Release of calcium ions from particulate monosodium titanates for dental mineralization applications.

PURPOSE: The calcium ion [Ca(II)] release from monosodium titanates (MST) complexed to calcium ions [Ca(II)], referred to as MST-Ca(II), was examined under varying incubation times, pH conditions, and ion equilibrium disruptions. METHODS: Sample supernatants were analyzed for Ca(II) using the QuantiChrom Calcium Assay Kit. RESULTS: No Ca(II) was detected in native MST (control) supernatants but was detected in MST-Ca(II) supernatants. At pH 7, Ca(II) release increased from 0 to 2.5 mg/dL over 3 days (P< 0.05 compared to MST control), remaining constant over the completed incubation times. At pH 5, 15 mg/dL of Ca(II) was immediately released with no further release. When the pH was modulated pH 4 to pH 9, Ca(II) concentration dropped from 25 mg/dL to ~0 mg/dL. Finally, when equilibrium was disrupted by partial replacement of the supernatant with sterile water, Ca(II) release was ongoing, reaching a cumulative total of 20 mg/dL over 35 days. CLINICAL SIGNIFICANCE: The current results suggest that particulate MST-Ca(II) complexes exhibit sustained release of calcium, and that release might be customized by conditions of pH and ionic strength. Thus, these complexes appear promising for biological applications where calcium-mediated mineralization or re-mineralization are desired.

Zirconia in biomedical applications.

INTRODUCTION: The use of zirconia in medicine and dentistry has rapidly expanded over the past decade, driven by its advantageous physical, biological, esthetic, and corrosion properties. Zirconia orthopedic hip replacements have shown superior wear-resistance over other systems; however, risk of catastrophic fracture remains a concern. In dentistry, zirconia has been widely adopted for endosseous implants, implant abutments, and all-ceramic crowns. Because of an increasing demand for esthetically pleasing dental restorations, zirconia-based ceramic restorations have become one of the dominant restorative choices. Areas covered: This review provides an updated overview of the applications of zirconia in medicine and dentistry with a focus on dental applications. The MEDLINE electronic database (via PubMed) was searched, and relevant original and review articles from 2010 to 2016 were included. Expert commentary: Recent data suggest that zirconia performs favorably in both orthopedic and dental applications, but quality long-term clinical data remain scarce. Concerns about the effects of wear, crystalline degradation, crack propagation, and catastrophic fracture are still debated. The future of zirconia in biomedical applications will depend on the generation of these data to resolve concerns.

Cytotoxicity of Titanate-Calcium Complexes to MC3T3 Osteoblast-Like Cells.

Monosodium titanates (MST) are a relatively novel form of particulate titanium dioxide that have been proposed for biological use as metal sorbents or delivery agents, most recently calcium (II). In these roles, the toxicity of the titanate or its metal complex is crucial to its biological utility. The aim of this study was to determine the cytotoxicity of MST and MST-calcium complexes with MC3T3 osteoblast-like cells; MST-Ca(II) complexes could be useful to promote bone formation in various hard tissue applications. MC3T3 cells were exposed to native MST or MST-Ca(II) complexes for 24-72 h. A CellTiter-Blue® assay was employed to assess the metabolic activity of the cells. The results showed that MST and MST-Ca(II) suppressed MC3T3 metabolic activity significantly in a dose-, time-, and cell-density-dependent fashion. MST-Ca(II) suppressed MC3T3 metabolism in a statistically identical manner as native MST at all concentrations. We concluded that MST and MST-Ca(II) are significantly cytotoxic to MC3T3 cells through a mechanism yet unknown; this is a potential problem to the biological utility of these complexes.

Genotype-specific regulation of oral innate immunity by T2R38 taste receptor.

The bitter taste receptor T2R38 has been shown to regulate mucosal innate immune responses in the upper airway epithelium. Furthermore, SNPs in T2R38 influence the sensitivity to 6-n-propylthiouracil (PROP) and are associated with caries risk/protection. However, no study has been reported on the role of T2R38 in the innate immune responses to oral bacteria. We hypothesize that T2R38 regulates oral innate immunity and that this regulation is genotype-specific. Primary gingival epithelial cells carrying three common genotypes, PAV/PAV (PROP super-taster), AVI/PAV (intermediate) and AVI/AVI (non-taster) were stimulated with cariogenic bacteria Streptococcus mutans, periodontal pathogen Porphyromonas gingivalis or non-pathogen Fusobacterium nucleatum. QRT-PCR analyzed T2R38 mRNA, and T2R38-specific siRNA and ELISA were utilized to evaluate induction of hBD-2 (antimicrobial peptide), IL-1α and IL-8 in various donor-lines. Experiments were set up in duplicate and repeated three times. T2R38 mRNA induction in response to S. mutans was highest in PAV/PAV (4.3-fold above the unstimulated controls; p<0.05), while lowest in AVI/AVI (1.2-fold). In PAV/PAV, hBD-2 secretion in response to S. mutans was decreased by 77% when T2R38 was silenced. IL-1α secretion was higher in PAV/PAV compared to AVI/PAV or AVI/AVI with S. mutans stimulation, but it was reduced by half when T2R38 was silenced (p<0.05). In response to P. gingivalis, AVI/AVI showed 4.4-fold increase (p<0.05) in T2R38 expression, whereas the levels in PAV/PAV and AVI/PAV remained close to that of the controls. Secretion levels of IL-1α and IL-8 decreased in AVI/AVI in response to P. gingivalis when T2R38 was silenced (p<0.05), while the changes were not significant in PAV/PAV. Our data suggest that the regulation of gingival innate immunity by T2R38 is genotype-dependent and that the ability to induce a high level of hBD-2 by PAV/PAV carriers may be a reason for protection against caries in this group.

Titanates and Titanate-Metal Compounds in Biological Contexts.

Metal ions are notorious environmental contaminants, some causing toxicity at exquisitely low (ppm-level) concentrations. Yet, the redox properties of metal ions make them attractive candidates for bio-therapeutics. Titanates are insoluble particulate compounds of titanium and oxygen with crystalline surfaces that bind metal ions; these compounds offer a means to scavenge metal ions in environmental contexts or deliver them in therapeutic contexts while limiting systemic exposure and toxicity. In either application, the toxicological properties of titanates are crucial. To date, the accurate measurement of the in vitro toxicity of titanates has been complicated by their particulate nature, which interferes with many assays that are optical density (OD)-dependent, and at present, little to no in vivo titanate toxicity data exist. Compatibility data garnered thus far for native titanates in vitro are inconsistent and lacking in mechanistic understanding. These data suggest that native titanates have little toxicity toward several oral and skin bacteria species, but do suppress mammalian cell metabolism in a cells-pecific manner. Titanate compounds bind several types of metal ions, including some common environmental toxins, and enhance delivery to bacteria or cells. Substantial work remains to address the practical applicability of titanates. Nevertheless, titanates have promise to serve as novel vehicles for metal-based therapeutics or as a new class of metal scavengers for environmental applications.

DNA methylation differentially regulates cytokine secretion in gingival epithelia in response to bacterial challenges.

Epigenetic modifications are changes in gene expression without altering DNA sequence. We previously reported that bacteria-specific innate immune responses are regulated by epigenetic modifications. Our hypothesis is that DNA methylation affects gingival cytokine secretion in response to bacterial stimulation. Gingival epithelial cells (GECs) were treated with DNMT-1 inhibitors prior to Porphyromonas gingivalis (Pg) or Fusobacterium nucleatum (Fn) exposure. Protein secretion was assessed using ELISA. Gene expression was quantified using qRT-PCR. The ability of bacteria to invade inhibitor pretreated GECs was assessed utilizing flow cytometry. Changes were compared to unstimulated GECs. GEC upregulation of IL-6 and CXCL1 by Pg or Fn stimulation was significantly diminished by inhibitor pretreatment. Pg stimulated IL-1α secretion and inhibitor pretreatment significantly enhanced this upregulation, while Fn alone or with inhibitor pretreatment had no effect on IL-1α expression. GEC upregulation of human beta-definsin-2 in response to Pg and Fn exposure was enhanced following the inhibitor pretreatment. GEC susceptibility to bacterial invasion was unaltered. These results suggest that DNA methylation differentially affects gingival cytokine secretion in response to Pg or Fn. Our data provide basis for better understanding of how epigenetic modifications, brought on by exposure to oral bacteria, will subsequently affect host susceptibility to oral diseases.

In vitro biological response of micro- and nano-sized monosodium titanates and titanate-metal compounds.

Previous studies report that microsized monosodium titanates (MSTs) deliver metal ions and species to mammalian cells and bacteria with cell-specific and metal-specific effects. In this study, we explored the use of MST and a new synthesized nanosized monosodium titanate (nMST) to deliver gold(III), cisplatin, or platinum(IV) to two human cell lines with different population doubling times, in vitro. The effect was measured using a fluorescent mitochondrial activity assay (CellTiter-Blue(®) Assay). This fluorescence assay was implemented to mitigate optical density measurement errors owing to particulate titanate interference and allowed for the studies to be extended to higher titanate concentrations than previously possible. Overall, native MST significantly (p < 0.05) decreased mitochondrial activity of both cell types by 50% at concentrations of >50 mg/L. Native nMST significantly suppressed the rapidly dividing cell line (by 50%) over untreated cultures, but had no effect on the more slowly dividing cells. For both cell types, increased titanate concentrations resulted in increased effects from delivered metals. However, there was no difference in the effect of metal delivered from micro- versus nano-sized MST.

Nickel alloys in the oral environment.

The use of nickel casting alloys for long-term restorations in dentistry has long been controversial. A 'tug-of-war' between economic, engineering and biological considerations is central to this controversy; nickel-casting alloys have low costs and favorable physical properties, but are corrosion-prone in the oral environment. Clinicians and researchers have questioned the safety of nickel-containing dental alloys because several nickel compounds are known to cause adverse biological effects in vivo and in vitro in contexts outside of dentistry. The debate revolves around the extent to which corrosion products from oral restorations cause intraoral or systemic biological problems. Current evidence suggests that nickel alloys may be used successfully and safely in dentistry if clinical risks are taken into account. However, these alloys may cause significant clinical problems, primarily allergenic and inflammatory, if the risks are ignored.

Effect of Ni(II) on inflammatory gene expression in THP1 monocytic cells.

Nickel-containing alloys are in common use for dental restorations, but tend to corrode and release Ni(II) in service. Ni(II) increases secretion of several inflammatory cytokines from activated monocytic cells, suggesting that nickel alloys may exaggerate inflammatory responses in adjacent periodontal tissues. In this work, the effects of Ni(II) on expression of inflammatory cytokine and receptor genes as well as nuclear factor-kappa B (NFκB)-related genes were assessed using quantitative real-time polymerase chain reaction (PCR) and PCR-based arrays in the human THP1 monocytic cell line pre-exposed to Ni(II) for 72 h, then activated by lipopolysaccharide. The expression of 10 inflammatory genes was down-regulated ≥50% by Ni(II) versus non-Ni(II) controls, whereas some genes like IL8 were up-regulated significantly by Ni(II). Expression of seven NFκB-related genes was up-regulated by Ni(II) by ≥50%, and HMOX1 expression, a redox protein regulated by NRF2, was increased by >500%. The current results suggest that Ni(II) has diverse effects on inflammatory gene expression, which may partly account for previous reports of Ni(II)-induced changes in inflammatory cytokine secretion from monocytes and alterations in NFκB regulation. Further work is needed to verify these effects in primary cells and to ascertain how Ni(II) alters gene expression.

Cellular cross-linking of peptide modified hydrogels.

Peptide modification of hydrogel-forming materials is being widely explored as a means to regulate the phenotype of cells immobilized within the gels. Alternatively, we hypothesized that the adhesive interactions between cells and peptides coupled to the gel-forming materials would also enhance the overall mechanical properties of the gels. To test this hypothesis, alginate polymers were modified with RGDSP-containing peptides and the resultant polymer was used to encapsulate C2C12 myoblasts. The mechanical properties of these gels were then assessed as a function of both peptide and cell density using compression and tensile tests. Overall, it was found that above a critical peptide and cell density, encapsulated myoblasts were able to provide additional mechanical integrity to hydrogels composed of peptide-modified alginate. This occurred presumably by means of cell-peptide cross-linking of the alginate polymers, in addition to the usual Ca++ cross-linking. These results are potentially applicable to other polymer systems and important for a range of tissue engineering applications.

The tensile properties of alginate hydrogels.

Alginate hydrogels are currently being employed and explored for a broad range of medical applications including cell encapsulation, drug delivery, and tissue engineering. In these capacities, knowledge of the mechanical and material properties of the hydrogels and the properties that govern and influence them is necessary to adequately design and effectively use these systems. Although much is known about the mechanical properties of alginate in compression and shear, little is known about the tensile characteristics. Thus, an extensive tensile assessment of alginate hydrogels was completed as a function of alginate type, formulation, gelling conditions, incubation, and strain rate. In general, the initial tensile behavior and properties of alginate hydrogels were highly dependent on the choice of the alginate polymer and how it was processed. Specifically, high guluronic acid containing alginate polymers yielded stronger, more ductile hydrogels than high mannuronic acid containing alginates. The ultimate stress, ultimate strain, and tensile modulus were decreased by increased phosphate concentrations, solution reconstitution with phosphate buffered saline instead of culture media, and peptide modification. Incubation of hydrogels for at least 7 days diminished many of the initial tensile property differences associated with formulation and gelling conditions. Overall, by controlling the specific alginate polymer and processing methods, a wide range of tensile properties are available from these hydrogels.

Hydrogels for tissue engineering: scaffold design variables and applications.

Polymer scaffolds have many different functions in the field of tissue engineering. They are applied as space filling agents, as delivery vehicles for bioactive molecules, and as three-dimensional structures that organize cells and present stimuli to direct the formation of a desired tissue. Much of the success of scaffolds in these roles hinges on finding an appropriate material to address the critical physical, mass transport, and biological design variables inherent to each application. Hydrogels are an appealing scaffold material because they are structurally similar to the extracellular matrix of many tissues, can often be processed under relatively mild conditions, and may be delivered in a minimally invasive manner. Consequently, hydrogels have been utilized as scaffold materials for drug and growth factor delivery, engineering tissue replacements, and a variety of other applications.