Biofilms

Example 1

119 Dicty strains were screened for their ability to feed on Dickeya (Dd) or Pectobacterium (Pcc) at 10° C. This assay was performed by inoculating Dd or Pcc on a low nutrient medium (SM2 agar) that supports both bacterial and Dicty growth. Dicty spores from individual strains were then inoculated on top of the bacterial growth and incubated at 10° C. to mimic potato storage temperatures. Dicty strains that successfully fed on Dd or Pcc created visible clearings in the lawn of bacterial growth and ultimately produced sporangia (fruiting bodies) that rose from the agar surface. An example of the phenotype that was considered successful clearing of bacteria is shown in FIG. 3A. From this initial screen, 36 Dicty strains that were capable of feeding on both Dd and Pcc at 10° C. were identified (FIG. 1B).

Of the 36 strains capable of feeding on both Dd and Pcc, 34 came from the Group 4 Dictyostelids (FIG. 1). This group includes D. discoideum, D. giganteum, D. minutum, D. mucoroides, D. purpureum, and D. sphaerocephalum (72). The results indicate that this group is particularly enriched in Dd and Pcc-feeding strains.

A further experiment was performed to identify Dicty species capable of feeding on biofilms of Dd and Pcc. Microporous polycarbonate membranes (MPMs) are widely reported to support biofilm formation of numerous Enterobacteriaceae species (2, 63, 70, 71). It was determined if Dd and Pcc formed biofilms on MPMs and determined if Dicty strains were capable of feeding on these biofilms. Membranes were placed on top of SM2 agar to provide Dd and Pcc with nutrients for growth. Bacteria were then inoculated on the surface of the MPMs and growth was monitored over the course of 1 week by washing bacteria off the membranes and performing dilution plating for colony counting. Growth of both bacterial strains plateaued around 4 dpi (FIG. 2).

From these results, it was determined that the best time to collect inoculated MPMs for biofilm analysis was at 2 dpi. Scanning electron microscopy (SEM) is commonly used to confirm biofilm formation by detecting extracellular polymeric substance (EPS) that forms the biofilm matrix (2). Samples of Dd and Pcc after 2 days of growth on MPMs in the presence and absence of Dicty are analyzed using SEM.

19 Dicty strains identified as active were tested for their ability to feed on Dd and Pcc growing on MPMs. These experiments were performed by establishing Dd and Pcc growth on MPMs overlaid on SM2 agar at 37° C. for 24 hr. Dicty spores were then applied to the center of bacterial growth in a 5 uL drop containing 1000 spores. Bacteria and Dicty were incubated at 10° C. for 2 weeks before remaining bacteria were washed off and colonies were counted. Representative images of Dicty growing on Dd and Pcc on MPMs are shown in FIG. 3A.

No Dicty strains produced a statistically significant reduction in Dd viability compared to the non-treated control. However, treating Dd lawns with Cohen 36, Cohen 9, WS-15, WS-20, and WS-69 consistently reduced the number of viable bacteria by approximately 100,000-fold compared to the non-treated control (FIG. 3B). Cohen 9 was the only Dicty strain that produced a statistically significant reduction in viability of Pcc compared to the non-treated control (FIG. 3C). Other Dicty strains capable of reducing the number of viable Pcc by at least 100,000-fold were Cohen 35, Cohen 36, WS-647, and WS-69 (FIG. 3C).

It was observed that Dicty strains Cohen 9, Cohen 36, and WS-69 were capable of feeding on both Dd and Pcc when these bacteria were cultured on SM2 agar and MPMs (FIGS. 1 and 3). These strains were also particularly effective feeders as all three reduced the number of viable Dd and Pcc on MPMs at 10° C. by 100,000-fold compared to the non-treated control (FIGS. 3B and 3C).

To determine if these strains could suppress soft rot development on seed potato tubers, tubers were tab-inoculated with Dd or Pcc and treated with spores from each Dicty strain. Seed potatoes were surface-sterilized and punctured using a sterile screw to a depth of 1.5 mm. Overnight cultures of Dd and Pcc were suspended in 10 mM potassium phosphate buffer, diluted to an OD600 of approximately 0.003, and administered as a 5 μL drop into the wound. Next, 5 of a Dicty spore suspension (100,000 spores) was added to the wound. Inoculated seed potatoes were placed in a plastic container with moist paper towels and were misted with water twice a day to maintain a high humidity. After 3 days at room temperature, seed potatoes were sliced in half and the area of macerated tissue was quantified using ImageJ.

All three strains reduced the severity of soft rot caused by Dd and Pcc (FIG. 4). Cohen 36 was the most effective strain on both Dd and Pcc: reducing the area of tissue maceration by 60% and 35%, respectively (FIG. 4B). Treating seed potatoes with WS-69 reduced the area of tissue maceration by 50% and 30% for Dd and Pcc, respectively (FIG. 4B). Finally, Cohen 9 was the least effective, but still able to reduce tissue maceration caused by Dd and Pcc by 25% and 20%, respectively (FIG. 4B).

FIG. 7 shows that three Dicty isolates control Dd and Pcc in seed tubers (at 25° C.). Two sets of data from different weeks were normalized to the Dickeya or Pectobacterium only bacterial control. The average area of macerated potato tissue measured in mm² was set as “1” or “100%”. The average of all the other treatments including Dicty were divided by bacteria only control and multiplied by 100 to obtain a percentage. Each set contained 5 tubers per treatment.

Dicty should be capable of sporulating at temperatures as cold as 10° C. on a potato surface if they are applied as a one-time pre-planting or post-harvest treatment. Sporulation was assessed by inoculating small potato discs (5×6 mm) with 10 μL of Dd or Pcc suspensions at an OD600 of 3×10⁻⁵ and Dicty spores at a concentration of 1×10⁷ spores/mL. Potato discs were kept in a covered 96-well plate for two weeks at 10° C. followed by visual inspection for son using a dissecting microscope. Representative images of a strain producing many sori (WS-517) and a strain producing few sori (WS-69) are shown in FIG. 5. Of the 11 strains evaluated, only Cohen 9 and WS-20 were unable to sporulate in the presence of both pathogens (Table 1).

Example 2

This example describes the use of a high throughput screening assay to identify Dicty strains from Alaska (e.g., BAC10A, BAF6A, BAC3A, NW2, KB4A (ATCC® MYA-4262™) SO8B, SO3A, BAF9B, IC2A (ATCC® MYA-4259™), AK1A1 (ATCC® MYA-4272™) PBF4B (ATCC® MYA-4263), PBF8B, BSB1A, SO5B (ATCC® MYA-4249), PBF3C, PBF6B, NW2B, NW10B (ATCC® MYA-4271™), PBF9A, IC5A (ATCC® MYA-4256TH), ABC8A (ATCC® MYA-4260), NW16B, ABC10B, ABB6B (ATCC® MYA-4261), BA4A (ATCC® MYA-4252), AKK5A, AKK52C, HP4 (ATCC® MYA-4286), HP8 (ATCC® MYA-4284), or NW9A) that feed on Dd and Pcc at 10° C. on potatoes.

Results from 11 Dicty strains screened against Dd at 10° C. are presented in FIG. 6. Data was analyzed for significance using a one-way analysis of variance (ANOVA; alpha =0.05) with Tukey's honest significant difference (HSD) test to compare means between the treatments and the No Dicty control. A reduction in Dd proliferation when potato discs were treated with Dicty strains Cohen 9, Cohen 36, WS-15, Maryland 18a, BAF6A, NW2, and SO3A.

The Alaskan Dicty strains, and those identified in Example 1, are further tested against coinfections of Dd and Pcc. It is useful to identify Dicty strains that can suppress Dd and Pcc coinfections as these two pathogens have been isolated together from diseased potatoes (15). The ability of Dicty strains with different feeding preferences (Dd vs. Pcc) to complement each other when administered as a cotreatment is assayed.

Example 4

The antibacterial efficacy of unaltered and experimental (doped) dental adhesive resins against non-disrupted cariogenic (caries producing) biofilms was further assessed in terms of relative luminescence units (RLUs) using a real-time luciferase-based bioluminescence assay. Toward this end, experimental dental adhesive resins containing either N—TiO₂ NPs (5%-30%, v/v), N—F—TiO₂ NPs (30%, v/v) and N—Ag—TiO₂ NPs (30%, v/v) were synthesized by dispersing the nanoparticles in OBSP adhesive resin using a sonicator (4 cycles of 1 min, intervals of 15-sec between cycles; Q700, QSonica, USA). Two non-antibacterial (OBSP, and Scotchbond Multipurpose, 3M ESPE, USA) and one antibacterial (Clearfil SE Protect, Kuraray, Noritake Dental Inc., Japan) commercially available dental adhesive resins were also tested for antibacterial functionalities. Streptococcus mutans biofilms were grown (UA 159-ldh, JM 10; 37° C., microaerophilic) on the surfaces of disk-shaped specimens (n=18/group, d=6.0 mm, t=0.5 mm) for either 24 or 48 hours with or without continuous visible light irradiation (405±15 nm). One set of specimens was fabricated with OBSP and was treated with Chlorhexidine 2% (2 min) that served as our control group. Results for the antibacterial efficacies of both unaltered and experimental dental adhesive resins containing either doped or co-doped TiO₂ NPs under continuous visible light irradiation for either 24 or 48 hours, demonstrated that all groups tested displayed similar antibacterial behaviors under continuous visible light irradiation. Such findings suggest that under the conditions investigated (wavelength and power intensity), visible light irradiation had a very strong antibacterial behavior that took place independently of the antibacterial activity of the substrate where biofilms were grown (either antibacterial or not). Such impact made impossible the determination of the materials' real antibacterial efficacies under such light irradiation conditions.

Experiments were then conducted under dark conditions; bacteria were grown in dark conditions for either 24 and 48 hours. The results indicated that the TiO₂-containing adhesive resins were more antibacterial than commercially available non-antibacterial dental adhesive resins (such as OptiBond Solo Plus and Scotchbond Multipurpose). The experimental dental adhesive resins containing 30% (v/v) of nanoparticles (N—TiO₂ NPs, N—F—TiO₂ NPs and N—Ag—TiO₂ NPs) displayed antibacterial efficacies in dark conditions that were similar to Clearfil SE Protect (Fluoride-releasing material, Kuraray, Noritake Dental Inc., Japan). S. mutans biofilms grown on specimens treated with chlorhexidine 2% (2 min) displayed the lowest RLU values amongst all groups investigated, thereby confirming the strong antibacterial behavior of non-immobilized chlorhexidine. In addition, the antibacterial effect was demonstrated to be concentration-dependent, wherein experimental adhesive resins containing higher concentrations of antibacterial nanoparticles (either doped or co-doped) displayed stronger antibacterial effects against non-disrupted S. mutans biofilms. Since long intra-oral irradiation periods (24-hour and 48-hour) are impractical and clinically not feasible, associated with the fact that these materials are intended to be used in the oral cavity's dark conditions, these results were considered of paramount importance and clinically relevant for the commercialization pathway of recently developed antibacterial and bioactive nano-filled dental adhesive resins.

Optical and mechanical properties of both unaltered and experimental dental adhesive resins containing 5%-30% (v/v, 5% increments) of N—TiO₂ NPs were assessed in terms of color stability and biaxial flexure strength. Color stability (n=5) and biaxial flexure strength (n=8) specimens (d=6.0 mm, t=0.5 mm) were fabricated and tested using a color analysis software (ScanWhite, Darwin Syst., Brazil) and an Instron universal testing machine (cross-head rate=1.27 mm/min), respectively. Color stability measurements were performed immediately after specimen fabrication and after water storage (1, 2, 3, 4, 5, 6 months; 37° C.). The color stability results demonstrated that specimens fabricated using either unaltered or experimental dental adhesive resins containing N—TiO₂ NPs (5%-30%, v/v) were subjected to color changes induced by long-term water storage. The highest color variations were observed at two months of water storage (37° C.) for specimens pertaining to experimental groups containing either 5% or 10% of N—TiO₂ NPs. Specimens fabricated with unaltered OptiBond Solo Plus have demonstrated color variations that were similar to the color variations observed for the experimental group containing 20% N—TiO₂ NPs. Specimens fabricated with 30% N—TiO₂ NP-containing dental adhesive resins have shown the least amount of color variation throughout the investigation time (6-mo), and therefore, were considered as the most color stable amongst all materials investigated. From the esthetic standpoint, the human eye can only detect differences in color above a certain threshold (ΔE≥3).

In at least one embodiment, dental composition specimens fabricated with at 30% N—TiO₂ NPs displayed color variations that were either lower than or close to the human eye detection capability, thereby corroborating the long-term use of these highly esthetic experimental dental adhesive resins. In at least certain embodiments, the dental compositions contain at least 5% to 80% (v/v) of doped-TiO₂ NPs as disclosed herein, such as at least 5% (v/v), at least 6% (v/v), at least 7% (v/v), at least 8% (v/v), at least 9% (v/v), at least 10% (v/v), at least 11% (v/v), at least 12% (v/v), at least 13% (v/v), at least 14% (v/v), at least 15% (v/v), at least 16% (v/v), at least 17% (v/v), at least 18% (v/v), at least 19% (v/v), at least 20% (v/v), at least 21% (v/v), at least 22% (v/v), at least 23% (v/v), at least 24% (v/v), at least 25% (v/v), at least 26% (v/v), at least 27% (v/v), at least 28% (v/v), at least 29% (v/v), at least 30% (v/v), at least 31% (v/v), at least 32% (v/v), at least 33% (v/v), at least 34% (v/v), at least 35% (v/v), at least 36% (v/v), at least 37% (v/v), at least 38% (v/v), at least 39% (v/v), at least 40% (v/v), at least 41% (v/v), at least 42% (v/v), at least 43% (v/v), at least 44% (v/v), at least 45% (v/v), at least 46% (v/v), at least 47% (v/v), at least 48% (v/v), at least 49% (v/v), at least 50% (v/v), at least 51% (v/v), at least 52% (v/v), at least 53% (v/v), at least 54% (v/v), at least 55% (v/v), at least 56% (v/v), at least 57% (v/v), at least 58% (v/v), at least 59% (v/v), at least 60% (v/v), at least 61% (v/v), at least 62% (v/v), at least 63% (v/v), at least 64% (v/v), at least 65% (v/v), at least 66% (v/v), at least 67% (v/v), at least 68% (v/v), at least 69% (v/v), at least 70% (v/v), at least 71% (v/v), at least 72% (v/v), at least 73% (v/v), at least 74% (v/v), at least 75% (v/v), at least 76% (v/v), at least 77% (v/v), at least 78% (v/v), at least 79% (v/v), or at least 80% (v/v), with the balance comprising the curable adhesive resin material, and optionally other components as described elsewhere herein.

The present results demonstrate that experimental dental adhesive resins containing varying concentrations of N—TiO₂ NPs display biaxial flexure strengths that are either similar or better than the strength observed for specimens fabricated with the unaltered OBSP. No differences were observed among the flexure strengths of experimental groups, thereby indicating that the presently disclosed materials can behave very similar to commercially available materials when subjected to masticatory forces.

Specimens (d=6.0 mm, t=0.5 mm) of the unaltered resins and experimental dental adhesive resins containing 30% N—TiO₂ NPs, 30% N—F—TiO₂ NPs and 30% N—Ag—TiO₂ NPs were fabricated and characterized using the state of the art scanning electron microscope. This dual focused ion-beam microscope (Dual-FIB SEM/EDS) is capable, through a destructive process, to characterize and map the chemical composition and distribution of elements in three dimensions. The 3-D characterization and localization of components clearly demonstrated that experimental materials containing co-doped nanoparticles (e.g., 30% v/v, N—F—TiO₂ NPs) displayed an optimized dispersion of filler particles (part of the original composition) when compared to the filler particle distribution observed on specimens fabricated with the unaltered dental adhesive resin. The 3-D images demonstrated that the experimental adhesive resins had more filler particles per unit volume with a more homogeneous size distribution than the filler fraction and size distribution observed on OptiBond Solo Plus samples. In addition, results showed that larger and more agglomerated filler particles tend to result in a polymer matrix containing more pores per unit volume. This finding was corroborated by the pore-size distribution calculated for the unaltered samples and experimental dental adhesive resin samples, where it is possible to observe that the quantity and sizes of pores formed in experimental materials were smaller when compared to the unaltered OptiBond Solo Plus samples.

In at least one embodiment, the present disclosure includes a dental composition, comprising doped and/or coated TiO₂ NPs, and a curable resin material, wherein the curable resin material comprises a polymer precursor component. The TiO₂ NPs may comprise at least one dopant or coating selected from the group consisting of N (nitrogen), Ag (silver), F (fluorine), P (phosphorus), and PO₄ (phosphate). As noted above, in non-limiting embodiments, the dental composition may comprise a volume to volume ratio of doped TiO₂ NPs to curable resin material in a range of 1% to 80% (v/v), 5% to 50% (v/v), or 10% to 40% (v/v), for example. The polymer precursor component may be photocurable. The polymer precursor may be selected from the group consisting of acrylates, methacrylates, dimethacrylates, epoxies, vinyls and thiols. The polymer precursor may be selected from the group consisting of ethylenedimethacrylate (“EDMA”), bisphenol A glycidyl methacrylate (“BisGMA”), triethyleneglycol dimethacrylate (“TEGDMA”), 1,6-bis(methacryloxy-2-ethoxycarbonylamino)-2,4,4-trimethylhexane (UDMA), pyromellitic glycerol dimethacrylate (PMGDM), and 2-hydroxyethyl methacrylate (HEMA). The dental composition may comprise at least one solvent. The at least one solvent may be selected from the group consisting of water, ethanol, methanol, toluene, ethyl ether, cyclohexane, isopropanol, chloroform, ethyl acetate, acetone, hexane, and heptanes. The dental composition may comprise a polymerization initiator. The dental composition may comprise a filler. The dental composition may be selected from the group consisting of dental resins, dental bonding agents, dental adhesives, dental cements, dental restoratives, dentals coatings, dental sealants, acrylic resins, and denture teeth. The dental composition may comprise bioactive and/or antibacterial activity in the absence of visible or ultraviolet light. The dental composition may be used to form a hardened dental article after a photocuring step. In at least one embodiment, the disclosure includes an in vivo dental process, comprising applying the dental composition to at least one of a dental restorative and a dental substrate, and causing the dental restorative to be bonded to the dental substrate via the dental composition after a step of photocuring the dental composition.

Accordingly, the present disclosure is directed to at least the following non-limiting embodiments:

Clause 1. In at least one embodiment the present disclosure includes a dental composition, comprising doped TiO₂ nanoparticles, and a curable resin material, wherein the curable resin material comprises a polymer precursor component.

Clause 2. The dental composition of clause 1, wherein the doped TiO₂ nanoparticles comprise at least one dopant selected from the group consisting of N (nitrogen), Ag (silver), F (fluorine), P (phosphorus), and PO₄ (phosphate).

Clause 3. The dental composition of clause 1 or 2, wherein the doped TiO₂ nanoparticles further comprise at least one second dopant selected from the group consisting of N, Ag, F, P, and PO₄.

Clause 4. The dental composition of any one of clauses 1-3, comprising a volume to volume ratio of doped TiO₂ nanoparticles to curable resin material in a range of 1% to 80% (v/v).

Clause 5. The dental composition of any one of clauses 1-4, comprising a volume to volume ratio of doped TiO₂ nanoparticles to curable resin material in a range of 5% to 50% (v/v).

Clause 6. The dental composition of any one of clauses 1-5, comprising a volume to volume ratio of doped TiO₂ nanoparticles to curable resin material in a range of 10% to 40% (v/v).

Clause 7. The dental composition of any one of clauses 1-6, wherein the polymer precursor component is photocurable.

Clause 8. The dental composition of any one of clauses 1-7, wherein the polymer precursor is selected from the group consisting of acrylates, methacrylates, dimethacrylates, epoxies, vinyls and thiols.

Clause 9. The dental composition of any one of clauses 1-8, wherein the polymer precursor is at least one selected from the group consisting of ethylenedimethacrylate (EDMA), bisphenol A glycidyl methacrylate (BisGMA), triethyleneglycol dimethacrylate (TEGDMA), 1,6-bis(methacryloxy-2-ethoxycarbonylamino)-2,4,4-trimethylhexane (UDMA), pyromellitic glycerol dimethacrylate (PMGDM), and 2-hydroxyethyl methacrylate (HEMA).

Clause 10. The dental composition of any one of clauses 1-9, further comprising at least one solvent.

Clause 11. The dental composition of any one of clauses 1-10, further comprising a solvent selected from the group consisting of water, ethanol, methanol, acetone, toluene, ethyl ether, cyclohexane, isopropanol, chloroform, ethyl acetate, hexane, and heptanes.

Clause 12. The dental composition of any one of clauses 1-11, further comprising a polymerization initiator.

Clause 13. The dental composition of any one of clauses 1-12, further comprising a filler.

Clause 14. The dental composition of any one of clauses 1-13, wherein the curable resin material is selected from the group consisting of dental resins, dental bonding agents, dental adhesives, dental cements, dental restoratives, dentals coatings, dental sealants, acrylic resins, and denture teeth.

Clause 15. The dental composition of any one of clauses 1-14, comprising bioactive and/or antibacterial activity in the absence of visible or ultraviolet light.

Clause 16. A kit for forming a dental composition, the kit comprising doped TiO₂ nanoparticles, and a curable resin material, wherein the curable resin material comprises a polymer precursor component.

Clause 17. The kit of clause 16, wherein the doped TiO₂ nanoparticles comprise at least one dopant selected from the group consisting of N (nitrogen), Ag (silver), F (fluorine), P (phosphorus), and PO₄ (phosphate).

Clause 18. The kit of clause 16 or 17, wherein the doped TiO₂ nanoparticles further comprise at least one second dopant selected from the group consisting of N, Ag, F, P, and PO₄.

Clause 19. The kit of any one of clauses 16-18, comprising sufficient doped TiO₂ nanoparticles and curable resin material such that the dental composition comprises a volume to volume ratio of doped TiO₂ nanoparticles to curable resin material in a range of 1% to 80% (v/v).

Clause 20. The kit of any one of clauses 16-19, comprising sufficient doped TiO₂ nanoparticles and curable resin material such that the dental composition comprises a volume to volume ratio of doped TiO₂ nanoparticles to curable resin material in a range of 5% to 50% (v/v).

Clause 21. The kit of any one of clauses 16-20, comprising sufficient doped TiO₂ nanoparticles and curable resin material such that the dental composition comprises a volume to volume ratio of doped TiO₂ nanoparticles to curable resin material in a range of 10% to 40% (v/v).

Clause 22. The kit of any one of clauses 16-21, wherein the polymer precursor component is photocurable.

Clause 23. The kit of any one of clauses 16-22, wherein the polymer precursor is selected from the group consisting of acrylates, methacrylates, dimethacrylates, epoxies, vinyls and thiols.

Clause 24. The kit of any one of clauses 16-23, wherein the polymer precursor is at least one selected from the group consisting of ethylenedimethacrylate (EDMA), bisphenol A glycidyl methacrylate (BisGMA), triethyleneglycol dimethacrylate (TEGDMA), 1,6-bis(methacryloxy-2-ethoxycarbonylamino)-2,4,4-trimethylhexane (UDMA), pyromellitic glycerol dimethacrylate (PMGDM), and 2-hydroxyethyl methacrylate (HEMA).

Clause 25. The kit of any one of clauses 16-24, further comprising at least one solvent.

Clause 26. The kit of any one of clauses 16-25, further comprising a solvent selected from the group consisting of water, ethanol, methanol, acetone, toluene, ethyl ether, cyclohexane, isopropanol, chloroform, ethyl acetate, hexane, and heptanes.

Clause 27. The kit of any one of clauses 16-26, further comprising a polymerization initiator for combining with the doped TiO₂ nanoparticles, and curable resin material.

Clause 28. The kit of any one of clauses 16-27, further comprising a filler for combining with the doped TiO₂ nanoparticles, and curable resin material.

Clause 29. The kit of any one of clauses 16-28, wherein the curable resin material is selected from the group consisting of dental resins, dental bonding agents, dental adhesives, dental cements, dental restoratives, dentals coatings, dental sealants, acrylic resins, and denture teeth.

Clause 30. The kit of any one of clauses 16-29, wherein the dental composition has bioactive and/or antibacterial activity in the absence of visible or ultraviolet light.

Clause 31. A hardened dental article formed from the dental composition of any one of clauses 1-15, after the dental composition has been photocured.

Clause 32. An in vivo dental process, comprising: applying a dental composition to a dental surface, the dental composition comprising doped TiO₂ nanoparticles, and a curable resin material, wherein the curable resin material comprises a polymer precursor component; and causing the dental composition to be bonded to the dental surface by photocuring the dental composition.

Clause 33. The dental process of clause 32, wherein the dental surface is at least one of a dental restorative and a dental substrate.

Clause 34. The dental process of clause 32 or 33, wherein the doped TiO₂ nanoparticles comprise at least one dopant selected from the group consisting of N (nitrogen), Ag (silver), F (fluorine), P (phosphorus), and PO₄ (phosphate).

Clause 35. The dental process of any one of clauses 32-34, wherein the doped TiO₂ nanoparticles further comprise at least one second dopant selected from the group consisting of N, Ag, F, P, and PO₄.

Clause 36. The dental process of any one of clauses 32-35, wherein the dental composition comprises a volume to volume ratio of doped TiO₂ nanoparticles to curable resin material in a range of 1% to 80% (v/v).

Clause 37. The dental process of any one of clauses 32-36, wherein the dental composition comprises a volume to volume ratio of doped TiO₂ nanoparticles to curable resin material in a range of 5% to 50% (v/v).

Clause 38. The dental process of any one of clauses 32-37, wherein the dental composition comprises a volume to volume ratio of doped TiO₂ nanoparticles to curable resin material in a range of 10% to 40% (v/v).

Clause 39. The dental process of any one of clauses 32-38, wherein the polymer precursor component is photocurable.

Clause 40. The dental process of any one of clauses 32-39, wherein the polymer precursor is selected from the group consisting of acrylates, methacrylates, dimethacrylates, epoxies, vinyls and thiols.

Clause 41. The dental process of any one of clauses 32-40, wherein the polymer precursor is at least one selected from the group consisting of ethylenedimethacrylate (EDMA), bisphenol A glycidyl methacrylate (BisGMA), triethyleneglycol dimethacrylate (TEGDMA), 1,6-bis(methacryloxy-2-ethoxycarbonylamino)-2,4,4-trimethylhexane (UDMA), pyromellitic glycerol dimethacrylate (PMGDM), and 2-hydroxyethyl methacrylate (HEMA).

Clause 42. The dental process of any one of clauses 32-41, wherein the dental composition further comprises at least one solvent.

Clause 43. The dental process of any one of clauses 32-42, further comprising a solvent selected from the group consisting of water, ethanol, methanol, acetone, toluene, ethyl ether, cyclohexane, isopropanol, chloroform, ethyl acetate, hexane, and heptanes.

Clause 44. The dental process of any one of clauses 32-43, wherein the dental composition further comprises a polymerization initiator.

Clause 45. The dental process of any one of clauses 32-44, wherein the dental composition further comprises a filler.

Clause 46. The dental process of any one of clauses 32-45, wherein the curable resin material is selected from the group consisting of dental resins, dental bonding agents, dental adhesives, dental cements, dental restoratives, dentals coatings, dental sealants, acrylic resins, and denture teeth.

Clause 47. The dental process of any one of clauses 32-46, wherein after curing, the dental composition has bioactive and/or antibacterial activity in the absence of visible or ultraviolet light.

Clause 48. The dental process of any one of clauses 32-47, wherein the dental surface has been acid-etched prior to the application of the dental composition thereon.

While the present disclosure has been described herein in connection with certain embodiments so that aspects thereof may be more fully understood and appreciated, it is not intended that the present disclosure be limited to these particular embodiments. On the contrary, it is intended that all alternatives, modifications and equivalents are included within the scope of the present disclosure as defined herein. Thus the examples described above, which include particular embodiments, will serve to illustrate the practice of the inventive concepts of the present disclosure, it being understood that the particulars shown are by way of example and for purposes of illustrative discussion of particular embodiments only and are presented in the cause of providing what is believed to be the most useful and readily understood description of procedures as well as of the principles and conceptual aspects of the present disclosure. Changes may be made in the formulation of the various compositions described herein, the methods described herein or in the steps or the sequence of steps of the methods described herein without departing from the spirit and scope of the present disclosure. Further, while various embodiments of the present disclosure have been described in claims herein below, it is not intended that the present disclosure be limited to these particular claims.

Example 2

Antimicrobial activity of the compositions according to the invention has been compared with compositions comprising either only the modified clay particle comprising an antimicrobial compound (‘CPC’, prepared as in Ex. 1), or only a nonionic triblock copolymer (‘pluronic’). Salivary flora and actives (according to Table 1 below) were co-incubated overnight and at the end of incubation biofilm was stained with crystal violet. Detailed protocol as mentioned below:

Treatment and Biofilm Formation

Early morning saliva samples before brushing was collected from 4-5 people, pooled together and washed twice in saline. Absorbance was set to 0.2 OD620 nm in ultra-filtered tryptone yeast extract broth (2% sucrose) and used for experiments as mentioned in further steps. 2 ml of set culture was added into 24/12 well plate to which test actives at varying concentrations were added into each of the wells. The plate was incubated anaerobically overnight at 37° C.

Staining Protocol

At the end overnight incubation, decant the plate out over a biohazard bag to remove all the planktonic bacteria. Rinse the plate in a tray of water and decant the water out over the tray. This step was done once to remove the loosely adhered biofilm. Place the plate on a blotting paper/paper towel over the bench top. Stain all the test wells with 1 ml of 1% Crystal violet stain (CV) for 10 min. This step was done using a pipette. Decant the plate out over the biohazard discard bag to remove all the stain. Rinse the plate in a tray of water and pour the water out over the tray. This step was done thrice consecutively, in three separate trays of water. (Each tray procedure was repeated thrice-total 9 rinse). Cover the bench top with more blotting paper/paper towel and hit the plate against the bench top until all the wells are free of any liquid. This step was done to ensure that only CV remaining is bound to a biofilm at the bottom of a well. Leave the plate face up on the bench top at room temperature (23+2° C.) until it dries completely. Add 1 ml of 33% glacial acetic acid to the test wells to solubilize the biofilm bound CV stain. Allow the acetic acid to sit for 10 mins. Pipette up and down the mix of acetic acid and CV in the wells.

Transfer 10 μl of above solution mix to 90 ul of 33% acetic acid in a well of flat bottom 96 well plate. Mix the solution well and absorbance is taken at 540 nm. All the test actives were done in duplicates.