Noninvasive dentistry: a dream or reality?

Various caries prevention and repair strategies are reviewed in this article ranging from the use of fluoride to nanohydroxyapatite particles. Several of the strategies which combine fluoride and calcium and phosphate treatments have both in vitro and in vivo data showing them to be efficacious if the surface integrity of the lesion is not breached. Once this has occurred, the rationale for cutting off the nutrient supplies to the pathogenic bacteria without the removal of the infected dentine, a noninvasive restorative technique, is discussed using existing clinical studies as examples. Finally two novel noninvasive restorative techniques using fluorohydroxyapatite crystals are described. The need for clinical data in support of emerging caries-preventive and restorative strategies is emphasized.

Biological and Mechanical Evaluation of Novel Prototype Dental Composites.

The breakdown of the polymeric component of contemporary composite dental restorative materials compromises their longevity, while leachable compounds from these materials have cellular consequences. Thus, a new generation of composite materials needed to be designed to have a longer service life and ensure that any leachable compounds are not harmful to appropriate cell lines. To accomplish this, we have developed concurrent thiol-ene-based polymerization and allyl sulfide-based addition-fragmentation chain transfer chemistries to afford cross-linked polymeric resins that demonstrate low shrinkage and low shrinkage stress. In the past, the filler used in dental composites mainly consisted of glass, which is biologically inert. In several of our prototype composites, we introduced fluorapatite (FA) crystals, which resemble enamel crystals and are bioactive. These novel prototype composites were benchmarked against similarly filled methacrylate-based bisphenol A diglycidyl ether dimethacrylate / triethylene glycol dimethacrylate (bisGMA/TEGDMA) composite for their cytotoxicity, mechanical properties, biofilm formation, and fluoride release. The leachables at pH 7 from all the composites were nontoxic to dental pulp stem cells. There was a trend toward an increase in total toughness of the glass-only-filled prototype composites as compared with the similarly filled bisGMA/TEGDMA composite. Other mechanical properties of the glass-only-filled prototype composites were comparable to the similarly filled bisGMA/TEGDMA composite. Incorporation of the FA reduced the mechanical properties of the prototype and bisGMA/TEGDMA composite. Biofilm mass and colony-forming units per milliliter were reduced on the glass-only-filled prototype composites as compared with the glass-only-filled bisGMA/TEGDMA composite and were significantly reduced by the addition of FA to all composites. Fluoride release at pH 7 was greatest after 24 h for the bisGMA/TEGDMA glass + FA composite as compared with the similarly filled prototypes, but overall the F- release was marginal and not at a concentration to affect bacterial metabolism.

Signals in Stem Cell Differentiation on Fluorapatite-Modified Scaffolds.

Previously, we reported that the fluorapatite (FA)-modified polycaprolactone (PCL) nanofiber could be an odontogenic/osteogenic inductive tissue-engineering scaffold by inducing stem cell differentiation and mineralization. The present study aimed to explore which of the signal pathways affected this differentiation and mineralization process. The Human Signal Transduction PathwayFinder RT2 Profiler PCR Array was used to analyze the involvement of potential signal transduction pathways during human dental pulp stem cell (DPSCs) osteogenic differentiation induced by FA-modified PCL nanofiber scaffolds. Based on the results, perturbation studies of the signaling pathways hedgehog, insulin, and Wnt were performed. Moreover, the autophagy process was studied, as indicated by the expression of the microtubule-associated protein 1 light chain 3A/B-II (LC3-II) and the cell osteogenic phenotypic changes. In a comparison of the cells grown on PCL + FA scaffolds and those on PCL-only scaffolds, the transcript expression of BMP2, BMP4, FOXA2, PTCH1, WNT1, and WNT2 (PCR array-labeled signal proteins of the hedgehog pathway); CEBPB, FASN, and HK2 (PCR array-labeled signal proteins of the insulin pathway); and CCND1, JUN, MYC, TCF7, and WISP1 (PCR array-labeled signal proteins of the Wnt pathway) doubled at day 14 when obvious cell osteogenic differentiation occurred. Phenotypically, in all the perturbation groups at day 14, ALP activity, OPN, and autophagy marker LC3-II expression were coincidently decreased. Consistently, no positive alizarin red staining or von Kossa staining was observed in the specimens from these perturbation groups at day 28. The results showed that when obvious cell differentiation occurred at day 14 on PCL + FA control groups, the inhibition of the hedgehog, insulin, and Wnt pathways significantly decreased DPSC osteogenic differentiation and mineralization. The osteogenic differentiation of DPSCs grown on FA-modified PCL scaffolds appeared to be positively modulated by the hedgehog, insulin, and Wnt signal pathways, which were coordinated with and/or mediated by the cell autophagy process.

Effects of systemic fluoride and in vitro fluoride treatment on enamel crystals.

Systemically administered fluoride at a concentration of 75 ppm increases the surface roughness of developing enamel crystals in rats, which may be significant in advancing our understanding of the biological mechanism of fluorosis. Thus, the aim of this study was to investigate whether the increased surface roughness may be a result of surface restructuring by the direct action of fluoride at the crystal surface. We examined the fluoride dose-dependent roughening of enamel crystal surfaces in vivo, in the rat, and whether this roughening could be mimicked by the in vitro treatment of rat enamel crystals with neutral pH fluoride solutions. Our results showed that enamel crystal surface roughness increased after treatment with increasing fluoride ion concentrations, whether applied in vitro or administered systemically. This suggests a mechanism, alongside others, for the increased surface roughness of crystals in fluorotic enamel.

Autophagy Modulates Cell Mineralization on Fluorapatite-Modified Scaffolds.

As a major intracellular degradation and recycling machinery, autophagy plays an important role in maintaining cellular homeostasis and remodeling during normal development. Our previous study showed that fluorapatite (FA) crystal-coated electrospun polycaprolactone (PCL) was capable of inducing differentiation and mineralization of human dental pulp stem cells. However, how autophagy changes and whether autophagy plays a vital role during these processes is still unknown. In this study, we seeded STEMPRO human adipose-derived stem cells (ASCs) on both PCL+FA and PCL scaffolds to investigate the osteogenic inductive ability of FA crystals and we observed the autophagy changes of these cells. Scanning electron microscopy and fluorescence microscopy images, along with DNA quantitation, showed that both PCL+FA and PCL scaffolds could sustain ASC growth but only the PCL+FA scaffold could sustain cell mineralization. This was confirmed by alkaline phosphatase activity and Alizarin red and Von Kossa staining results. The autophagy RT2 Profiler polymerase chain reaction array analysis showed many autophagy-related genes changes during ASC differentiation. Western blot analysis indicated that several autophagy-related proteins fluctuated during the procedure. Among them, the microtubule-associated protein 1 light chain 3 (LC3)-II protein changes of the ASCs grown on the 2- or 3-dimensional environments at 6 h, 12 h, 1 d, 3 d, 7 d, 14 d, and 21 d reached a peak value at day 7 during osteogenesis. At earlier stages (from day 0 to day 3), the addition of autophagy inhibitors (3-mathyladenine, bafilomycin A1, and NH4Cl) attenuated the expression of osteogenic related markers (osteopontin, alkaline phosphatase activity, Alizarin red, and Von Kossa) compared with the control group. All data indicated that autophagy played an important role in ASC differentiation on the PCL+FA scaffold. Inhibition of autophagy before day 3 strongly inhibited osteogenic differentiation and mineralization of ASCs in the 3-dimensional model. This observation further elucidates the mechanism of autophagy in mesenchymal stem cell osteogenic differentiation.

VEGF promotes osteogenic differentiation of ASCs on ordered fluorapatite surfaces.

Vascular endothelial growth factor (VEGF) has been reported to mediate both osteogenesis and angiogenesis in bone regeneration. We previously found an upregulation of VEGF in adipose-derived stem cells (ASCs) when obvious mineralization occurred on a novel fluorapatite (FA)-coated surfaces. This study investigated the effect of FA and VEGF on the growth, differentiation and mineralization of (ASC) grown on ordered FA surfaces. Cells grown on FA and treated with VEGF demonstrated osteogenic differentiation as measured with ALP staining, and obvious mineralization as measured by Alizarin red staining. A combined stimulating effect of FA and VEGF was seen using both indicators. VEGF signaling pathway perturbation using a specific VEGF receptor inhibitor showed the lowest levels of ALP and Alizarin red staining, which was partially rescued when the cells were grown on FA and/or treated with the addition of VEGF. The osteogenic differentiation of ASCs stimulated by these FA surfaces as well as VEGF has been shown to be mediated through, but probably not only, the VEGF signaling pathway. The enhancement of osteogenic differentiation and mineralization supports the potential use of therapeutic VEGF and FA coatings in bone regeneration.

The effect of metal/fluoride complexes on fluoride uptake and caries-like lesion formation in enamel.

Pretreatment of enamel with TiCl3 and ZrOCL2-8H2O solutions prior to APF application was shown to increase fluoride uptake and reduce caries-like lesion formation to a greater extent than APF alone. The increase in fluoride uptake by enamel was not solely due to the low pH of the pretreatment solutions.

The effect of polyvalent metal ion mordanting on caries-like lesion progression in enamel.

The ability of metal ion mordanting followed by APF application to inhibit the progression of caries-like lesions was compared with APF treatment alone. The progression of the lesion was investigated in vitro using an acidified gel system, and the lesions were compared using polarized light microscopy. Both ferric and zirconyl pre-treatments, but not titanium, were shown to inhibit lesion progression to a greater extent than did APF. This inhibition was thought to be due to the presence of an amorphous-type coating similar to those produced on sound enamel by metal ion mordanting.

A model for producing caries-like lesions in enamel and dentin using oral bacteria in vitro.

Subsurface enamel lesions and root surface caries-like lesions were consistently produced in vitro using Streptococcus mutans FA1 cultured in thioglycollate broth containing 3.5% w/v dextrose and 2% w/v gelatin. When viewed in polarized light and after imbibition in water, the enamel lesions had a negatively birefringent surface zone and positively birefringent body of the lesion. Those lesions produced after six weeks, after imbibition in quinoline, exhibited a dark zone. The root surface caries-like lesions exhibited a less-radiolucent surface zone above a heavily demineralized body of the lesion. However, no reactionary dentin was seen in the in vitro lesions.

Fluoride distribution in enamel after in vitro caries-like lesion formation.

Using a microdissection technique to obtain samples from carious and adjacent sound enamel, we compared the fluoride concentrations in surface and subsurface enamel. Undialyzed and dialyzed gels were used to create lesions with and without negatively birefringent surface zones, respectively. The fluoride concentration in the surface enamel of the lesions was always greater than that in the corresponding sound enamel, as was the subsurface fluoride concentrations of caries lesions compared with adjacent sound enamel at the same depth.

Microprobe and SEM analysis of surface coatings on caries-like lesions in enamel after metal ion mordanting and APF application.

The production of tenacious surface coatings on enamel after metal ion mordanting followed by APF has been reported to prevent caries-like lesion initiation in vitro. In this study, similar surface coatings, which may be responsible for the inhibition of lesion progression, were examined using SEM and microprobe analysis. Relatively thick, even surface coatings which were in intimate contact with the enamel surface of caries-like lesions were seen after zirconium and iron (ferric) pre-treatment, but a thinner and unevenly distributed coating was seen after titanium pre-treatment. This latter coating was similar to that seen after APF treatment alone. Fluoride concentrations on the surface and in the subsurface of caries-like lesions were similar for all treatment groups. The surface coatings after zirconium and iron mordanting may be responsible for the greater inhibition of lesion progression than that seen after both titanium pre-treatment and APF treatment alone.

In vitro caries-like lesion production by Streptococcus mutans and Actinomyces viscosus using sucrose and starch.

We investigated the formation of caries-like lesions on root and enamel sections by S. mutans and A. viscosus when grown in four different carbohydrate substrates. The substrates were: sucrose, starch + alpha-amylase, and a combination of starch and sucrose with and without alpha-amylase. Twenty-four sections with exposed windows on both the root and enamel surfaces were exposed to the individual bacterial species in each of the four substrates for three weeks. At three weeks, the sections were removed and lesion depths measured. When grown in sucrose alone, S. mutans produced significantly deeper lesions in both root and enamel surfaces when compared with A. viscosus. However, S. mutans failed to produce lesions when grown in "limited" sucrose with starch added, whereas A. viscosus in this same substrate produced lesions in the enamel and root surfaces. A viscosus also produced either similar or significantly deeper lesions in both root and enamel surfaces, when compared with S. mutans grown in the two substrates with amylase. Thus, under these in vitro conditions, it was demonstrated that A. viscosus could utilize starch, whereas utilization of starch by S. mutans was alpha-amylase-dependent.

Interaction of dendrimers (artificial proteins) with biological hydroxyapatite crystals.

This investigation sets out to mimic protein-crystal interaction during biomineralization with the use of artificial proteins (dendrimers). It is hypothesized that these interactions depend on the surface charge of hydroxyapatite crystals. This was investigated with the use of dendrimers with capped surfaces of different charges to probe the surface. We used AFM images of crystal-bound dendrimers to determine the distribution of the surface charge, and its magnitude was correlated to the binding capacity of the dendrimers to the surface. The binding capacity of the dendrimers in ascending order at pH 7.4 was: acetamide-capped, -NHC(O)CH3, neutral charge; carboxylic-acid-capped, -COOH, negative charge; and amine-capped, -NH2, positive charge. AFM images of the crystals showed dendrimers spaced equally along the crystal. The results suggest that the crystal surface has alternating bands of positive and negative charge or a differential charge array, i.e., alternating bands of either more or less positive or negative charge.

Effects of phosphoprotein on collagen fibril formation in vitro.

Transmission electron microscopy showed that when either dentinal phosphoprotein or calcium-treated phosphoprotein or phosvitin were introduced during type I collagen fibrillogenesis the fibrils formed were significantly wider and the cross-banding was more distinct than in the absence of phosphoprotein. The collagen fibril width also increased with increasing concentrations of these molecules. When either bovine serum albumin (BSA) or dephosphorylated dentine phosphoprotein were used, no differences in the fibril characteristics were seen when compared to the controls that contained no phosphoprotein or BSA. When these dialysed matrices were placed into mineralizing solutions, no mineral was observed in any of the samples.

Comparison of phosphoprotein isolated from mature and immature human tooth roots.

Mature (average patient age = 29.5 yr, closed apical foramen) and immature (average patient age = 17.5 yr, open apical foramen) root shards were placed in dialysis tubing and demineralized to completion using either 10% disodium EDTA plus protease inhibitors or 0.6 N HCl. The demineralized shards were re-extracted (five times) with 0.05 M tris-HCl, 1.0 M NaCl and then collagenase digested. No major differences were observed in chromatograms of extracts, re-extracts or collagenase digests from root shards demineralized in either way. In contrast, chromatograms of immature and mature roots showed qualitative differences. Chromatograms of mature roots demineralized in either way showed broader protein peaks and less organic phosphorus than those from immature tooth roots. A distinct band amid degraded phosphoprotein (150 K) was found in SDS-PAGE gels (7.5%) from EDTA-extracted immature tooth roots but not from mature tooth roots. Electroelution of this band revealed a typical phosphoprotein amino-acid profile containing increased aspartic acid and serine residues. Comparison of the total phosphoprotein and amino acid composition of extracts, re-extracts and collagenase digests revealed that phosphoprotein, serine and to a lesser extent aspartic acid were recovered in greater quantities from immature roots than mature tooth roots. These data suggest that the degree of maturation is crucial to the isolation of an intact phosphoprotein and provides additional evidence that human dentine phosphoprotein undergoes amino acid compositional changes during maturation.

Phosphoprotein extraction from the dentine/cementum complex of human tooth roots.

Root shards were placed in dialysis tubing and demineralized to completion in either 10% disodium EDTA, pH 7.4, 0.6 M HCl, 0.1 M HCl, 0.5 M acetic or 75 mM-25 mM lactic-acetic acids. The demineralized shards were then re-extracted with 0.05 M tris-HCl, 1.0 M NaCl. DEAE chromatography revealed that the major peak of the 0.6 M CHl and EDTA extracts contained organic phosphorus, whereas much less organic phosphorus was found in the major peak of the 0.1 M HCl extract. Analysis of the re-extracts gave a pattern opposite to that obtained from the initial extractions. Measurements of protein and organic phosphorus released during extraction and re-extraction confirmed these results. Staining of SDS-PAGE gels for phosphoprotein with Stains-All resulted in a blue smear in fractions containing organic phosphorus. Thus the extraction of phosphoproteins from human tooth roots differed depending upon the demineralizing conditions. This ability to remove phosphoprotein differentially will allow further investigation of the role of phosphoprotein in mineralization and remineralization.

Fluoride release and re-uptake in direct tooth colored restorative materials.

OBJECTIVES: Glass ionomers may be "recharged" through topical fluoride (F-) treatments; however, this reported "recharging," may be attributed to surface changes after F- treatment. This study examined differences in F- release and re-uptake among dual-cured and chemically-cured glass ionomers, and a photo-cured F- releasing composite. A secondary goal was to determine if tensile strength or surface roughness changed due to F- release, or F- re-uptake and re-release. METHODS: In Phase 1, initial surface roughness and diametral tensile strength were measured. F- release was measured for 30 days. Strength and roughness were then remeasured. In Phase 2, surface roughness was measured, then materials were treated with a 5000 ppm neutral F- gel, the same gel without F-, or phosphoric acid. F- release was measured for 30 days, then final surface roughness and strength were determined. RESULTS: Significant differences were found in amount and rate of F- release, and F- re-uptake and re-release among study materials and enamel controls (p < 0.001). The amount and rate of F- re-release after NaF treatment differed significantly from F- release after acid treatment in glass ionomers, although both groups showed increased F- release after surface treatment (p < 0.001). There were no significant changes in tensile strength or surface roughness after F- release or F- re-uptake and re-release as determined by ANOVA. SIGNIFICANCE: The results of this in vitro study indicate that applications of neutral 5000 ppm F- gel to aged glass ionomer restorations results in a significant fluoride uptake and subsequent release. The data suggest that the application of neutral fluoride gel to glass ionomer restorations in situ may result in increases in oral fluoride concentrations, without affecting the restoration's surface roughness or tensile strength.

Introduction to cariology.

This article examines the discipline of cariology. A brief history is presented, and an overview of the origin and management of caries is discussed. This article frames the picture that the other authors in this issue paint.

Emerging methods used in the prevention and repair of carious tissues.

A systematic review was undertaken to investigate four emerging methodologies that might be used in the prevention of caries and/or repair of carious tissues. These included a partitioned dentifrice, laser technology, fluoride-releasing dental materials, and for deep carious lesions, bone morphogenic protein (BMP) therapy. The search strategy was to review articles written in English, indexed in MEDLINE and EMBASE databases and published since 1976. Over two hundred articles were read but because of the inclusion and exclusion criteria, only thirty-three were included in the evidence tables. The review of partitioned toothpaste showed either a greater remineralizing effect or a greater increase in the resistance to demineralization of both enamel and dentin, with the exception of its lack of effectiveness on coronal caries in the only clinical trial. Five of the six in vitro studies on enamel and the one study on dentin reported that lased tissue was less soluble than nonlased. Six clinical and four in situ studies were reviewed in answering the question as to whether fluoride-releasing restorative materials increase the remineralization or resistance to demineralization of human enamel and dentin. Eight of these reported positive findings. Six animal studies investigating BMP were reviewed, and all showed the ability of BMP to induce tubular dentin formation. Although the laboratory, animal, and limited clinical trials report encouraging results, independent, randomized, controlled clinical trials need to be carried out before these emerging technologies can be recommended for use in general practice.

Fluorapatite-modified scaffold on dental pulp stem cell mineralization.

In previous studies, fluorapatite (FA) crystal-coated surfaces have been shown to stimulate the differentiation and mineralization of human dental pulp stem cells (DPSCs) in two-dimensional cell culture. However, whether the FA surface can recapitulate these properties in three-dimensional culture is still unknown. This study examined the differences in behavior of human DPSCs cultured on electrospun polycaprolactone (PCL) NanoECM nanofibers with or without the FA crystals. Under near-physiologic conditions, the FA crystals were synthesized on the PCL nanofiber scaffolds. The FA crystals were evenly distributed on the scaffolds. DPSCs were cultured on the PCL+FA or the PCL scaffolds for up to 28 days. Scanning electron microscope images showed that DPSCs attached well to both scaffolds after the initial seeding. However, it appeared that more multicellular aggregates formed on the PCL+FA scaffolds. After 14 days, the cell proliferation on the PCL+FA was slower than that on the PCL-only scaffolds. Interestingly, even without any induction of mineralization, from day 7, the upregulation of several pro-osteogenic molecules (dmp1, dspp, runx2, ocn, spp1, col1a1) was detected in cells seeded on the PCL+FA scaffolds. A significant increase in alkaline phosphatase activity was also seen on FA-coated scaffolds compared with the PCL-only scaffolds at days 14 and 21. At the protein level, osteocalcin expression was induced only in the DPSCs on the PCL+FA surfaces at day 21 and then significantly enhanced at day 28. A similar pattern was observed in those specimens stained with Alizarin red and Von Kossa after 21 and 28 days. These data suggest that the incorporation of FA crystals within the three-dimensional PCL nanofiber scaffolds provided a favorable extracellular matrix microenvironment for the growth, differentiation, and mineralization of human DPSCs. This FA-modified PCL nanofiber scaffold shows promising potential for future bone, dental, and orthopedic regenerative applications.