Antimicrobial photodynamic therapy against a dual-species cariogenic biofilm using a ruthenium-loaded resin-based dental material.

BACKGROUND: Streptococcus mutans and Candida albicans are associated with caries recurrence. Therefore, this study evaluated the combination of a Ru(II)-loaded resin-based dental material (RDM) and antimicrobial photodynamic therapy (aPDT) against a dual-species biofilm of S. mutans and C. albicans. METHODS: An aPDT protocol was established evaluating Ru(II)'s photocatalytic activity and antimicrobial potential under blue LED irradiation (440-460 nm, 22.55 mW/cm2) at different energy densities (0.00, 6.25, 20.25, 40.50 J/cm2). This evaluation involved singlet oxygen quantification and determination of minimum inhibitory concentration (MIC) and minimum bactericidal/fungicidal concentration (MBC/MFC). The biofilm was grown (72 h) on resin disks prepared with Ru(II)-doped RDM (0.00, 0.56, or 1.12 %) and samples were exposed to aPDT or dark conditions. The biofilm was then harvested to analyze cell viability (CFU counts) and formation of soluble and insoluble exopolysaccharides. RESULTS: The photocatalytic activity of Ru(II) was concentration and energy density dependent (p < 0.05), and MIC/MBC values were reduced for the microorganisms after LED irradiation (40.5 J/cm2); therefor, this energy density was chosen for aPDT. Although incorporation of Ru(II) into RDM reduced the biofilm growth compared to Ru(II)-free RDM for both species in dark conditions (p < 0.05), aPDT combined with an Ru(II)-loaded RDM (0.56 or 1.12 %) potentialized CFU reductions (p < 0.05). Conversely, only 1.12 % Ru(II) with LED irradiation showed lower levels of both soluble and insoluble exopolysaccharides compared to Ru(II)-free samples in dark conditions (p < 0.05). CONCLUSIONS: When the Ru(II)-loaded RDM was associated with blue LED, aPDT reduced cell viability and lower soluble and insoluble exopolysaccharides were found in the cariogenic dual-species biofilm.

A Systematic Evaluation of Curcumin Concentrations and Blue Light Parameters towards Antimicrobial Photodynamic Therapy against Cariogenic Microorganisms.

Dental caries is a highly preventable and costly disease. Unfortunately, the current management strategies are inadequate at reducing the incidence and new minimally invasive strategies are needed. In this study, a systematic evaluation of specific light parameters and aqueous curcumin concentrations for antimicrobial photodynamic therapy (aPDT) was conducted. Aqueous solutions of curcumin were first prepared and evaluated for their light absorbance after applying different ~56 mW/cm2 blue light treatments in a continuous application mode. Next, these same light treatments as well as different application modes were applied to the curcumin solutions and the molar absorptivity coefficient, reactive oxygen species (ROS) release, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) for Streptococcus mutans and the MIC and minimum fungicidal concentration (MFC) for Candida albicans were measured. After up to 1 min of light treatment, the molar absorptivity of curcumin when added to culture media was lower than that for water only; however, at higher energy levels, this difference was not apparent. There was a noted dependence on both ROS type and cariogenic microorganism species on the sensitivity to both blue light treatment and application mode. In conclusion, this study provides new information towards improving the agonistic potential of aPDT associated with curcumin against cariogenic microorganisms.

Impact of curcumin loading on the physicochemical, mechanical and antimicrobial properties of a methacrylate-based experimental dental resin.

Oral biofilms are directly linked to one of the most common chronic human diseases, dental caries. Resin-based dental materials have significant potential to replace amalgam, however they lack sufficient antimicrobial power. This innovative study investigates a curcumin-loaded dental resin which can be utilized in an antimicrobial photodynamic therapy (aPDT) approach. The study evaluated the effects of curcumin loading on resin physicochemical, mechanical, and adhesive properties, as well as the antimicrobial response associated with blue light activation. Preliminary tests involving degree of conversion (DC) and sample integrity determined the optimal loading of curcumin to be restricted to 0.05 and 0.10 wt%. These optimal loadings were tested for flexural strength (FS), water sorption (WS) and solubility (SL), shear bond strength to dentin (SBS), and viability of Streptococcus mutans under 14.6 J/cm2 blue light or dark conditions, in 6 h and 24 h biofilms. The results demonstrated that 0.10 wt% curcumin had minimal impact on either FS or SBS, but detectably increased WS and SL. A 2 log10 (CFU/mL) reduction in S. mutans after light application in both 6 h and 24 h biofilms were corroborated by CLSM imaging and highlighted the significant potential of this novel aPDT approach with resin-based dental materials.

Exploring the Physicochemical, Mechanical, and Photocatalytic Antibacterial Properties of a Methacrylate-Based Dental Material Loaded with ZnO Nanoparticles.

While resin-based materials meet the many requirements of a restorative material, they lack adequate, long-lasting antimicrobial power. This study investigated a zinc oxide nanoparticle (ZnO NP)-loaded resin-blend (RB) toward a new antimicrobial photodynamic therapy (aPDT)-based approach for managing dental caries. The results confirmed that up to 20 wt% ZnO NPs could be added without compromising the degree of conversion (DC) of the original blend. The DC achieved for the 20 wt% ZnO NP blend has been the highest reported. The effects on flexural strength (FS), shear bond strength to dentin (SBS), water sorption (WS), solubility (SL), and viability of Streptococcus mutans under 1.35 J/cm2 blue light or dark conditions were limited to ≤20 wt% ZnO NP loading. The addition of up to 20 wt% ZnO NPs had a minimal impact on FS or SBS, while a reduction in the bacteria count was observed. The maximum loading resulted in an increase in SL. Furthermore, 28-day aging in 37 °C water increased the FS for all groups, while it sustained the reduction in bacteria count for the 20 wt% resin blends. Overall, the ZnO NP-loaded resin-based restorative material presents significant potential for use in aPDT.

Exploring the use of a Ruthenium complex incorporated into a methacrylate-based dental material for antimicrobial photodynamic therapy.

OBJECTIVES: To evaluate the effects of a blue light photosensitizer (PS), Ruthenium II complex (Ru), on the chemical, physical, mechanical, and antimicrobial properties of experimental dental resin blends. METHODS: The experimental resin (BisEMA, TEEGDMA, HPMA, ethanol, and photoinitiator) was loaded with Ru at 0.00%, 0.07%, 0.14%, 0.28%, 0.56%, 1.12%, 1.2%, 1.5%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% w/w. Samples were evaluated for the degree of conversion (DC) after 30 and 60 s curing-time (n = 6). Selected formulations (0.00%, 0.28%, 0.56%, 1.12%) were further tested for shear bond strength (SBS) (n = 15); flexural strength (FS) (n = 12); and antimicrobial properties (CFUs), in dark and light conditions. These latter tests were performed on specimens stored for 24-h or 2-month in 37°C water. Water sorption (WS) and solubility (SL) tests were also performed (n = 12). Data were analyzed either by a one- or two-factor general linear model (α = 0.05). RESULTS: Overall, Ru concentration above 1.2% resulted in reduced DC. In SBS results, only the 1.12%Ru resin blend samples had statistically lower values compared to the 0.00%Ru resin blend at 24-h storage (p = 0.004). In addition, no differences in SBS were detected among the experimental groups after 2-month storage in water. Meanwhile, FS increased for all experimental groups under similar aging conditions (p < 0.001). Antimicrobial properties were improved upon inclusion of Ru and application of light (p < 0.001 for both) at 24-h and 2-month storage. Lastly, no detectable changes in WS or SL were observed for the Ru-added resins compared to the 0.00%Ru resin blend. However, the 0.28% Ru blend presented significantly higher WS compared to the 0.56% Ru blend (p = 0.007). CONCLUSIONS: Stable SBS, improved FS, and sustained antimicrobial properties after aging gives significant credence to our approach of adding the Ruthenium II complex into dental adhesive resin blends intended for an aPDT approach.

The antimicrobial, physical, and chemical properties of a riboflavin-loaded dental resin intended for antimicrobial photodynamic therapy.

BACKGROUND: Dental caries remain a significant global health challenge. Unfortunately, current dental materials lack sufficient antimicrobial power to address the pathogenic species involved in this disease. In this study the potential to load a dental resin blend (RB) with riboflavin (B2) for use in an antimicrobial photodynamic therapy (aPDT) approach was investigated. METHODS: B2 was added to our experimental RB (0.1 - 10 wt%). Upon investigating the degree of conversion and specimen integrity of the RB as a function of B2 concentration, it was determined that loading should be restricted to 0.1, 1.0, and 1.5 wt%. Subsequent investigation included water sorption (WS) and solubility (SL), as well as shear bond strength (SBS) and flexural strength (FS) of the specimens after 24 h and 28-day water storage. Lastly, the antimicrobial response of Streptococcus mutans (S. mutans) biofilm following 6 h growth and 60 s of blue LED light (1.3 J/cm2) in an aPDT-based approach was measured. RESULTS AND CONCLUSIONS: Adding up to 1.5 wt% B2 had minimal impact on the FS or SBS of the RB. However, aging for 28-days notably increased the FS by as much as 50% for the 1.5 wt% B2-loaded RB. In addition, adding 1.5 wt% B2 resulted in a significant reduction in WS/SL of the RB. Lastly, while adding B2 did not change the antimicrobial response, this was an initial study under these conditions and future investigation will seek to optimize light parameters to produce a more agonistic response. Overall, a riboflavin-loaded dental resin shows significant promise for utilization in restorative dentistry with aPDT.

Acrylated epoxidized soybean oil/hydroxyapatite-based nanocomposite scaffolds prepared by additive manufacturing for bone tissue engineering.

The mechanical properties and biocompatibility of nanocomposites composed of Acrylated Epoxidized Soybean Oil (AESO), nano-Hydroxyapatite (nHA) rods and either 2-Hydroxyethyl Acrylate (HEA) or Polyethylene Glycol Diacrylate (PEGDA) and 3D printed using extrusion-based additive manufacturing methods were investigated. The effects of addition of HEA or PEGDA on the rheological, mechanical properties and cell-biomaterial interactions were studied. AESO, PEGDA (or HEA), and nHA were composited using an ultrasonic homogenizer and scaffolds were 3D printed using a metal syringe on an extrusion-based 3D printer while simultaneously UV cured during layer-by-layer deposition. Nanocomposite inks were characterized for their viscosity before curing, and dispersion of the nHA particles and tensile mechanical properties after curing. Proliferation and differentiation of human bone marrow-derived mesenchymal stem cells (BM-MSCs) were studied by seeding cells onto the scaffolds and culturing in osteogenic differentiation medium for 7, 14 and 21 days. Overall, each of the scaffolds types demonstrated controlled morphology resulting from the printability of nanocomposite inks, well-dispersed nHA particles within the polymer matrices, and were shown to support cell proliferation and osteogenic differentiation after 14 and 21 days of culture. However, the nature of the functional groups present in each ink detectably affected the mechanical properties and cytocompatibility of the scaffolds. For example, while the incorporation of HEA reduced nHA dispersion and tensile strength of the final nanocomposite, it successfully enhanced shear yield strength, and printability, as well as cell adhesion, proliferation and osteogenic differentiation, establishing a positive effect perhaps due to additional hydrogen bonding.

Effect of sterilization treatment on mechanical properties, biodegradation, bioactivity and printability of GelMA hydrogels.

Gelatin methacryloyl (GelMA) hydrogel scaffolds and GelMA-based bioinks are widely used in tissue engineering and bioprinting due to their ability to support cellular functions and new tissue development. Unfortunately, while terminal sterilization of the GelMA is a critical step for translational tissue engineering applications, it can potentially cause thermal or chemical modifications of GelMA. Thus, understanding the effect of terminal sterilization on GelMA properties is an important, though often overlooked, aspect of material design for translational tissue engineering applications. To this end, we characterized the effects of FDA-approved terminal sterilization methods (autoclaving, ethylene oxide treatment, and gamma (γ)-irradiation) on GelMA prepolymer (bioink) and GelMA hydrogels in terms of the relevant properties for biomedical applications, including mechanical strength, biodegradation rate, cell culture in 2D and 3D, and printability. Autoclaving and ethylene oxide treatment of the GelMA decreased the stiffness of the hydrogel, but the treatments did not modify the biodegradation rate of the hydrogel; meanwhile, γ-irradiation increased the stiffness, reduced the pore size and significantly slowed the biodegradation rate. None of the terminal sterilization methods changed the 2D fibroblast or endothelial cell adhesion and spreading. However, ethylene oxide treatment significantly lowered the fibroblast viability in 3D cell culture. Strikingly, γ-irradiation led to significantly reduced ability of the GelMA prepolymer to undergo sol-gel transition. Furthermore, printability studies showed that the bioinks prepared from γ-irradiated GelMA had significantly reduced printability as compared to the GelMA bioinks prepared from autoclaved or ethylene oxide treated GelMA. These results reveal that the choice of the terminal sterilization method can strongly influence important properties of GelMA bioink and hydrogel. Overall, this study provides further insight into GelMA-based material design with consideration of the effect of terminal sterilization.

Impact of Side Chain Polarity on Non-Stoichiometric Nano-Hydroxyapatite Surface Functionalization with Amino Acids.

In this study the affinity of three amino acids for the surface of non-stoichiometric hydroxyapatite nanoparticles (ns-nHA) was investigated under different reaction conditions. The amino acids investigated were chosen based on their differences in side chain polarity and potential impact on this surface affinity. While calcium pre-saturation of the calcium-deficient ns-nHA was not found to improve attachment of any of the amino acids studied, the polarity and fraction of ionized functional side groups was found to have a significant impact on this attachment. Overall, amino acid attachment to ns-nHA was not solely reliant on carboxyl groups. In fact, it seems that amine groups also notably interacted with the negative ns-nHA surface and increased the degree of surface binding achieved. As a result, glycine and lysine had greater attachment to ns-nHA than aspartic acid under the reaction conditions studied. Lastly, our results suggest that a layer of each amino acid forms at the surface of ns-nHA, with aspartic acid attachment the most stable and its surface coverage the least of the three amino acids studied.

A two-stage cold isostatic pressing and gelling approach for fabricating a therapeutically loaded amorphous calcium polyphosphate local delivery system.

Local delivery systems have taken on a greater clinical focus for osteomyelitis therapy owing to their ability to overcome many disadvantages of systemic delivery. This study reports for the first time the capacity to fabricate strontium- and vancomycin-doped calcium polyphosphate beads using a two-stage cold isostatic pressing and gelling approach. The fabricated beads were of uniform shape and diameter, and upon gelling exhibited reduced porosity. Of greatest significance in the subsequent in vitro study was the improvement of bead long-term structural stability upon vancomycin incorporation; a characteristic that further encourages the extended release of therapeutically relevant levels of antibiotic. Overall, this study provides support for the inclusion of a cold isostatic pressing step in the fabrication of a therapeutically loaded calcium polyphosphate bead-based local delivery system intended for osteomyelitis treatment.

In vivo evaluation of calcium polyphosphate for bone regeneration.

Current problems associated with bone allografts include risk of disease transmission, limited availability, and cost. Synthetic scaffolds have been proposed as substitute graft materials to address these issues. Calcium polyphosphate is a novel synthetic scaffold material that has shown good mechanical properties and biocompatibility. Here, we evaluated calcium polyphosphate in terms of its ability to support cell proliferation and differentiation in vivo. Calcium polyphosphate, morsellized cancellous bone, and hydroxyapatite/tricalcium phosphate particles were seeded with marrow stromal cells and implanted subcutaneously in the back of NOD/Scid mice. At 7, 14, and 28 days the samples were harvested and the proliferation characteristics and gene expression were analyzed. All tested graft materials had similar proliferation characteristics and gene expression. The subcutaneous environment had a stronger impact on the proliferation and differentiation of the cells than the scaffold material itself. However, it was shown that calcium polyphosphate is superior to hydroxyapatite/tricalcium phosphate and bone in its ability to support cell survival in vivo. The study confirmed that calcium polyphosphate has potential for replacing morsellized cancellous bone as a graft material for bone regeneration.