B.H. Clarkson

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.

Adhesion and growth of dental pulp stem cells on enamel-like fluorapatite surfaces

UNLABELLEDnTo study how apatite crystal alignment of an enamel-like substrate affects DPSC cellular adhesion and growth as a precursor to produce an in vitro enamel/dentin superstructure for future studies. The cells were subcultured in 10% FBS DMEM up to seven weeks on the two surfaces. Specimens were observed under SEM, counted, and analyzed using the human pathway-focused matrix and adhesion PCR array. After three days, the cell number on ordered FA surface was significantly higher than on the disordered surface. Of the 84 focused pathway genes, a total of 20 genes were either up or down regulated in the cells on ordered FA surface compared to the disordered surface. More interestingly, of the cell-matrix adhesion molecules, integrin alpha 7 and 8 (ITGA 7 and 8), integrin beta 3 and 4 (ITGB3 and 4), and the vitronectin receptor-integrin alpha V (ITGAV) and the key adhesion protein-fibronectin1 (FN1) were up-regulated. In SEM, both surfaces showed good biocompatibility and supported long term growth of DPSC cells but with functional cell-matrix interaction on the ordered FA surfaces.nnnSIGNIFICANCEnThe enhanced cellular response of DPSC cell to the ordered FA crystal surface involves a set of delicately regulated matrix and adhesion molecules which could be manipulated by treating the cells with a dentin extract, to produce a dentin/enamel superstructure.

Synthesis of a Potentially Bioactive, Hydroxyapatite-Nucleating Molecule

A human phosphophoryn (PP) cDNA was previously cloned from immature root apex total RNA in our laboratory. This cDNA comprises 2,364 bp, encoding 788 amino acids. More than 80% of the sequences are arranged as (DSS)n (n = 1–16), DS, and NSS motifs. We hypothesize that the capability of PP to bind Ca2+ and nucleate hydroxyapatite may depend on these repeated sequences. Two polypeptides were synthesized based on the human PP cDNA sequence to test the hypothesis. One polypeptide has the amino acid sequence DDPNSSDESNGNDD (synthetic polypeptide 1, SP1), which is from the N-terminal end of PP; the other polypeptide, DSKSDSSKSESDSS (synthetic polypeptide 2, SP2), is the PP repeated sequence motif. Phosphorylation of the polypeptides was accomplished by reacting them with adenosine triphosphate and casein kinases I and II. The ability of these molecules to cause mineralization was tested in a steady-state agarose gel system. The results show that phosphorylated SP2 (P-SP2) precipitated approximately 60% of the total Ca + PO4 precipitated by PP. P-SP1 precipitated about 23% of that precipitated by PP and was similar to the amount precipitated in the control gel, that is, without added peptides. Transmission electron microscopy and X-ray diffraction analysis showed that the precipitate formed in the P-SP2-containing gel was hydroxyapatite. The capability of P-SP2 to nucleate Ca + PO4 and precipitate hydroxyapatite is a result of the repeated sequence motif, which contains a high percentage of phosphorylated serine. This molecule could be used in the repair and regeneration of dental tissue.

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.

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.

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.

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.

In vitro anti-caries effect of fluoridated hydroxyapatite-coated preformed metal crowns

AimTo synthesise fluoridated hydroxyapatite (FA) crystals directly on preformed metal crowns (PMCs) and evaluate the anti-cariogenic properties in an in vitro model.MethodsFA crystals were grown on etched PMCs and stainless steel discs and characterised by SEM. FA-coated discs allowed fluoride release to be assessed from a known surface area of FA crystals. Discs were divided into four groups (nxa0=xa06/group) and exposed to solutions at pH 4–7. Fluoride levels in solution were measured after each exposure. Twelve FA-coated and 12 non-coated PMCs were cemented onto human molars using glass ionomer (GI) or unfilled resin, making four groups of six teeth; FA-coatedxa0+xa0GI, FA-coatedxa0+xa0resin; non-coatedxa0+xa0GI and non-coatedxa0+xa0resin. Teeth were exposed to acidified gelatin (pHxa0=xa04.3) for 9xa0weeks.ResultsSEM showed FA crystal growth on interior and exterior of the crowns. Average fluoride release from FA-coated discs was 0.16xa0mg/L/cm² at pH < 5.0. Teeth were sectioned through the lesion. Polarised microscopic examination revealed significantly smaller lesions in FA-coated crown groups compared to non-coated crown groups.ConclusionFA-coated PMCs demonstrated carious lesion preventing effects, i.e. fluoride release and reduction of demineralisation at crown/tooth interface. FA-coated crowns could be an aesthetic, inexpensive and caries preventive alternative in clinical dentistry.

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.