Brian H. Clarkson

Remineralization of natural and artificial lesions in human dental enamel in vitro. Effect of calcium concentration of the calcifying fluid

Exposure of both small carious lesions and artificial caries-like lesions to a synthetic calcifying fluid in vitro produced a significant degree of ‘healing’ or remineralization of the lesion. Changing the calcium concentration of the calcifying fluid had a marked effect on the degree of remineralization produced. When a low calcium concentration of 1 mM was employed, remineralization occurred throughout the entire depth of a lesion. Under these conditions, there was a mean reduction of 69% in area of the body of the lesion and a mean increase of 40% in orientated mineral. The dark zone at the advancing front of the lesion showed a dramatic increase in area of 526% and was much closer to the enamel surface relative to the control. When higher calcium ion concentrations of 3 mM were used, remineralization occurred but was limited to the surface of the lesion. Under these conditions, the mean reduction in area of the body of the lesion was 20%, brought about by a mean increase in orientated mineral of 17%. Although changes were found in relation to the dark zone, these were much smaller than those found for the 1-mM fluid, the increase in area being 38%. With respect to exposure times, results obtained using ten consecutive 24-hour exposures to the synthetic calcifying fluid were similar to those obtained after ten consecutive 1-hour exposures. Remineralization, therefore, occurred within each 1-hour exposure increment. Scanning electron microscopy showed that crystal diameters for sound enamel were in the range 35–40 nm. In the body of the lesion crystal diameters were reduced and found to be in the range 10–30 nm. In lesions remineralized with the high calcium-calcifying fluid containing 3 mM calcium, crystal diameters were larger than those found in either control lesions or in sound enamel, being in the range 50–75 nm. When the low calcium-calcifying fluid was used, remineralized lesions showed crystal diameters in the range 50–150 nm with a small number of crystals having diameters of 200 nm. Calculation of the supersaturation of the calcifying fluids revealed that the low calcium-calcifying fluid having 1 mM calcium favors crystal growth as opposed to nucleation.

IN VITRO DIFFERENTIATION AND MINERALIZATION OF HUMAN DENTAL PULP CELLS INDUCED BY DENTIN EXTRACT

SummaryIn this study, the progenitor cells isolated from the human dental pulp were used to study the effects of ethylenediaminetetraacetic acid-soluble dentin extract (DE) on their differentiation and mineralization to better understand tissue injury and repair in the tooth. Mineralization of the matrix was increasingly evident at 14, 21, and 28 d after treatment with a mineralization supplement (MS) (ascorbic acid [AA], β-glycerophosphate [β-GP]) and MS+DE. Real-time polymerase chain reaction results showed type I collagen upregulation after the addition of MS+DE at 7 d. Alkaline phosphatase was downregulated after the mineralization became obvious at 14 d. Bone sialoprotein was shown to be upregulated in the mineralized cell groups at all time points and dentin sialophosphoprotein after 7 d. Core binding factor a 1 was upregulated by the treatment of MS and DE at 7, 14, and 21 d. These results indicated that the MS of AA, β-GP, and DE synergistically induced cell differentiation of pulp progenitor cells into odontoblast-like cells and induced in vitro mineralization.

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 TGFβ 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.

CHARACTERIZATION AND IDENTIFICATION OF A HUMAN DENTIN PHOSPHOPHORYN

The present study further characterizes an extract from immature, human tooth apicies from which an intact dentin phosphoprotein has been identified. Third molar apicies from developing roots were decalcified in 10% EDTA until Ca2+ was undetectable in the decalcifying solution. The crude extract was run on 7.5% SDS-PAGE and stained with “Stains-All.” Four distinct bands were found and the molecular weights were 140,60, 50, and 34 k. When run on a SDS-PAGE under nonreducing conditions the 60, 50, and 34 k bands were absent. These results suggest that the lower molecular weight bands may be subunits of the larger protein. The extract was then further purified by adding CaCl2 and MgCl2 to precipitate the phosphoprotein. The precipitate was subjected to a DEAE-Sepharose CL6B column and eluted by 0–0.7 M NaCl gradient solution. The amino acid composition of the purified phosphoprotein was determined and the extract was found to be rich in serine and aspartic acid residues. The N-terminal peptide Asp-Asp-Pro was identified. The sequence of the three amino acids is identical to rat incisor phosphoprotein.

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 lessradiolucent surface zone above a heavily demineralized body of the lesion. However, no reactionary dentin was seen in the in vitro lesions.

Redistribution of Enamel Fluoride during White Spot Lesion Formation: an in vitro Study on Human Dental Enamel

In vitro fluoride by depth profiles were obtained from sound enamel windows and from adjacent enamel windows after caries-like enamel lesion formation. The caries-like lesions were

Dentin-pulp complex responses to carious lesions.

To understand the molecular events underlying the dentin-pulp complex responses to carious progression, we systematically analyzed tissue morphology and dentin matrix protein distribution in non-carious teeth and in teeth with enamel and dentin caries. Dentin matrix proteins analyzed included collagen type I, phosphophoryn (PP) and dentin sialoprotein (DSP), all of which play decisive roles in the dentin mineralization process. Human non-carious and carious third molar teeth were freshly collected, demineralized, and processed for hematoxylin and eosin staining. The ABC-peroxidase method was used for immunohistochemical staining of collagen type I, PP and DSP proteins using specific antibodies. In situ hybridization was also performed. In contrast to elongated odontoblasts in non-carious teeth, odontoblasts subjacent to dentin caries were cuboidal and fewer in number. The predentin zone was also dramatically reduced in teeth with dentin caries. The staining intensity for collagen type I, PP and DSP in the dentin-pulp complex increased progressively from non-carious teeth, to teeth with enamel and dentin caries. In situ hybridization studies showed DSP-PP mRNA expression in odontoblasts and dental pulp that was consistent with our immunohistochemical results. These results suggest that carious lesions stimulate the dentin-pulp complex to actively synthesize collagen type I, PP and DSP proteins. This response to carious lesions is likely to provide a basis for reparative and/or reactionary dentin formation.

Fibronectin adsorption studied using neutron reflectometry and complementary techniques

In implantology it is known that fibronectin affects cell-substrate adhesion, consequently, the structure and composition of the initially adsorbed fibronectin layer to a large extent determines the biological response to a biomaterial implanted into the body. In this study we have used neutron reflectometry and quartz-crystal microbalance with dissipation to investigate the amount of fibronectin adsorbed, the layer density, thickness and structure of films adsorbed to polished silicon oxide surfaces. We have cultured MG63 osteoblast-like cells on surfaces coated and uncoated with fibronectin and monitored the cellular response to these surfaces. The results show that at fibronectin concentrations in the range 0.01 to 0.1mg/ml a single highly hydrated layer of fibronectin approximately 40-50Å in thickness adsorbs to a polished silicon oxide surface and is likely to correspond to one diffuse monolayer of fibronectin arranged side-on. Cells cultured on this fibronectin layer have dramatically different morphology and growth to those grown on bare surfaces. Using a model silicon oxide surface has enabled us to study the substrate/protein interface, together with the impact of a fibronectin layer on the cellular response using consistent experimental conditions across a unique set of experimental techniques.

Titanium and Fluoride Concentrations in Titanium Tetrafluoride and APF Treated Enamel

Greater fluoride concentrations were obtained in enamel treated with phosphate/fluoride solutions than in that treated with TiF4 at similar pHs (1.0) and F concentrations (0.6M F and 1.6M F). However, fluoride solution without added phosphate at pH 1.0 and 1.6M F concentration produced lower fluoride concentrations in enamel than in TiF4 treated enamel. It is proposed that in TiF4 treated enamel the fluoride uptake may be dependent upon the amount of Ti introduced into the enamel.

Chemical and Physical Evaluation of Dialyzed-Reconstituted Acidified Gelatin Surface Lesions of Human Enamel

Innate calcium, phosphate, and fluoride were removed from gelatin gels by dialysis. Gels were reconstituted to 25% (w/v) gelatin to contain 75 mM lactic acid, 25 m M acetic acid, des