Novel dental adhesive with triple benefits of calcium phosphate recharge, protein-repellent and antibacterial functions

Abstract

Objective

A new adhesive containing nanoparticles of amorphous calcium phosphate (NACP) with calcium (Ca) and phosphate (P) ion rechargeability was recently developed; however, it was not antibacterial. The objectives of this study were to: (1) develop a novel adhesive with triple benefits of Ca and P ion recharge, protein-repellent and antibacterial functions via dimethylaminohexadecyl methacrylate (DMAHDM) and 2-methacryloyloxyethyl phosphorylcholine (MPC); and (2) investigate dentin bond strength, protein adsorption, Ca and P ion concentration, microcosm biofilm response and pH properties.

Methods

MPC, DMAHDM and NACP were mixed into a resin consisting of ethoxylated bisphenol A dimethacrylate (EBPADMA), pyromellitic glycerol dimethacrylate (PMGDM), 2-hydroxyethyl methacrylate (HEMA) and bisphenol A glycidyl dimethacrylate (BisGMA). Protein adsorption was measured using a micro bicinchoninic acid method. A human saliva microcosm biofilm model was tested on resins. Colony-forming units (CFU), live/dead assay, metabolic activity, Ca and P ion concentration and biofilm culture medium pH were determined.

Results

The adhesive with 5% MPC + 5% DMAHDM + 30% NACP inhibited biofilm growth, reducing biofilm CFU by 4 log, compared to control (p < 0.05). Dentin shear bond strengths were similar (p > 0.1). Biofilm medium became a Ca and P ion reservoir having ion concentration increasing with NACP filler level. The adhesive with 5% MPC + 5% DMAHDM + 30% NACP maintained a safe pH > 6, while commercial adhesive had a cariogenic pH of 4.

Significance

The new adhesive with triple benefits of Ca and P ion recharge, protein-repellent and antibacterial functions substantially reduced biofilm growth, reducing biofilm CFU by 4 orders of magnitude, and yielding a much higher pH than commercial adhesive. This novel adhesive is promising to protect tooth structures from biofilm acids. The method of using NACP, MPC and DMAHDM is promising for application to other dental materials to combat caries.

Introduction

Because of their esthetics and direct-filling capability, composites and adhesives are widely used to restore tooth cavities . However, dental resins were shown to accumulate more biofilms and plaques than amalgams and glass ionomer restorations . The acid production by biofilms can decrease the local pH to a cariogenic range of 5–4, which could lead to tooth structure demineralization and secondary caries formation . Recurrent caries is the main reason for restoration failures, and replacement of the failed restorations accounts for 50–70% of all restorations performed . The tooth-restoration bonded interface has been identified as the weak link .

A strong and durable adhesion to dental hard tissues is a key factor in the success of the restoration . The mechanism of dentin bonding involves the infiltration of adhesive monomers into a demineralized dentin collagen matrix and the formation of the hybrid layer (HL) . The adhesive is not only a connection between the tooth structure and the restorative composite, it also serves as a barrier to protect the demineralized collagen scaffold from the acidic and enzymatic attacks of the oral bacteria, enzymes and fluids . Clinically, residual bacteria could exist in the prepared tooth cavity. In addition, microleakage could allow bacteria to invade the tooth-restoration interfaces. Therefore, it is desirable for the adhesive to be antibacterial to inhibit recurrent caries at the margins . For this purpose, quaternary ammonium methacrylates (QAMs) were incorporated into dental resins to achieve antibacterial activities to combat biofilm growth and acid production . Resins containing 12-methacryloyloxydodecylpyridinium bromide (MDPB) had a potent antibacterial function . Recently, a new dimethylaminohexadecyl methacrylate (DMAHDM) was synthesized and incorporated into composites and bonding agents, achieving strong inhibition against oral biofilms . In addition, oral bacteria attach to dental resins through a layer of adsorbed salivary proteins on the resin surface, which is a prerequisite for bacterial adhesion and biofilm growth . Therefore, rendering the resin protein-repellent would help to repel bacteria attachment. Indeed, studies showed that a protein-repellent agent 2-methacryloyloxyethyl phosphorylcholine (MPC) could be incorporated into resins to repel proteins and bacteria .

Another approach for caries-inhibition is to incorporate calcium phosphate (CaP) particles into resins to promote remineralization and suppress demineralization . Adhesives containing CaP particles could remineralize the remnants of tooth lesions in the cavity as well as the acid-etched dentin, and hence are promising to improve the longevity of the restorations . Recently, bonding agents containing nanoparticles of amorphous calcium phosphate (NACP) were developed . These bonding agents could release high levels of Ca and P ions to induce remineralization and combat caries . The addition of NACP did not negatively affect the dentin bond strength . Due to their small particle sizes, the NACP readily flowed with bonding agent into dentinal tubules to form resin tags . The NACP adhesive was “smart” because it could substantially increase the Ca and P ion release at a low cariogenic pH when these ions would be most needed to combat caries . For both total-etch and self-etch bonding systems, the bonding stability is limited by the degradation of the HL . The Ca and P ion release from adhesive may be highly beneficial and can serve as seed crystals to facilitate remineralization in HL and at the tooth-restoration margins . Thus, the CaP adhesive may protect the exposed collagen within the bonded interface and improve the bonding stability and durability . Therefore, the NACP-containing adhesive with Ca and P ion release could be meritorious in protecting the weak link of the tooth restoration. However, the Ca and P ion release from CaP resins lasted for only a couple of months and then diminished over time . Recently, a rechargeable CaP resin was developed with long-term Ca and P ion release for the first time . Its Ca and P ion recharge and re-release were sustained, showing no decrease in ion release with increasing the number of recharge/re-release cycles. However, while NACP resins have remineralization and acid neutralizing capabilities, they are not antibacterial . To date, there has been no report on the incorporation of DMAHDM and MPC into the rechargeable NACP adhesive to achieve both protein-repellent, antibacterial and long-term CaP ion recharge and re-release capabilities.

Therefore, the objectives of this study were to develop a novel bioactive adhesive for caries-inhibition by incorporating DMAHDM and MPC into a rechargeable NACP adhesive, and to investigate the dentin bond strength, protein adsorption, biofilm response and pH properties. It was hypothesized that: (1) Incorporating MPC, DMAHDM and NACP into the adhesive would yield dentin bond strength similar to a commercial control adhesive; (2) Incorporating MPC, DMAHDM into the rechargeable NACP adhesive would greatly decrease protein-adsorption, biofilm growth and viability; (3) Increasing NACP filler level in the resin would increase the pH and Ca and P ion concentrations in the biofilm culture medium.

Materials and methods

Development of bioactive bonding agents

The parent primer contained pyromellitic glycerol dimethacrylate (PMGDM) (Esstech, Essington, PA) and 2-hydroxyethyl methacrylate (HEMA) (Esstech) at a mass ratio 3.3/1, with 50% acetone solvent (all mass fractions) . This primer is referred to as “PM primer”. The adhesive consisted of 44.5% of PMGDM, 39.5% of ethoxylated bisphenol A dimethacrylate (EBPADMA) (Sigma-Aldrich, St, Louis, MO), 10% of HEMA and 5% of bisphenol A glycidyl dimethacrylate (BisGMA) (Esstech) . 1% of phenylbis (2,4,6-trimethylbenzoyl) phosphine oxide (Sigma–Aldrich) was added to enable light cure . PMGDM and EBPADMA were used because they had cytotoxicity similar to other dental dimethacrylates, but significantly less than the cytotoxicity of BisGMA . In addition, PMGDM is an acidic adhesive monomer , and can chelate with calcium ions from the recharging solution to render the resin rechargeable. HEMA was added to improve the flowablity and hydrophilicity, following a previous study . BisGMA was added because it could improve the cross-linkage of monomers and the bonding properties of the adhesive . This parent adhesive is referred to as “PEHB”.

DMAHDM was synthesized using a modified Menschutkin reaction where a tertiary amine group was reacted with an organo-halide . A benefit of this reaction is that there action products are generated at virtually quantitative amounts and require minimal purification. Briefly, 10 mmol of 2-(dimethylamino) ethyl methacrylate (DMAEMA, Sigma-Aldrich) and 10 mmol of 1-bromohexadecane (BHD, TCI America, Portland, OR) were combined with 3 g of ethanolin a 20 mL scintillation vial. The vial was stirred at 70 °C for 24 h. The solvent was then removed via evaporation, yielding DMAHDM as a clear, colorless, and viscous liquid . To make the primer antibacterial, DMAHDM was mixed at DMAHDM/(PM primer + DMAHDM) = 5% by mass. The 5% was selected following a previous study . Similarly, to make the adhesive antibacterial, DMAHDM was mixed into adhesive at DMAHDM/(PEHB + DMAHDM) = 5%.

MPC was obtained commercially (Sigma-Aldrich) which was synthesized as previously described . The PM primer was first mixed with DMAHDM as described above. Then 5% by mass of MPC was mixed with the PM-DMAHDM primer. Higher MPC mass fractions were not used due to a decrease in dentin bond strength when combined with DMAHDM in preliminary study. Similarly, 5% MPC was incorporated into the PEHB-DMAHDM adhesive.

NACP [Ca 3 (PO 4 ) 2 ] were synthesized via a spry-drying technique as previously described . Briefly, calcium carbonate and dicalcium phosphate anhydrous were dissolved into an acetic acid solution. The concentrations of Ca and P ions were 8 mmol/L and 5.333 mmol/L, respectively. The solution was sprayed into a heated chamber to evaporate the water and volatile acid. The dried NACP powders were collected using an electrostatic precipitator. This yielded NACP with a mean particle size of 116 nm . NACP were incorporated into the adhesive at 0%, 20%, 30% and 40% filler mass fractions, following previous studies . NACP levels greater than 40% were not used due to a decrease in dentin bond strength in preliminary study.

A commercial bonding agent Prime & Bond NT (Dentsply, Milford, DE) served as a comparative control (denoted “commercial control”). According to the manufacturer, NT was a total-etching one-bottle bonding system and contained 30% typical methacrylates, <10% methyl methacrylate, and 60% acetone. NT was combined with a self-cure activator (SCA) at 1:1 ratio to enable dual-cure. Six groups were tested:

  • (1)

    Commercial Prime & Bond NT (denoted “commercial control”).

  • (2)

    PM primer and PEHB adhesive, no MPC, no DMAHDM, no NACP (denoted “Experimental control”).

  • (3)

    Primer contained 5% MPC and 5% DMAHDM. Adhesive contained 5% MPC and 5% DMAHDM, no NACP (denoted “MPC+DMAHDM”).

  • (4)

    Primer contained 5% MPC and 5% DMAHDM. Adhesive contained 5% MPC and 5% DMAHDM, filled with 20% NACP (denoted “MPC+DMAHDM+20NACP”).

  • (5)

    Primer contained 5% MPC and 5% DMAHDM. Adhesive contained 5% MPC and 5% DMAHDM, filled with 30% NACP (denoted “MPC+DMAHDM+30NACP”).

  • (6)

    Primer contained 5% MPC and 5% DMAHDM. Adhesive contained 5% MPC and 5% DMAHDM, filled with 40% NACP (denoted “MPC+DMAHDM+40NACP”).

Dentin shear bond strength testing

Extracted human third molars were stored in 0.01% thymol solution at 4 °C. Each tooth was cut perpendicularly to the long axis of the tooth to expose the mid-coronal dentin using a low speed diamond saw (Isomet, Buehler, Lake Bluff, IL). The dentin surface was polished with 600-grit SiC paper. Then the dentin surface was etched with 37% phosphoric acid gel for 15 s and rinsed with water. A primer was applied with a brush-tipped applicator for 15 s, and the dentin was gently blown with air for 5 s to remove the solvent. An adhesive was then applied and light-cured for 10 s with an Optilux curing unit (VCL 401, Demeron Kerr, Danbury, CT). A stainless-steel cylindrical mold (inner diameter = 4 mm, thickness = 1.5 mm) was placed on the adhesive-treated dentin surface. A composite (TPH, Caulk/Dentsply, Milford, DE) was filled into the mold and light-cured for 60 s. The bonded specimens were stored in distilled water at 37 °C for 24 h. A chisel on a Universal Testing Machine (MTS, Eden Prairie, MN) was aligned to be parallel to the composite-dentin interface . Load was applied at a cross-head speed of 0.5 mm/min until the bond failed. Dentin shear bond strength = 4P/(πd 2 ), where P is the load at failure, and d is the diameter of the composite . Ten teeth were tested for each group.

Measurement of protein adsorption onto resin surface

The cover of a sterile 96-well plate (Costar, Corning Inc., Corning, NY) was used as molds to fabricate resin disks following a previous study . Briefly, 10 μL of a primer was placed in the bottom of each dent of the 96-well plate. After drying with a stream of air, 20 μL of adhesive was applied to the dent and photo-polymerized for 30 s (Optilux), using a Mylar strip covering to obtain a disk of approximately 8 mm in diameter and 0.5 mm in thickness. The cured resin disks were immersed in 200 mL of distilled water and magnetically stirred with a bar at a speed of 100 rpm for 1 h to remove any uncured monomers . The disks were then sterilized with ethylene oxide (Anprolene AN 74i, Andersen, Haw River, NC) and de-gassed for 3 days .

Protein adsorption on resin disks was determined using a micro bicinchoninic acid (BCA) method . Each disk was immersed in phosphate buffered saline (PBS) for 2 h, and then immersed in 4.5 g/L bovine serum albumin (BSA, Sigma-Aldrich) solution at 37 °C for 2 h . The disks were rinsed with fresh PBS by stirring at a speed of 300 rpm for 5 min, and then immersed in a solution of 1% sodium dodecylsulfate (SDS) in PBS and sonicated for 20 min to detach the BSA adsorbed on the disk . A protein analysis kit (micro BCA protein assay kit, Fisher Scientific, Pittsburgh, PA) was used to determine the BSA concentration in the SDS solution. Briefly, 25 μL of the SDS solution and 200 μL of the BCA reagent were mixed into the wells of a 96-well plate and incubated at 60 °C for 30 min . The absorbance at 562 nm was measured via a microplate reader (SpectraMax M5, Molecular Devices, Sunnyvale, CA). Standard curves were prepared using the BSA standard .

Saliva collection and dental plaque microcosm biofilm formation

The dental plaque microcosm model was approved by University of Maryland Institutional Review Board. Saliva is ideal for growing microcosm biofilms in vitro , with the advantage of maintaining much of the complexity and heterogeneity of dental plaque in vivo . Saliva was collected from ten healthy donors having natural dentition without active caries, and not having used antibiotics within the past 3 months. The donors did not brush teeth for 24 h and abstained from food and drink intake for 2 h prior to donating saliva . An equal volume of saliva from each of the ten donors was combined to form the saliva sample. The saliva was diluted in sterile glycerol to a concentration of 70%, and stored at −80 °C for subsequent use .

The saliva–glycerol stock was added, with 1:50 final dilution, into a McBain artificial saliva growth medium as inoculum . This medium contained mucin (Type II, porcine, gastric) at a concentration of 2.5 g/L; bacteriological peptone, 2.0 g/L; tryptone, 2.0 g/L; yeast extract, 1.0 g/L; NaCl, 0.35 g/L, KCl, 0.2 g/L; CaCl 2 , 0.2 g/L; cysteine hydrochloride, 0.1 g/L; hemin, 0.001 g/L; vitamin K 1 , 0.0002 g/L, at pH 7 . 2% sucrose was added to this medium. 1.5 mL of inoculum was added to each well of 24-well plates with a resin disk, and incubated at 37 °C in 5% CO 2 for 8 h. Then, the disks were transferred to new 24-well plates with fresh medium and incubated for 16 h. Then, the disks were transferred to new 24-well plates with fresh medium and incubated for another 24 h. This totaled 48 h of culture, which was previously shown to form relatively mature dental plaque microcosm biofilms on resins .

Live/dead staining of biofilms

Resin disks with 2-day biofilms were washed with PBS and stained using the BacLight live/dead kit (Molecular Probes, Eugene, OR) . Live bacteria were stained with Syto 9 to produce a green fluorescence. Bacteria with compromised membranes were stained with propidium iodide to produce a red fluorescence. The stained disks were examined using an inverted epifluorescence microscope (Eclipse TE2000-S, Nikon, Melville, NY).

MTT assay of metabolic activity of biofilms

The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay was used to evaluate the metabolic activity of biofilms on the disks . MTT is a colorimetric assay that measures the enzymatic reduction of MTT, a yellow tetrazole, to formazan. Disks with 2-day biofilms were transferred to a new 24-well plate, and 1 mL of MTT dye was added to each well and incubated at 37 °C in 5% CO 2 for 1 h. Then the disks were transferred to a new 24-well plate, and 1 mL of dimethyl sulfoxide (DMSO) was added to resolve the formazan crystals. The plate was incubated for 20 min with gentle mixing in the dark. Then, 200 mL of the DMSO solution from each well was collected, and its absorbance at 540 nm was measured via a microplate reader (SpectraMax M5). A higher absorbance is related to a higher formazan concentration, which indicates a higher metabolic activity in the biofilm on the disk .

Colony-forming unit (CFU) counts

Disks with 2-day biofilms were transferred into tubes with 2 mL of CPW, and the biofilms were harvested by sonication and vortexing . Three types of agar plates were prepared. First, tryptic soy blood agar culture plates were used to determine the total microorganisms . Second, mitis salivarius agar (MSA) culture plates containing 15% sucrose were used to determine the total streptococci . This is because MSA contains selective agents including crystal violet, potassium tellurite and trypan blue, which inhibit most Gram-negative bacilli and most Gram-positive bacteria except streptococci, thus enabling the streptococci to grow . Third, MSA agar culture plates plus 0.2 units of bacitracin per mL was used to determine the mutans streptococci . The bacterial suspensions were serially diluted, spread onto agar plates and incubated at 37 °C in 5% CO 2 for 24 h. The number of colonies that grew was counted and used, along with the dilution factor, to calculate the CFU on each resin disk .

Ca and P ion concentration measurement of the biofilm culture medium

The experimental control and commercial control groups were not included in Ca and P ion concentration measurements because they did not release Ca and P ions. The biofilm culture media after 72 h of incubation for the other four groups were collected and centrifuged at 12000 rpm for 5 min (Eppendorf Centrifuge 5415, Brinkmann, Westbury, NY). Then, 1 mL supernatant was used and analyzed for Ca and P concentrations via a spectrophotometric method (DMS-80 UV–vis, Varian, Palo Alto, CA) using known standards and calibration curves .

pH of biofilm culture medium

Each resin disk was placed in a well and 1.5 mL of inoculum was added to each well of 24-well plates. They were incubated at 37 °C in 5% CO 2 for 24 h as described above. Then, the disks with adherent biofilms were transferred to new 24-well plates with fresh medium, and the pH measurement was started. The pH of the culture medium was measured from 24 h to 72 h using a pH meter (Accumet Excel XL25, Fisher, Pittsburgh, PA) . The pH measurements were not collected for the initial 0–24 h of culture, because the planktonic bacteria in the medium would interfere with the pH. By placing disks with adherent biofilms in new wells with fresh medium and measuring the pH from 24 h to 72 h, it enabled the measured pH to be related to the biofilm on the resin . The pH data were recorded once every hour from 24 h to 32 h of the incubation. Then no pH measurement was made at night for 16 h. The pH measurement was re-started the next morning every two hours from 48 h to 60 h of the incubation. The last pH measurement was made in the next morning at 72 h of the incubation.

Statistical analysis

One-way and two-way analyses of variance (ANOVA) were performed to detect the significant effects of the variables. Tukey’s multiple comparison tests was used to compare the data at a p values of 0.05.

Materials and methods

Development of bioactive bonding agents

The parent primer contained pyromellitic glycerol dimethacrylate (PMGDM) (Esstech, Essington, PA) and 2-hydroxyethyl methacrylate (HEMA) (Esstech) at a mass ratio 3.3/1, with 50% acetone solvent (all mass fractions) . This primer is referred to as “PM primer”. The adhesive consisted of 44.5% of PMGDM, 39.5% of ethoxylated bisphenol A dimethacrylate (EBPADMA) (Sigma-Aldrich, St, Louis, MO), 10% of HEMA and 5% of bisphenol A glycidyl dimethacrylate (BisGMA) (Esstech) . 1% of phenylbis (2,4,6-trimethylbenzoyl) phosphine oxide (Sigma–Aldrich) was added to enable light cure . PMGDM and EBPADMA were used because they had cytotoxicity similar to other dental dimethacrylates, but significantly less than the cytotoxicity of BisGMA . In addition, PMGDM is an acidic adhesive monomer , and can chelate with calcium ions from the recharging solution to render the resin rechargeable. HEMA was added to improve the flowablity and hydrophilicity, following a previous study . BisGMA was added because it could improve the cross-linkage of monomers and the bonding properties of the adhesive . This parent adhesive is referred to as “PEHB”.

DMAHDM was synthesized using a modified Menschutkin reaction where a tertiary amine group was reacted with an organo-halide . A benefit of this reaction is that there action products are generated at virtually quantitative amounts and require minimal purification. Briefly, 10 mmol of 2-(dimethylamino) ethyl methacrylate (DMAEMA, Sigma-Aldrich) and 10 mmol of 1-bromohexadecane (BHD, TCI America, Portland, OR) were combined with 3 g of ethanolin a 20 mL scintillation vial. The vial was stirred at 70 °C for 24 h. The solvent was then removed via evaporation, yielding DMAHDM as a clear, colorless, and viscous liquid . To make the primer antibacterial, DMAHDM was mixed at DMAHDM/(PM primer + DMAHDM) = 5% by mass. The 5% was selected following a previous study . Similarly, to make the adhesive antibacterial, DMAHDM was mixed into adhesive at DMAHDM/(PEHB + DMAHDM) = 5%.

MPC was obtained commercially (Sigma-Aldrich) which was synthesized as previously described . The PM primer was first mixed with DMAHDM as described above. Then 5% by mass of MPC was mixed with the PM-DMAHDM primer. Higher MPC mass fractions were not used due to a decrease in dentin bond strength when combined with DMAHDM in preliminary study. Similarly, 5% MPC was incorporated into the PEHB-DMAHDM adhesive.

NACP [Ca 3 (PO 4 ) 2 ] were synthesized via a spry-drying technique as previously described . Briefly, calcium carbonate and dicalcium phosphate anhydrous were dissolved into an acetic acid solution. The concentrations of Ca and P ions were 8 mmol/L and 5.333 mmol/L, respectively. The solution was sprayed into a heated chamber to evaporate the water and volatile acid. The dried NACP powders were collected using an electrostatic precipitator. This yielded NACP with a mean particle size of 116 nm . NACP were incorporated into the adhesive at 0%, 20%, 30% and 40% filler mass fractions, following previous studies . NACP levels greater than 40% were not used due to a decrease in dentin bond strength in preliminary study.

A commercial bonding agent Prime & Bond NT (Dentsply, Milford, DE) served as a comparative control (denoted “commercial control”). According to the manufacturer, NT was a total-etching one-bottle bonding system and contained 30% typical methacrylates, <10% methyl methacrylate, and 60% acetone. NT was combined with a self-cure activator (SCA) at 1:1 ratio to enable dual-cure. Six groups were tested:

  • (1)

    Commercial Prime & Bond NT (denoted “commercial control”).

  • (2)

    PM primer and PEHB adhesive, no MPC, no DMAHDM, no NACP (denoted “Experimental control”).

  • (3)

    Primer contained 5% MPC and 5% DMAHDM. Adhesive contained 5% MPC and 5% DMAHDM, no NACP (denoted “MPC+DMAHDM”).

  • (4)

    Primer contained 5% MPC and 5% DMAHDM. Adhesive contained 5% MPC and 5% DMAHDM, filled with 20% NACP (denoted “MPC+DMAHDM+20NACP”).

  • (5)

    Primer contained 5% MPC and 5% DMAHDM. Adhesive contained 5% MPC and 5% DMAHDM, filled with 30% NACP (denoted “MPC+DMAHDM+30NACP”).

  • (6)

    Primer contained 5% MPC and 5% DMAHDM. Adhesive contained 5% MPC and 5% DMAHDM, filled with 40% NACP (denoted “MPC+DMAHDM+40NACP”).

Dentin shear bond strength testing

Extracted human third molars were stored in 0.01% thymol solution at 4 °C. Each tooth was cut perpendicularly to the long axis of the tooth to expose the mid-coronal dentin using a low speed diamond saw (Isomet, Buehler, Lake Bluff, IL). The dentin surface was polished with 600-grit SiC paper. Then the dentin surface was etched with 37% phosphoric acid gel for 15 s and rinsed with water. A primer was applied with a brush-tipped applicator for 15 s, and the dentin was gently blown with air for 5 s to remove the solvent. An adhesive was then applied and light-cured for 10 s with an Optilux curing unit (VCL 401, Demeron Kerr, Danbury, CT). A stainless-steel cylindrical mold (inner diameter = 4 mm, thickness = 1.5 mm) was placed on the adhesive-treated dentin surface. A composite (TPH, Caulk/Dentsply, Milford, DE) was filled into the mold and light-cured for 60 s. The bonded specimens were stored in distilled water at 37 °C for 24 h. A chisel on a Universal Testing Machine (MTS, Eden Prairie, MN) was aligned to be parallel to the composite-dentin interface . Load was applied at a cross-head speed of 0.5 mm/min until the bond failed. Dentin shear bond strength = 4P/(πd 2 ), where P is the load at failure, and d is the diameter of the composite . Ten teeth were tested for each group.

Measurement of protein adsorption onto resin surface

The cover of a sterile 96-well plate (Costar, Corning Inc., Corning, NY) was used as molds to fabricate resin disks following a previous study . Briefly, 10 μL of a primer was placed in the bottom of each dent of the 96-well plate. After drying with a stream of air, 20 μL of adhesive was applied to the dent and photo-polymerized for 30 s (Optilux), using a Mylar strip covering to obtain a disk of approximately 8 mm in diameter and 0.5 mm in thickness. The cured resin disks were immersed in 200 mL of distilled water and magnetically stirred with a bar at a speed of 100 rpm for 1 h to remove any uncured monomers . The disks were then sterilized with ethylene oxide (Anprolene AN 74i, Andersen, Haw River, NC) and de-gassed for 3 days .

Protein adsorption on resin disks was determined using a micro bicinchoninic acid (BCA) method . Each disk was immersed in phosphate buffered saline (PBS) for 2 h, and then immersed in 4.5 g/L bovine serum albumin (BSA, Sigma-Aldrich) solution at 37 °C for 2 h . The disks were rinsed with fresh PBS by stirring at a speed of 300 rpm for 5 min, and then immersed in a solution of 1% sodium dodecylsulfate (SDS) in PBS and sonicated for 20 min to detach the BSA adsorbed on the disk . A protein analysis kit (micro BCA protein assay kit, Fisher Scientific, Pittsburgh, PA) was used to determine the BSA concentration in the SDS solution. Briefly, 25 μL of the SDS solution and 200 μL of the BCA reagent were mixed into the wells of a 96-well plate and incubated at 60 °C for 30 min . The absorbance at 562 nm was measured via a microplate reader (SpectraMax M5, Molecular Devices, Sunnyvale, CA). Standard curves were prepared using the BSA standard .

Saliva collection and dental plaque microcosm biofilm formation

The dental plaque microcosm model was approved by University of Maryland Institutional Review Board. Saliva is ideal for growing microcosm biofilms in vitro , with the advantage of maintaining much of the complexity and heterogeneity of dental plaque in vivo . Saliva was collected from ten healthy donors having natural dentition without active caries, and not having used antibiotics within the past 3 months. The donors did not brush teeth for 24 h and abstained from food and drink intake for 2 h prior to donating saliva . An equal volume of saliva from each of the ten donors was combined to form the saliva sample. The saliva was diluted in sterile glycerol to a concentration of 70%, and stored at −80 °C for subsequent use .

The saliva–glycerol stock was added, with 1:50 final dilution, into a McBain artificial saliva growth medium as inoculum . This medium contained mucin (Type II, porcine, gastric) at a concentration of 2.5 g/L; bacteriological peptone, 2.0 g/L; tryptone, 2.0 g/L; yeast extract, 1.0 g/L; NaCl, 0.35 g/L, KCl, 0.2 g/L; CaCl 2 , 0.2 g/L; cysteine hydrochloride, 0.1 g/L; hemin, 0.001 g/L; vitamin K 1 , 0.0002 g/L, at pH 7 . 2% sucrose was added to this medium. 1.5 mL of inoculum was added to each well of 24-well plates with a resin disk, and incubated at 37 °C in 5% CO 2 for 8 h. Then, the disks were transferred to new 24-well plates with fresh medium and incubated for 16 h. Then, the disks were transferred to new 24-well plates with fresh medium and incubated for another 24 h. This totaled 48 h of culture, which was previously shown to form relatively mature dental plaque microcosm biofilms on resins .

Live/dead staining of biofilms

Resin disks with 2-day biofilms were washed with PBS and stained using the BacLight live/dead kit (Molecular Probes, Eugene, OR) . Live bacteria were stained with Syto 9 to produce a green fluorescence. Bacteria with compromised membranes were stained with propidium iodide to produce a red fluorescence. The stained disks were examined using an inverted epifluorescence microscope (Eclipse TE2000-S, Nikon, Melville, NY).

MTT assay of metabolic activity of biofilms

The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay was used to evaluate the metabolic activity of biofilms on the disks . MTT is a colorimetric assay that measures the enzymatic reduction of MTT, a yellow tetrazole, to formazan. Disks with 2-day biofilms were transferred to a new 24-well plate, and 1 mL of MTT dye was added to each well and incubated at 37 °C in 5% CO 2 for 1 h. Then the disks were transferred to a new 24-well plate, and 1 mL of dimethyl sulfoxide (DMSO) was added to resolve the formazan crystals. The plate was incubated for 20 min with gentle mixing in the dark. Then, 200 mL of the DMSO solution from each well was collected, and its absorbance at 540 nm was measured via a microplate reader (SpectraMax M5). A higher absorbance is related to a higher formazan concentration, which indicates a higher metabolic activity in the biofilm on the disk .

Colony-forming unit (CFU) counts

Disks with 2-day biofilms were transferred into tubes with 2 mL of CPW, and the biofilms were harvested by sonication and vortexing . Three types of agar plates were prepared. First, tryptic soy blood agar culture plates were used to determine the total microorganisms . Second, mitis salivarius agar (MSA) culture plates containing 15% sucrose were used to determine the total streptococci . This is because MSA contains selective agents including crystal violet, potassium tellurite and trypan blue, which inhibit most Gram-negative bacilli and most Gram-positive bacteria except streptococci, thus enabling the streptococci to grow . Third, MSA agar culture plates plus 0.2 units of bacitracin per mL was used to determine the mutans streptococci . The bacterial suspensions were serially diluted, spread onto agar plates and incubated at 37 °C in 5% CO 2 for 24 h. The number of colonies that grew was counted and used, along with the dilution factor, to calculate the CFU on each resin disk .

Ca and P ion concentration measurement of the biofilm culture medium

The experimental control and commercial control groups were not included in Ca and P ion concentration measurements because they did not release Ca and P ions. The biofilm culture media after 72 h of incubation for the other four groups were collected and centrifuged at 12000 rpm for 5 min (Eppendorf Centrifuge 5415, Brinkmann, Westbury, NY). Then, 1 mL supernatant was used and analyzed for Ca and P concentrations via a spectrophotometric method (DMS-80 UV–vis, Varian, Palo Alto, CA) using known standards and calibration curves .

pH of biofilm culture medium

Each resin disk was placed in a well and 1.5 mL of inoculum was added to each well of 24-well plates. They were incubated at 37 °C in 5% CO 2 for 24 h as described above. Then, the disks with adherent biofilms were transferred to new 24-well plates with fresh medium, and the pH measurement was started. The pH of the culture medium was measured from 24 h to 72 h using a pH meter (Accumet Excel XL25, Fisher, Pittsburgh, PA) . The pH measurements were not collected for the initial 0–24 h of culture, because the planktonic bacteria in the medium would interfere with the pH. By placing disks with adherent biofilms in new wells with fresh medium and measuring the pH from 24 h to 72 h, it enabled the measured pH to be related to the biofilm on the resin . The pH data were recorded once every hour from 24 h to 32 h of the incubation. Then no pH measurement was made at night for 16 h. The pH measurement was re-started the next morning every two hours from 48 h to 60 h of the incubation. The last pH measurement was made in the next morning at 72 h of the incubation.

Statistical analysis

One-way and two-way analyses of variance (ANOVA) were performed to detect the significant effects of the variables. Tukey’s multiple comparison tests was used to compare the data at a p values of 0.05.

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Nov 22, 2017 | Posted by in Dental Materials | Comments Off on Novel dental adhesive with triple benefits of calcium phosphate recharge, protein-repellent and antibacterial functions
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