Patent Application
20240293289
The present invention relates to the fields of dentistry, oral diseases, and drug delivery systems. More particularly, the present invention relates to microemulsions as magnetic responsive nanoplatforms for the delivery of formulations to pathogenic microbes in the oral environment during photodynamic therapy.
The longevity of resin composite dental restorations relies heavily on the effectiveness of dental adhesives in bonding the tooth-colored material to the dentin, the inner part of the tooth (1,2). This bonding interface forms a hybrid layer, combining dental adhesive with dentin components like collagen, water, and hydroxyapatite crystals. However, this interface is subjected to mechanical stress, temperature changes, and bacterial acid attacks in the mouth. Evidence indicates this interface weakens over time, leading to poor sealing between the restorative material and dentin (3). This can result in issues like microleakage, tooth sensitivity, and cavities around restorations, necessitating costly replacement (4).
Superparamagnetic iron oxide nanoparticles (SPIONs) have shown promise in dentistry as reinforcing fillers that can exhibit antibacterial properties and strengthen resins (5). These particles, in particular, have garnered significant interest in dental adhesion due to their unique properties, such as enhanced adhesive strength and the ability to deliver drugs by magnetic motion, making them a highly desired material for advancing dental restoration (6).
Core-shell technology, where superparamagnetic iron oxide nanoparticles are encapsulated, can mitigate these particles' aggregation and dark coloration (7). The controlled release of chlorhexidine (CHX) from core-shell particles could be beneficial to protect collagen fibrils from degradation (8). CHX has dual benefits: it acts as an antibacterial agent against dental plaque and inhibits collagen-degrading enzymes (9).
Particles like superparamagnetic iron oxide nanoparticles require surface modification through silanization to enhance bonding performance (10). This chemical modification should not compromise the particle's properties, including saturation magnetization (11). For dental applications, organosilicons play a crucial role in creating a strong interphase between filler and resin, contributing to the strength and durability of dental bonds (12). Furthermore, organosilicon quaternary ammonium salt monomers have long-lasting antibacterial effects (13).
The prior art is deficient in multifunctional dental adhesives. The present invention fulfills this long-standing need and desire in the art.
The present invention is directed to a biomedical composition for dental restoration. The biomedical composition has a plurality of magnetic microparticles, an antibiotic and a mesoporous silica encapsulating the plurality of magnetic microparticles and the antibiotic with an antibacterial shell disposed therearound.
The present invention is further directed to a dental formulation for a dental bonding restoration. The dental formulation has a dental adhesive material and the biomedical composition described herein incorporated therein.
The present invention is directed further to a method for increasing longevity of a dental restoration in a subject. In this method, the dental formulation described herein is prepared and is applied to an area of interest in an oral cavity of the subject. The dental formulation is bonded to dentin in the area of interest, where the magnetic microparticles, the antibiotic and the antibacterial shell of the biomedical composition incorporated therein increase the longevity of the dental restoration in the subject.
The present invention is directed further still to a dental adhesive. The dental adhesive has a biomedical adhesive and a magnetic core-shell platform incorporated therein, where the dental adhesive is pharmacologically effective for a tooth restoration.
The present invention is directed further still to a method for restoring a tooth in a subject in need thereof. In this method, a dental adhesive comprising a dental adhesive material and containing a multifunctional magnetic platform therein is formulated. The tooth is prepared for restoration, the dental adhesive is applied to a dentin layer on the tooth and the tooth is restored.
Other and further aspects, features, benefits, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention given for the purpose of disclosure.
The appended drawings have been included herein so that the above-recited features, advantages, and objects of the invention will become clear and can be understood in detail. These drawings form a part of the specification. It is to be noted, however, that the appended drawings illustrate preferred embodiments of the invention and should not be considered to limit the scope of the invention.
As used herein, the term “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” Some embodiments of the invention may consist of or consist essentially of one or more elements, method steps, and/or methods of the invention. It is contemplated that any method described herein can be implemented with respect to any other method described herein.
As used herein, the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.”
As used herein, “comprise” and its variations, such as “comprises” and “comprising,” will be understood to imply the inclusion of a stated item, element or step or group of items, elements or steps but not the exclusion of any other item, element or step or group of items, elements or steps unless the context requires otherwise. Similarly, “another” or “other” may mean at least a second or more of the same or different claim element or components thereof.
As used herein, “biomedical composition”, “magnetic core-shell platform”, and “multifunctional magnetic platform” are used interchangeably.
As used herein, “biomedical adhesive material” and “dental adhesive material” are used interchangeably.
As used herein, “pharmacologically effective” refers to an amount or concentration of the dental adhesive and/or the multifunctional magnetic platform contained therein that results in an increase in an interfacial bond formed between the dental adhesive and a dentin layer on a tooth being restored. A person having ordinary skill in this art would understand that the pharmacologically effective amount may improve the stability of the bond and increase resistance to degradation thereof and may comprise an antibacterial efficacy for the subject's restored tooth, but may not completely eliminate the problems present with dental adhesives known and standard in the art.
As used herein, the term “subject” refers to any recipient of a dental restoration utilizing the multifunctional magnetic platforms and dental adhesives provided herein.
In one embodiment of the present invention, there is provided a biomedical composition for dental restoration, comprising a plurality of magnetic microparticles; an antibiotic; and a mesoporous silica encapsulating the plurality of magnetic microparticles and the antibiotic with an antibacterial shell disposed therearound.
In this embodiment, the plurality of magnetic microparticles may comprise iron oxide Fe3O4. Further in this embodiment, the antibiotic may be chlorhexidine. In addition, the antibacterial shell may be an antibacterial quaternary ammonium silane. Further in this embodiment, the biomedical composition may have a formula CHX@SiQuac@Fe3O4@m-SiO2.
In another embodiment of the present invention, there is provided a dental formulation for a dental bonding restoration, comprising a dental adhesive material; and the biomedical composition as described supra incorporated therein.
In yet another embodiment of the present invention, there is provided a method for increasing longevity of a dental restoration in a subject, comprising preparing the dental formulation as described supra, applying the dental formulation to an area of interest in an oral cavity of the subject; and bonding the dental formulation to dentin in the area of interest, wherein the magnetic microparticles, the antibiotic and the antibacterial shell of the biomedical composition incorporated therein increase the longevity of the dental restoration in the subject. In this embodiment the biomedical composition may strengthen an interfacial bond between the dental adhesive and the dentin.
In yet another embodiment of the present invention, there is provided a dental adhesive, comprising a biomedical adhesive material; and a magnetic core-shell platform incorporated therein, the dental adhesive pharmacologically effective for a tooth restoration.
In this embodiment, the magnetic core-shell platform may comprise a core of a plurality of magnetic microparticles doped with an antibiotic and coated with a mesoporous silane shell that is functionalized with an antibacterial agent.
In an aspect of this embodiment, the plurality of magnetic microparticles may be iron oxide Fe3O4. In another aspect, the plurality of magnetic microparticles may be doped with chlorhexidine. In yet another aspect, the antibacterial agent may be an antibacterial quaternary ammonium silane. In this embodiment and all aspects thereof, the magnetic core-shell platform may have a formula of CHX@SiQuac@Fe3O4@m-SiO2.
In yet another embodiment of the present invention, there is provided a method for restoring a tooth in a subject in need thereof, comprising formulating a dental adhesive comprising a dental adhesive material and containing a multifunctional magnetic platform therein; preparing the tooth for restoration; applying the dental adhesive to a dentin layer on the tooth; and restoring the tooth.
In this embodiment, the multifunctional magnetic platform may have a core-shell structure comprising a core formed of a plurality of magnetic microparticles and an antibiotic doping agent; and an antibacterial silane shell coating the core. In an aspect thereof, the core may be formed of a plurality of iron oxide Fe3O4 microparticles and doped with chlorhexidine. In another aspect, the antibacterial silane shell may be a mesoporous silane comprising an antibacterial quaternary ammonium silane. In this embodiment and aspects thereof, the multifunctional magnetic platform has a core-shell structure of CHX@SiQuac@Fe3O4@m-SiO2. Also in this embodiment and aspects thereof, the multifunctional magnetic platform contained in the dental adhesive upon forming an interfacial hybrid bond with the dentin layer during restoration may increase longevity thereof via increasing bonding stability via magnetic interactions; resisting degradation of the hybrid bond by matrix metalloproteinases; or inhibiting bacterial growth; or a combination thereof.
Provided herein are the synthesis, characterization, and assessments of a multifunctional magnetic platform incorporated into a dental adhesive to provide 1) filler reinforcement and magnetic motion, 2) a combinatorial antibacterial effect, and 3) an anti-bond degradation strategy via protease inhibitor such as matrix metalloproteinases. This multifunctional platform, denoted CHX@SiQuac@Fe3O4@m-SiO2, comprises Fe3O4 microparticles and chlorhexidine (CHX) encapsulated within the mesoporous silica, which is capped by an antibacterial quaternary ammonium silane (SiQuac).
This combinatorial strategy of incorporating silane-coated magnetic core-shell particles, doped with chlorhexidine and functionalized with an antibacterial agent enables the platform to simultaneously target bacterial inhibition, stability of the hybrid layer, and filler reinforcement. The resulting dental adhesive with CHX@SiQuac@Fe3O4@m-SiO2 offers multiple benefits, including bacterial inhibition, bonding stability of the hybrid layer, resistance to degradation, and improved filler infiltration through magnetic motion (
The present invention addresses the critical issue of adhesive interfacial breakdown, which significantly reduces the service life of composite restorations. The multifunctional platform is effective in several aspects. The silanization process enhances the specific surface area and pore size contributing to the improved performance of the material. CHX loading into the framework exhibits increased cumulative release thereof and MMP inhibition, further enhancing the antibacterial and anti-MMP activities without affecting human cell viability.
Also provided herein are methods for increasing longevity of a dental restoration in a subject and for restoring a tooth in a subject in need thereof utilizing the dental adhesives containing the microfunctional magnetic platform described herein. One of ordinary skill in the art is well-able to determine a pharmacologically effective amount of the dental adhesives provided herein for use in a dental restoration procedure or other procedure requiring the same.
The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion.
Synthesis of Fe3O4@m-SiO2
The mesoporous silica oxide shell with iron oxide core (Fe3O4@m-SiO2) was synthesized by a sol-gel method (14). Chemicals, including Iron (II,III) oxide nanopowder, TEOS, CHX, dichloromethane, N-propylamine, cyclohexane, and NaOH were procured from Sigma-Aldrich, Saint Louis, MO, USA. Cetyltrimethylammonium bromide CTAB was sourced from EMD Millipore Crop, Burlington, MA, USA, and SiQuac from Gelest Inc, Morrisville, PA, USA. Briefly, 0.007 g of Fe3O4, 0.0029 g of CTAB, and 116 μL of TEOS at 20% were dissolved in 5 mL of distilled water with sonication at 40% power, 24 KHz frequency, 37° C. in pulse mode (10 seconds on/10 seconds off) for 80 min. The pH was adjusted to 8 by adding 0.1 M NaOH, and the solution was sonicated again following the previous parameters (66). The solution was kept for 20 h at room temperature. The purification was performed via sonication (same parameters, 12 min), centrifugation (7,000 RCF for 15 mins at 23° C.), and removal of the supernatant. The sedimented particles were redispersed ten times with 25 mL of ethanol and four times with 25 ml of water. The particles were dispersed in 5 ml of water and vacuum dried at 75° C. overnight.
Chlorhexidine Loading into Fe3O4@m-SiO2
Chlorhexidine (CHX) and Fe3O4@m-SiO2 (50 mg each) were dissolved in 5 mL of dichloromethane by sonication for 12 min, followed by shaking at 100 RPM at room temperature for 24 h. The particles were separated by centrifugation at 7,000 RCF for 10 min at 25° C. The supernatant was preserved to obtain the drug loading. The particles were rinsed with ethanol and water and vacuum dried at 75° C. overnight (
Surface Silanization with SiQuac
To functionalize the CHX@Fe3O4@m-SiO2, these particles (2 g) were added to a mix of octadecyl-dimethyl (3-trimethoxysilylpropyl) ammonium chloride (SiQuac) (0.08 mL), n-propylamine (0.04 mL), and cyclohexane (3 mL) (16). The mixture was stirred for 2 h at room temperature. The solution was placed into a rotary evaporator at 90° C. for 1 h, and the powder was dried at 80° C. for 1 h. Some of the CHX@Fe3O4@m-SiO2 was kept without silanization as a control group.
Fe3O4@m-SiO2 and CHX@SiQuac@Fe3O4@m-SiO2 were characterized via transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The images were acquired from 20,000X to 40,000X magnification and 12,500 KV for TEM and 500X to 5,000X magnification with 3.5 kV for SEM. Energy-dispersive X-ray analysis spectroscope (EDS) was used with TEM. The particles were analyzed for surface area and pore size distribution using N2 adsorption-desorption isotherms. Before the analysis, the particles were kept in a vacuum at 100° C. for 8 h to remove the adsorbed gas. X-ray diffraction (XRD) was used with Cu-Kα radiation (λ=1.54 Å), 40 kV, and 40 mA. The powders were also analyzed with attenuated total reflectance-Fourier transform infrared (ATR/FT-IR) spectroscopy with a diamond-ZnSe crystal. The spectra were acquired from 4000 to 550 cm−1, with a resolution of 0.5 cm−1, and 4 scans/s. The magnetic properties were tested with a vibrating sample magnetometer with a 3T electromagnet with 75 Hz, ±5 kOe (+0.5 T).
In vitro CHX Loading Analysis
The percentage of encapsulation-efficiency (EE) and drug-loading (DL) were calculated by determining the amount of free non-encapsulated CHX via Ultraviolet-Visible (UV-Vis) spectroscopy at 289 nm (17,18) The DL (%) and EE (%) were calculated according to the following equations:
The particles were also tested via thermogravimetric analysis (TGA) using 10 mg of SiQac@Fe3O4@m-SiO2 and Fe3O4@m-SiO2 heated in alumina crucibles from 30 to 800° C., heat flow rate of 10° C./min, under N2 atmosphere (19).
In vitro CHX Release Analysis
CHX solutions were measured to generate a standard curve via UV-Vis. CHX @Fe3O4@m-SiO2 and CHX@SiQuac@Fe3O4@m-SiO2 (50 mg each) were suspended in 5 mL of phosphate buffer saline (PBS) at room temperature under stirring (100 RPM). Two different pH values (5.5 and 7.4) of PBS were used to mimic the acidic and neutral environments (17). The samples were centrifuged at 7,000 RCF for 10 min, and fresh PBS replaced the solutions with the same pH values. The removed aliquots were analyzed for CHX content using UV-Vis at 289 nm and high-performance liquid chromatography (HPLC). The cumulative release of CHX was calculated according to the following equation 4):
For the MMPs inhibition assay, SENSOLYTE Generic MMP Assay Kit Colorimetric was used. The MMP thiopeptide substrate solution was diluted to 0.2 mM with the supplied assay buffer in a 1:50 volume ratio. A 96-well plate was used for the assay using four replicates per group. In each well, 40 μL of MMP-9 (20 ng/well), 10 μL of CHX (1 mg/mL) at serial dilutions, and 50 μL of thiopeptide substrate solution were mixed. The fluorescence intensity was measured using a plate reader (SpectraMax M5, Molecular Devices, Sunnyvale, CA, USA) at Ex/Em-490 nm/540 nm with a kinetic mode every 5 min for 60 min (20). The substrate control group was set as a background, where its absorbance was subtracted from other wells. The percentage of MMP inhibition was calculated according to equation 5:
Human gingival fibroblasts and dental pulp stem cells were used to analyze the particle's cytotoxicity. Human gingival fibroblasts, fibroblast medium, dental pulp stem cells, cell counting, and LIVE/DEAD imaging kits were sourced from Innoprot, Sciencell Research Laboratories, Lonza, Dojindo, and ThermoFisher Scientific, respectively. Human gingival fibroblasts were cultured in a fibroblast medium supplemented with 2% fetal bovine serum, 1% fibroblast growth supplement, 100 IU/mL penicillin, and 100 IU/mL streptomycin. Dental pulp stem cells were cultured in basal medium supplemented with 2% fetal bovine serum, 2 mM L-glutamine, GA-1000, 100 mM ascorbic acid solution, 100 IU/mL penicillin, and 100 IU/mL streptomycin. Different concentrations of CHX@Fe3O4@m-SiO2 and CHX@SiQuac@Fe3O4@m-SiO2 were dissolved in 4 mL of each medium and incubated for 24 h at 37° C. The cells were seeded at 5000 cell/well in a 96-well plate, incubated overnight, and exposed to CHX@Fe3O4@m-SiO2 and CHX@SiQuac@Fe3O4@m-SiO2 for 24 h. CCK-8 solution (10 μL/well) was added, and the plates were incubated for 2 h at 37° C. The absorbance was measured at 450 nm. The cell viability was normalized against the cell viability of untreated cells. The results were expressed as a percentage of viability. For the live/dead assay, HGF and DPSC were seeded in the wells of a 24-well plate 10,000 cells/well), and the medium was changed to allow the viability to be above 90%. Cells were then exposed to CHX@Fe3O4@m-SiO2 and CHX@SiQuac@Fe3O4@m-SiO2 for 24 h at the ratio of 1:1. Live/Dead Cell imaging kit was used to stain the cells and visualized using a fluorescence microscope.
The antibacterial activity of CHX@Fe3O4@m-SiO2 and CHX@SiQuac@Fe3O4@m-SiO2 was analyzed using S. mutans cultures. Streptococcus mutans (UA159) and various laboratory reagents including Phosphate Buffered Saline (PBS), Brain Heart Infusion broth and agar, ethanol, Dimethyl Sulfoxide (DMSO), and MTT were sourced from Quality Biological, Research Products International, and Sigma-Aldrich. S. mutans was cultured for 24 h, and its optical density was adjusted using brain heart infusion (BHI) broth to 0.1 at 600 nm. In a 96-well microtiter plate, the concentrations of 1, 2, 3, 4, and 5 wt. % of the particles were added with equal volumes of the diluted S. mutans in BHI broth. The plate was incubated at 37° C. for 24 h in an anaerobic incubator. The total absorbance was measured at 595 nm. The planktonic cells were removed from the plate, leaving the attached biofilm, and 200 μL of 10% formaldehyde was added to each well for 30 minutes to fix the cells. Next, formaldehyde was removed, and the biofilm cells were washed three times with deionized water. 200 μL of 0.5% crystal violet dye was added to each well, and the cells were stained for 30 min. The wells were rinsed three times with deionized water, and 200 μL of 2-isopropanol was added into each well for 1 h to lyse the cells and extract the crystal violet. After that, the plate was read at 490 nm to measure the biofilm formation. The “Initial CFU” stands for “Initial Colony Forming Units.” This term refers to the number of Colony Forming Units in the control group, which was not exposed to the particles investigated in our study. It serves as a baseline measurement to gauge the initial microbial load before any treatment or exposure occurs.
In the context herein, the percentage reduction of S. mutans biofilm growth was evaluated. To do so, the CFU count after the exposure to particles with the Initial CFU count in the control group was compared. The formula for calculating the percentage reduction is as follows:
A base resin was formulated with bisphenol-A-glycidyl methacrylate (BisGMA) and 2-hydroxyethyl methacrylate (HEMA) combined at 66.66 wt. % and 33.33 wt. %, respectively, to formulate the dental adhesives. Camphorquinone, Ethyl 4-dimethylaminobenzoate, and Butylated hydroxytoluene were procured from Sigma-Aldrich, and Bisphenol-A-glycidyl methacrylate (BisGMA) and 2-hydroxyethyl methacrylate (HEMA) from Esstech, all used as received without further purification. Camphorquinone, ethyl 4-dimethylamino benzoate, and butylated hydroxytoluene were added at 1 mol %, 1 mol %, and 0.01 wt. %, respectively (21). The multifunctional platform (denoted CHX@SiQuac@Fe3O4@m-SiO2) was composed of iron oxide (Fe3O4) microparticles (core), mesoporous silica (SiO2, shell), and chlorhexidine (CHX) encapsulated by the mesoporous silica. The particles were functionalized by an antibacterial quaternary ammonium silane (SiQuac). The particles were added into the adhesive at 2, 3, 4, and 5 wt. %. An adhesive without the particles was assigned as a control group. The dental adhesives were mixed using a mechanical mixer (DAC 150 SpeedMixer, Flacktek, Landrum, SC, USA) at 2800 RPM for 1 min. Groups are described as follows:
The uncured samples (n=3) (21) were directly dispensed on the ATR (Golden Gate, Specac, Fort Washington, PA, USA) of FT-IR (Nexus 670, Thermo Scientific, Madison, WI, USA) using a silicon mold with 1 mm heigh to contain the adhesive. The samples were photoactivated using a light-emitting diode with 1000 mW/cm2 (VALO Cordless, Ultradent Products, South Jordan, Utah, USA) for 20 s. The spectra were acquired before and after the photoactivation using Opus 6.5 Software and Blackman Harris 3-Term apodization with 4 cm−1 resolution and 4 scans/s. The degree of conversion (DC %) was calculated using the high peaks at 1640 cm−1 and 1610 cm−1. The following equation was employed (21):
The contact angle between the water drops and the polymerized adhesives and the contact angle between the uncured adhesives and the dentin surface was tested. For the first test, the sessile drop method was used to measure the static contact angle of water by dropping 10 mL on the polymerized disc-shaped samples of adhesives (n=3, 8 mm in diameter and 1 mm in thickness) (22). After 2 s from the drop placement, three images were captured using goniometer software (Sessile drop method). For the second test, a flat dentin surface was prepared (22), and an adhesive drop of 10 mL was placed on this surface following the sessile drop method.
Disc-shaped adhesive samples (n=6, 8 mm and thickness of 0.5 mm (23) were prepared, immersed in water, and agitated for 1 h to remove uncured monomers. Fluorescein Sodium Salt, Cetyltrimethyl ammonium chloride (CTMAC), and both forms of Sodium phosphate were sourced from Sigma-Aldrich, Saint Louis, MO, USA.
The samples were immersed in a 48-well plate containing 200 ml of fluorescein sodium salt at 100 mg/mL in deionized water. The plate was shaken at 60 RPM for 10 min in the dark at room temperature. The samples were washed three times with deionized water and transferred to a new 48-well plate that contained 200 ml of 0.1 wt. % cetyltrimethylammonium chloride solution in deionized water supplemented with 10 vol % of 100 mM phosphate buffer at pH 8. The 48-well plate was shaken at 60 rpm for 20 min in the dark at room temperature to desorb complex fluorescein dye. The absorbance value was read at 501 nm using the plate reader to yield the concentration of fluorescein dye. Then, fluorescein concentration was calculated using Brees Law and an extinction coefficient of 77 mM−1 cm−1 by performing the following equation:
where [Dye] is the extracted fluorescein concentration, Abs501 is the absorbance value at 501 nm, e501 is the extinction coefficient that equals 77 mM−1 cm−1 for fluorescein, and L is the length of polystyrene cuvette (1 cm) traversed by spectrophotometer light. The cationic charge density of adhesive discs was calculated by using the following equation:
where V is the extraction solution volume, N is Avogadro's number (6.023×1023), and A is the sample's surface area. Results are then expressed as the total charge molecules per exposed surface area (24).
The water sorption (WS) and solubility (SL) were performed according to ISO 4049:2009 (25). Disc-shaped samples of adhesives (n=6, 15 mm diameter, 1 mm thickness) (26) were prepared. The samples were placed in a silica gel desiccator at 37° C. for 24 h and measured till constant mass (m1) was obtained using an analytical balance (VWRâ A-Series, Radnor, PA, USA). The diameter and thickness of each sample were measured using a digital caliper to calculate the volume (v) in mm3. The samples were immersed in 10 mL of distilled water in sealed vials at 37° C. for seven days. Then, the samples were washed under running water and gently wiped with absorbent paper. They were weighed (m2) and returned to the desiccator for 24 hours. Lastly, the samples were weighed till a constant weight was obtained to record the final mass (m3). The WS and SL were calculated according to the following equations:
Hourglass-shaped samples were prepared (n=5, 8 mm long, 2 mm wide, 1 mm thick, 1 mm2 cross-sectional area) (27) using a metallic mold and kept in distilled water at 37° C. for 24 h. The samples were fixed in metallic jigs using cyanoacrylate resin and tested under tension with a universal testing machine (EZ-SX Series, Shimadzu, Kyoto, Japan) at 1 mm/min crosshead speed. The samples were fractured at the constricted area; the values were acquired in newton (N) and divided by the constriction area (mm2) to be expressed in megapascal (MPa).
Micro Shear Bond Strength (m-SBS)
Extracted human third molars (n=12) were used after the approval of the institutional board of this University (HP-00088564). The roots were separated at the cementoenamel junction under running water, the crowns were individually embedded in self-curing acrylic resin, and the dentin was exposed and flattened with a silicon carbide sandpaper (600-grit) for 30 s with distilled water irrigation. The samples were rinsed for over 1 min, dried, and the dentin was etched (37% phosphoric acid for 15 s), rinsed for 30 s, and gently dried using absorbent paper. A primer (Scotch Bond Multipurpose, 3M EPSE, Monrovia, Calif., USA) was applied for 20 s using a micro brush and air-dried. The dental adhesives were applied with a micro brush and photoactivated for 20 seconds. A commercial resin composite (TPH Spectra ST LV, Dentsply Sirona, York, PA, USA) was used to fill polyethylene tubes placed on the adhesive-treated dentin surfaces with an adhesive area of 0.44 mm2 (TYGON Medical Tubing Formulations 54-HL, Saint Gobiain Performance Plastics, Akron, OH, USA) and covered by Mylar strips. The resin composites were photoactivated for 20 s on the top, and the samples were stored in distilled water for 24 h at 37° C. The samples were tested in a computer-controlled Universal Testing system (Insight 1, MTS, Eden Prairie, MN, USA) at 1 mm/min. The maximum force in Newtons (N) was obtained for each sample and then divided by the cross-sectional area of the resin composite cylinders to yield the data in megapascals (MPa) (28).
Microtensile Bond Strength (μ-TBS) under Magnetic Field in a Simulated Pulpal Pressure Model
Extracted human third molars (n=5) were used after the approval of the institutional board of this University (HP-00088564). The teeth were sectioned transversally with a slow-speed diamond saw (Isomet, Buehler, Lake Bluff, IL, USA) in two areas: 1) to expose the dentin and 2) to remove the roots (
For the magnetic field, a Neodymium magnet (NdFeB, NIB, or Neo magnet; grade N42 coated with Nickle-Copper-Nickel triple-layer, size 1.5″×1.5″) was fixed 12 mm away from the flat mid-coronal dentin using a custom 3D support (Polylactic acid-PLA filament, melted Extrusion Modeling, Creality Ender 3 Pro Printer).
The groups with the best performance outcomes in the μ-SBS data were tested for microtensile bond strength (μ-TBS) in addition to the control group at two different time points: baseline and aged. The dentin was treated according to the previously described μ-SBS for the bonding procedures. Two 2-mm increments of the commercial resin composite were placed on dentin and photoactivated for 20 seconds each. The samples were stored in distilled water for 24 h at 37° C. and sectioned into beams of 0.7×0.7 mm under distilled water (Isomet; #15HC blade 127×0.04 mm). The beams were fixed in metallic jigs using cyanoacrylate resin and tested under tension using the universal testing machine (Insight 1, MTS, Eden Prairie, MN, USA) at 1 mm/min until fracture. The values of beams fractured at the interface were recorded in N, divided by the bonding area, and expressed in MPa (28).
The failure pattern was analyzed and classified as adhesive (failure along the adhesive interface), cohesive in dentin (failure within dentin substrate), and cohesive in composite (failure within the resin composite) (29). Half of the beams per tooth were tested immediately after 24 hours from the restoration placement, and another half was tested after thermocycling aging (Odeme Thermocycling, OMC-250 L, Luzerna, Brazil) with computer-controlled water baths of 5 and 55° C. to simulate six months of clinical service. Each cycle consisted of 30 seconds of immersion (dwell time) and 5 seconds of transfer time.
Sample Preparation for the S. mutans Biofilm Model
Disc-shaped adhesive samples (n=6, 8 mm diameter, and 1 mm thickness) were prepared and stored for 24 h at 37° C. Samples were sterilized using ethylene oxide (Anprolene AN 74i, Anderson Products, Haw River, NC, USA) for 24 hours and degassed for seven days. S. mutans UA159 was used in the biofilm mode (30) after being overnight cultured for 18 h at 5% CO2 and 37° C. in brain heart infusion (BHI) broth. After that, the culture was supplemented by 1 wt. % of sucrose and added to BHI broth to prepare the inoculum. The adhesive samples were placed at the bottom of a 24-well plate, the inoculum was added to each well, and the plate was incubated for 24 hours at 5% CO2 and 37° C. After 24 hours, the medium was changed by a fresh BHI broth supplemented with 1 wt. % of sucrose and incubated for 24 hours to develop a 48-hour biofilm.
Each biofilm sample was placed in 1 mL of cysteine peptide water (CPW) in a glass vial to detach the biofilm by 10 s vortex, 1 m sonication (CGOLDENWALL Ultrasonic Homogenizer, SPW Industrial, Laguna Hills, CS, USA), and vortex again for 10 s. The bacterial suspension of each sample was serially diluted, and three drops of 10 μL from each dilution were plated onto BHI agar plates and incubated for 48 h at 5% CO2 and 37° C. The number of colonies was visually counted with a loupe (Reichert Quebec Darkfield Colony Counter, Depew, NY, USA), and the CFU/mL was calculated according to the dilution factor.
The biofilm samples grown over the tested adhesives (n=6) were transferred to a new 24-well plate containing 1 mL of tetrazolium dye and incubated for 1 h at 37° C. in 5% CO2. The samples were transferred to a new 24-well plate containing 1 ml of dimethyl sulfoxide for 20 min at room temperature in the dark to dissolve the formazan crystals. The solution from each well (200 μL) was transferred to a 96-well plate to measure the absorbance at 540 nm using a plate reader (31).
The biofilm samples grown over the tested adhesives (n=1) were rinsed with phosphate-buffered silane to remove non-adherent bacteria. Live/dead BacLight kit (Molecular Probes Eugene, OR) was used to stain samples by applying a mixture of 2.5 mM of each SYTO 9 and Propidium iodide for 15 min. Dead bacteria were stained by propidium iodide, emitting a red fluorescence, while live bacteria were stained by SYTO 9, emitting a green fluorescence. Samples were imaged at five random locations using an inverted fluorescence microscope (Eclipse TE2000-S, Nikon, Melville, NY).
Images were obtained to evaluate the load and morphology of S. mutans biofilm formed in 48 h on adhesive samples. Samples were washed with PBS to remove unattached biofilm, then fixed using 3% formaldehyde overnight. Biofilm was then dried using ethanol dilutions followed by 100% hexamethyldisilazane. Samples were sputter-coated with platinum; images were then captured using SEM at magnifications of X500, X3,000, and X8,000 with 3.5 kV (Quanta 200, FEI Company, Hillsboro, OR, USA).
Samples (n=6) were aged by thermocycling (Odeme Thermocycling, OMC-250 L, Luzerna, Brazil) with computer-controlled water baths of 5 and 55° C. and subjected 15,000 thermocycles to estimate an 18-month clinical service (32-34). Then, samples were sterilized as described. Antibacterial tests were performed as described, and the results were recorded as aged data.
The cytotoxicity was assessed using human gingival fibroblast (HGF) and dental pulp stem cells (DPSC). HGF (passage 7) was cultured in a fibroblast medium supplemented with 2 wt. % fetal bovine serum, 1 wt. % fibroblast growth supplement, 100 IU/mL penicillin, and 100 IU/mL streptomycin. DPSC (passage 6) were cultured in DPSC basal medium supplemented with 2 wt. % fetal bovine serum, 2 mM L-glutamine, GA-1000, 100 mM ascorbic acid solution, 100 IU/mL penicillin, and 100 IU/mL streptomycin.
Cells were seeded into a 96-well plate at 5,000 cells/well density in the corresponding medium. The disc-shaped adhesive samples (n=3, 4 mm diameter, and 1 mm thick) were prepared and sterilized as described for the antibacterial analyses. Each sample was immersed in a 4 mL medium for 24 h at 37° C. This procedure results in a surface area/solution ratio of 0.63 cm2/mL, following the recommendation of ISO 10993-12:2021 (0.5-6 cm2/mL range). Original extracts were diluted with fresh medium at 2-,4-,8-,16-,32-,64-, and 128-fold. The concentrations expressed in weight percentage (1 to 5 Wt. %), correspond to 10,000 to 50,000 mg/L. Then, 100 mL of each sample's original and diluted extracts were added to the cultured cells and incubated for 24 h at 37° C. with 5% CO2. After that, 10 mL of cell counting kit-8 (CCK-8) (WST®-8, Selleckchem, Houston, TX, USA) was added to each well and incubated for 2 h at the same previous conditions. Absorbance was measured at 450 nm to represent the cellular dehydrogenase activity of live cells in culture media, with the control group consisting of cells grown in the media alone.
In a 24-well plate, cells were seeded at 40,000 cells/well. Original and diluted extracts were added to the cultured cells and incubated for 24 h at 37° C. with 5% CO2. Live/Dead Cell Imaging Kit was used to stain the cells for 15 minutes, and then cells were visualized using an inverted epifluorescence microscope (Eclipse TE2000-S, Nikon, Melville, NY, USA).
All biological experiments were performed in triplicate; the average of the three experiments per sample was considered a statistical unit and expressed as means and standard deviation. Data normality was evaluated using the Shapiro-Wilk test SPSS, version 28.0 (SPSS Inc., Chicago, IL, USA). Experiments were assessed for significance using a One-way Analysis of Variance (ANOVA) followed by Tukey's post hoc test. Immediate and long-term comparisons were performed using paired t-tests within each group. A level of significance of 5% was applied.
TEM characterization evidenced the cubic Fe3O4 particles (mean size of 191±30 nm) with a non-normal distribution size (
The mean particle size for Fe3O4@m-SiO2 was 172±41 nm, while the shell thickness was 15.4±5 nm (
A significant finding from the EDS results is the approximately 46 wt. % carbon doped in the mesoporous silica. This high carbon content is indicative of the presence of chlorhexidine (CHX) (
In
An X-ray diffraction of both Fe3O4@m-SiO2 and CHX@SiQuac@Fe3O4@m-SiO2 exhibited characteristic peaks of a face-centered cubic system (41), typical of magnetic Fe3O4 with a spinel structure (42). The diffraction peaks corresponded to various crystallographic planes, with a dominant crystalline phase in Fe3O4@m-SiO2 and a more amorphous phase in CHX@SiQuac@Fe3O4@m-SiO2 due to SiQuac and CHX.
A M-H (magnetization=M and field strength=H) magnetic hysteresis loop of CHX@SiQuac@Fe3O4@m-SiO2 at room temperature displayed symmetric hysteresis and saturation magnetization, indicating ferrimagnetic properties. Results (not shown) showed symmetric hysteresis and a saturation magnetization of 61.3 emu/g, similar to 53-57 emu/g values as Balhaddad et al. (43) reported, using SPIONs. The superparamagnetic property, characterized by the absence of remanence or coercivity, suggests that the particles respond to external magnetic fields without retaining residual magnetism once the field is removed. This feature is crucial for potential controlled movement within dental resins, as it minimizes the risk of unwanted magnetic interactions post-treatment.
A characteristic absorption peak of the Fe3O4 core at 579 cm−1 related to Fe—O vibration (44), and 1085 cm−1 associated with Si—O—Si vibrational band was obtained by FTIR analysis. In CHX@SiQuac@Fe3O4@m-SiO2 spectra, the presence of SiQuac is confirmed by the characteristic vibrations of quaternary amine NR4+ at 950 cm−1. In contrast, the occurrence of multiple vibrations evidences the existence of CHX at 1093, 2947, and 3325 cm−1 associated with the bands C—N, C—H, and N—H, respectively (45).
The weight loss observed between 200-800° C. can be primarily attributed to the quantity of organic material attached to the nanoparticles. Notably, the weight loss in CHX@SiQuac@Fe3O4@m-SiO2 was higher than in Fe3O4@SiO2, indicating greater organic functionalization. This is a significant finding as it confirms the successful attachment of CHX and SiQuac to the Fe3O4@m-SiO2, which is a key factor in the antibacterial and drug-release properties of the nanoparticles. A study by Azadpour et al. (46) reported a similar trend in the thermal degradation of functionalized magnetic nanoparticles but with a slightly lower onset of degradation, suggesting a difference in the thermal stability of their functional groups.
In vitro CHX Loading and Release Profile
Differential thermal analysis (DTA) was utilized to determine the loading quantity of chlorhexidine (CHX) into the synthesized Fe3O4@m-SiO2. An initial weight loss (1.5 wt. %) was observed under 100° C., which can be attributed to the evaporation of physically absorbed solvents. There was almost no mass loss between 100° C. and 165° C. in the analysis of Fe3O4@m-SiO2 and between 100° C. and 200° C. in CHX@SiQuac@Fe3O4@m-SiO2. DTA curves showed a weak and broad peak at 140° C. in Fe3O4@m-SiO2 and 186° C. in CHX@SiQuac@Fe3O4@m-SiO2. CHX loading efficiency was 68%+3.7% (
For further analysis, CHX cumulative release was evaluated using UV-vis spectroscopy (
The rationale for employing UV-Vis spectroscopy and HPLC lies in their complementary strengths for analyzing drug release. UV-Vis spectroscopy is a widely used method for its rapidity and ease of use, providing a quick and reliable way to monitor the concentration of CHX in solution. On the other hand, HPLC offers a higher specificity and accuracy level. It can separate CHX from other components in the sample, thus providing a more precise measurement of its concentration. This is crucial in complex systems where multiple components might interfere with the measure. HPLC is especially valuable for confirming the release kinetics of CHX and ensuring the accuracy of the release data (47).
The selection of pH 7.4 and 5.0 for the release study reflects the physiological and cariogenic conditions of the oral cavity. pH 7.4 simulates the typical saliva environment, while pH 5.0 mimics the acidic conditions often related to tooth decay development. The enhanced release of CHX in the presence of SiQuac at both pH levels is a significant finding, indicating the material's versatility and effectiveness across a range of oral environments.
Interestingly, demonstrating efficient CHX release at pH 7.4, challenge the notion that drug release is typically more efficient at lower pH levels. This phenomenon is thought to be influenced by the presence of SiQuac, a quaternary ammonium compound, which typically shows resistance to acidic conditions. In neutral to alkaline conditions, where SiQuac is more stable, the release of CHX might be slower and more controlled. In contrast, under acidic conditions (lower pH), the stability of SiQuac could be slightly compromised, potentially leading to a different release profile.
Chlorhexidine, known for its effectiveness as a matrix metalloproteinase inhibitor, shows varying degrees of inhibition efficacy, ranging from partial to complete, particularly at concentrations between 0.2% and 2% (48). MMP-9 and MMP-2 are prevalent in dentin. The impact of CHX on MMP-9 was assessed using concentrations of 1, 0.5, 0.25, 0.125, and 0.0625 mg/mL (
Complementing these results,
Cytotoxicity Analysis of CHX@SiQuac@Fe3O4@m-SiO2
The culture extract testing method for cytotoxicity screening was used as an MTT assay, focusing on two cell types clinically relevant to the intended application (
Light microscopy was employed to observe the cell growth of dental pulp stem cells (DPSC) in contact with CHX@SiQuac@Fe3O4@m-SiO2, as shown in
When human gingival fibroblasts were exposed to the particles, the viability pattern mirrored that of DPSC cells, with statistically significant differences only observed at concentrations above 3%. Yet, all remained within the acceptable viability threshold (
The non-cytotoxic nature of CHX@Fe3O4@m-SiO2 and CHX@SiQuac@Fe3O4@m-SiO2, as demonstrated by their acceptance within the ISO 10993-5 standard, is indicative of their safety for clinical use. Reports of the cell viability of Fe3O4 (49), CHX (50), and SiQuac (51) have also shown a concentration-dependent profile, and results were within the ISO 10993-5 recommendations with viability higher than 70% (52). Their ability to enhance cell viability in both DPSC and HGF cells, especially at higher concentrations without inducing cytotoxicity, highlights their use as biocompatible materials suitable for use in areas close to the pulp and gingiva.
Antibacterial Assessment of CHX@SiQuac@Fe3O4@m-SiO2 Particles
The antibacterial assessment of the CHX@SiQuac@Fe3O4@m-SiO2 was conducted against Streptococcus mutans, a key pathogen in dental caries development, in two distinct stages of complexity: planktonic and biofilm stages. This dual-stage testing is crucial for evaluating the effectiveness of new formulations against the different forms of bacterial growth. Planktonic bacteria represent the free-floating individual bacterial cells, while biofilm represents a more complex, community-based stage where bacteria are embedded in a protective matrix. The ability of CHX@SiQuac@Fe3O4@m-SiO2 particles to combat S. mutans in both these stages is relevant for its efficacy in preventing dental caries. The isolated antibacterial performance of the particles was evaluated to illustrate their use as additives for dental adhesive formulations.
Regarding biofilm reduction, CHX@Fe3O4@m-SiO2 particles required concentrations above 3 wt. % to reduce the biofilm significantly by at least 60%. In contrast, CHX@SiQuac@Fe3O4@m-SiO2 particles, which include antibacterial silane, were more effective, achieving at least a 78% reduction in biofilm at concentrations over 3 wt. % (
The distinction between the antibacterial effects on planktonic and biofilm stages of S. mutans is decisive. The dental plaque that grows over the teeth and dental bonded restorations is mainly presented in a biofilm stage (53). Biofilms are inherently more resistant to antibacterial agents due to their protective matrix and the communal nature of bacterial cells (54). This increased resistance explains why lower concentrations (1 wt. % and 2 wt. %) of the dental adhesives were effective against planktonic cultures but less against biofilms. The higher concentration threshold for significant biofilm reduction highlights the challenge in developing effective dental materials against more resilient bacterial communities.
The antibacterial activity of CHX@SiQuac@Fe3O4@m-SiO2 particles is mainly attributed to the release of CHX and the positive charge density of the quaternary ammonium segment in the SiQuac structure without the release of these methacrylates derived from quaternary ammonium salts (13, 22, 55).
The initial chemical property assessed in the adhesive formulations was the degree of conversion (DC %). This parameter is critical as it directly impacts the adhesives' mechanical robustness and biological compatibility (56). Achieving a high DC % is vital for optimal performance; a suboptimal DC % can have several negative consequences (57). Firstly, it may release unreacted monomers, potentially compromising the adhesive's structural integrity. Secondly, these unconverted monomers can elevate cytotoxicity, posing a risk to the surrounding biological tissue. Thirdly, residual monomeric components have been shown to influence biofilm development through gene expression regulation, potentially exacerbating bacterial proliferation on the adhesive surface (58). Thus, ensuring a high DC % is integral to dental adhesive applications' overall efficacy and safety.
The adhesives formulated as a base resin herein were modeled after a gold-standard, commercial three-step etch-and-rinse adhesive consistently exhibited high conversion (DC %) across all tested particle concentrations. In the evaluation of particle incorporation into the dental resin, concentrations up to 4 wt. % showed no statistically significant difference in the degree of conversion (DC %) when compared to the control (p<0.05). However, incorporating CHX@SiQuac@Fe3O4@m-SiO2 at 5 wt. % resulted in a statistically significant decrease in DC % (p>0.05). The observed reduction in the degree of conversion at higher concentrations is likely attributable to impaired light penetration through the resin matrix.
The first chemical property analyzed for the adhesives was the degree of conversion (DC %). The achievement of a proper DC % is crucial because it affects the mechanical and biological properties of adhesives (3). A low DC % can lead to 1) the release of unconverted monomers, 2) an increase in cytotoxicity, and 3) the influence of biofilm development via gene regulation since monomer segments can increase bacterial growth (59).
The static contact angle measurement is commonly used to study surface wettability, adhesion, and spreading (60). A contact angle less than 90° indicates a wetting surface, a larger than 90° points to a non-wetting surface, and a zero-contact angle indicates complete wetting (60). The higher the particle concentration, the higher the observed contact angle, as shown in
In addition, these results can correlate to the amount of CHX released from the surface.
For water sorption (WS) and solubility (SL), significant differences were noted across the groups (p<0.05), as shown in
While the contact angle tests indicated an increase in surface hydrophobicity, prolonged water immersion, as simulated in the water sorption and solubility tests, revealed that higher concentrations of particles corresponded to increased water uptake and solubility. The authors suggest that water may permeate the microvoids within the material over time, and the interaction between chlorhexidine and the hydroxyl groups on the silica surface might play a more pivotal role than the initial hydrophobicity observed. This dynamic is thought to significantly influence the results seen in the water sorption and solubility tests.
Moreover, it was observed that despite the higher water sorption and solubility values in the groups with elevated particle concentration, the long-term micro-tensile bond strength (μ-TBS) for the 4% and 5% groups showed an improvement compared to the control. This suggests that the initial increase in hydrophobicity and the interactions between CHX and silica may contribute positively to the adhesive's bonding performance over time despite the higher propensity for water uptake.
The ultimate tensile strength (UTS) test is a critical measure for assessing the robustness and endurance of dental adhesives, as it determines the maximum stress a material can withstand before rupture. In the present invention, the UTS values for the dental adhesives showed a positive correlation with the concentration of the active component, ranging from 44.6 (±2.3) MPa in the 2% group to 56.9 (±2.3) MPa in the 5% group, with the increase being statistically significant (p<0.05) as illustrated in
The micro-shear bond strength (μ-SBS) outcomes, as illustrated in
In the micro-tensile bond strength (μ-TBS) testing, a model was used that simulates pulpal pressure in a real clinical scenario (68). Assessing the micro-tensile bond strength (μ-TBS) of dental adhesives immediately after application and over time is crucial for determining dental restorations' long-term effectiveness and reliability (69). The preparation steps for microtensile bond strength (μ-TBS) comprise 1) remotion of enamel and roots, 2) bonding procedure with pulpal pressure and magnetic field application, 3) restoration cut into beams, 4) beams fixing in metallic jigs, 5) beams under tension until fracture, and 6) fracture pattern analysis using a stereomicroscope. Immediate μ-TBS testing ensures the adhesive can withstand the stresses after its placement. At the same time, longitudinal assessments are essential to understand how the bond strength holds up against the daily mechanical stresses and environmental factors encountered inside the mouth over time. According to the previous results of the micro shear, the formulations containing CHX@SiQuac@Fe3O4@m-SiO2 particles at 4 wt. % and 5 wt. % reported the best bonding performance. Based on that, immediate and longitudinal (after artificial aging) μ-SBS was performed on groups containing CHX@SiQuac@Fe3O4@m-SiO2 particles at concentrations greater than 4 wt. %.
After aging, micro-tensile bond strength (μ-TBS) decreased by 66% and 50% for the 4% and 5% groups, respectively, in comparison to immediate (baseline) values. Despite this reduction, these groups outperformed the control group (p<0.05). This relative durability may be attributed to improved adhesive infiltration by the magnetic motion of particles into the dentin during the adhesive application, which could enhance hybridization and minimize the impact of simulated pulpal pressure on the bond strength (19). It has been hypothesized that magnetic particles might enhance resin infiltration at the bottom of the mixed layer, potentially leading to a superior hybrid layer formation (70). Furthermore, the magnetic field's force is thought to facilitate deeper penetration of Bis-GMA into the dentin, reducing phase separation between Bis-GMA and HEMA, which could subsequently strengthen the bond (71).
Fracture pattern analysis revealed that most fractures in all groups occurred at the adhesive interface (
Although significant, the observed decrease in micro-tensile bond strength (μ-TBS) after aging for both the 4% and 5% groups did not undermine the overall performance of these groups compared to the control. The fact that these groups with higher concentrations of CHX@SiQuac@Fe3O4@m-SiO2 particles still showed improved bond strength relative to the control, even after the aging process, suggests that the incorporation of these particles may confer long-term benefits to the adhesive's structural integrity. This could be particularly relevant in the stressful environment of the oral cavity, where restorations are subjected to various mechanical and biochemical challenges over time.
Utilizing a simulated pulpal pressure model herein significantly enhances the clinical relevance of the present invention. This model closely mimics the actual conditions within a living tooth, where the pulpal fluids exert an inward pressure that can influence the penetration and adhesion of dental restoratives (72, 73). In the absence of simulated pulpal pressure, the outcomes related to the movement of superparamagnetic iron oxide nanoparticles (SPIONs) toward the pulp could be overstated and not reflective of the actual behavior inside a living tooth. Simulated pulpal pressure is crucial to emulate the pulp's natural inward fluid dynamics, which counteracts adhesive penetration. Without this counterpressure, the magnetic attraction of SPIONs towards the pulp chamber might appear more pronounced than it would be under normal physiological conditions, potentially skewing the results and compromising the study's clinical translation potential (74).
By employing both microshear and micro-tensile bond strength (μ-TBS) tests, the adhesive capabilities of the materials were assessed thoroughly. The μ-TBS is especially noteworthy as it provides a more sensitive measure of bond strength, allowing for the detection of subtle differences in adhesive performance that might not be apparent with traditional macro-shear tests (75). This enhanced discriminative power is crucial for identifying the best-performing adhesive formulations that can withstand the rigors of the oral environment.
Moreover, the temporal perspective given by the longitudinal assessment of bond strength adds a vital dimension. While immediate bond strength is important for initial clinical success, understanding how adhesives hold up over time is critical for predicting long-term performance (74). By evaluating bond strength after an aging period, insights were provided into the durability of adhesive bonds and their potential to maintain integrity over the life of a dental restoration.
Antibacterial & Cytotoxicity Performance of the Adhesives containing CHX@SiQuac@Fe3O4@m-SiO2
Incorporating antibacterial and bioactive particles into dental resin formulations is critical to developing dental materials. However, the effectiveness of these particles can be significantly reduced when mixed with dental monomers due to interactions that may diminish their intended benefits (76). To address this, adhesives' antibacterial and cytotoxicity performance containing CHX@SiQuac@Fe3O4@m-SiO2, were assessed ensuring their incorporation maintains the desired antibacterial efficacy.
To ensure the long-term effectiveness and safety of the newly developed dental adhesives containing CHX@SiQuac@Fe3O4@m-SiO2, immediate and long-term assessments of their antibacterial efficacy and cell viability were conducted, (
The CFU analysis revealed a minimum of 17% reduction in bacterial growth of S. mutans biofilm for the 2% group compared to the control. The most significant decreases were observed adhesives formulated with CHX@SiQuac@Fe3O4@m-SiO2 at 4 and 5 wt. %, with around 3 and 6 log reductions in bacterial count (
After artificial aging, the antibacterial properties were maintained, with a more pronounced bacterial reduction observed for adhesives at 5 wt. % CHX@SiQuac@Fe3O4@m-SiO2. The bacterial reduction was lower after aging, significantly different from the baseline results (
The results following artificial aging of the dental adhesives containing 5 wt. % of CHX@SiQuac@Fe3O4@m-SiO2 demonstrate their enduring antibacterial properties. Notably, even after aging, these adhesives showed a significant bacterial reduction compared to the baseline results. This finding is critical as it suggests the durability of the antibacterial effectiveness over time, a key factor for long-term clinical success in dental restorative treatment. The antibacterial activity of CHX@SiQuac@Fe3O4@m-SiO2-containing adhesives is mainly attributed to the release of CHX and the positive charge density of the quaternary ammonium segment in the SiQuac structure without the release of these methacrylates derived from quaternary ammonium salts (13, 34, 35). Here, the material was challenged with an artificial aging proposed to simulate six months of material behavior inside the mouth. Factors such as leaching of the active ingredients, breakdown of the polymer matrix, and loss of mechanical integrity may contribute to a reduction in the antibacterial effectiveness of the material over time. Despite these challenges, the sustained antibacterial effect observed herein post-aging is noteworthy. The retention of antibacterial activity in the CHX@SiQuac@Fe3O4@m-SiO2-containing adhesives, even after being subjected to artificial aging, demonstrates their potential durability and effectiveness in a clinical setting.
Regarding quaternary ammonium-based components, it's important to note that not all materials containing these compounds maintain their antibacterial effectiveness over an extended period (77). The persistence of this effect largely depends on the ability of the antibacterial monomer to cross-polymerize effectively within the dental material (78). In the case of the SiQuac silanized particles used herein, the chemical bonding of SiQuac to the shell likely contributes to the retention of the antibacterial properties even after aging.
This non-provisional patent application claims benefit of priority under 35 U.S.C. §119(e) of provisional application U.S. Ser. No. 63/449,334, filed Mar. 2, 2023, the entirety of which is hereby incorporated by reference.
This invention was made with government support under grant number DE030562 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63449334 | Mar 2023 | US |
