating appeared to dominate the cell differentiation and mineralization process.

The effect of novel fluorapatite surfaces on osteoblast-like cell adhesion, growth, and mineralization.

There is increasing demand for biomedical implants to correct skeletal defects caused by trauma, disease, or genetic disorder. In this study, the MG-63 cells were grown on metals coated with ordered and disordered fluorapatite (FA) crystal surfaces to study the biocompatibility, initial cellular response, and the underlying mechanisms during this process. The long-term growth and mineralization of the cells were also investigated. After 3 days, the cell numbers on etched metal surface are significantly higher than those on the ordered and disordered FA surfaces, but the initial adherence of a greater number of cells did not lead to earlier mineral formation at the cell-implant interface. Of the 84 cell adhesion and matrix-focused pathway genes, an up- or down-regulation of a total of 15 genes such as integrin molecules, integrin alpha M and integrin alpha 7 and 8 was noted, suggesting a modulating effect on these adhesion molecules by the ordered FA surface compared with the disordered. Osteocalcin expression and the mineral nodule formation are most evident on the FA surfaces after osteogenic induction (OI) for 7 weeks. The binding of the ordered FA surfaces to the metal, with and without OI, was significantly higher than that of the disordered FA surfaces with OI. Most significantly, even without the OI supplement, the MG-63 cells grown on FA crystal surfaces start to differentiate and mineralize, suggesting that the FA crystal could be a simple and bioactive implant coating material.

The effect of the surface characteristics of various substrates on fluorapatite crystal growth, alignment, and spatial orientation.

BACKGROUND: Fluorapatite-coated materials have potential application for dental and medical implants. In tooth development, the prism-like structure in enamel is thought to be created by the cells and proteins involved in enamel formation and maturation. This study investigated whether the substrate itself on which the films are grown, without the inclusion of cells or proteins, may affect their crystal alignment and three-dimensional morphology. MATERIAL/METHODS: Human dentin/enamel junction (DEJ), dentin, stainless steel plates, etched stainless steel plates, etched titanium plates, glass, and mica were used as substrates to grow fluorapatite (FA) crystals using a hydrothermal method. FA crystal growth on these substrates was observed using scanning electron microscopy. RESULTS: Under hydrothermal conditions, enamel prism-like structure could not be seen when the crystals were grown on mica or glass. Organized bundles of FA were created using DEJ, but not dentin, although it supported crystal growth. Etched stainless steel and titanium supported the growth of aligned FA crystals, but without the prism structure. CONCLUSIONS: The surface properties of the substrate on which the nanocrystals grow appear to affect their alignment, organization, and consequently the formation of a prism structure.

Matrix and TGF-beta-related gene expression during human dental pulp stem cell (DPSC) mineralization.

We have recently reported the induction of dental pulp stem cells (DPSCs) into dentin-secreting odontoblast-like cells after stimulation by isolated dentin matrix components, thus mimicking the nature of tissue regeneration seen after tooth disease and injury. After confluency, the cells were further cultured for 21 d in the 10% fetal bovine serum (FBS) Dulbecco's modified Eagle's medium (DMEM) (control), and in this medium, with the addition of dentin extract (DE) and the mineralization supplement (MS) of ascorbic acid and beta-glycerophosphate (treatment). To identify genes associated with this process, specimens were analyzed with a HG-U133A human gene chip and Arrayassist software. A total of 425 genes, among them 21 matrix and eight TGF-beta-related genes, were either up- or downregulated in the experimental group in which the cells showed odontoblast-like differentiation and mineralization. Expression of selected genes was further confirmed by real-time polymerase chain reaction (PCR) analysis. Of the extracellular matrix (ECM)-related genes, two types of collagen genes were upregulated and seven others downregulated. Other ECM-related genes, for example fibulin-1, tenascin C, and particularly thrombospondin 1, were upregulated, and fibulin-2 was downregulated. Most noticeably, the matrix metalloproteinase 1 was induced by the treatment. In the TGF-beta superfamily, upregulation of the type II receptor, endoglin, and growth/differentiation factor 5 was coordinated with the downregulation of activin A, TGF-beta2, and TGF-beta1 itself. This study identifies the matrix and TGF-beta-related gene profiles during the DPSC cell mineralization in which several genes are reported for the first time to be associated with this process, thus greatly expanding our molecular knowledge of the induced disease repair process.