Protein-repellent and antibacterial functions of a calcium phosphate rechargeable nanocomposite

Abstract

Objectives

We recently developed a new rechargeable composite with nanoparticles of amorphous calcium phosphate (NACP) having long-term calcium (Ca) and phosphate (P) ion release; however, this composite was not antibacterial. The objectives of this study were to: (1) incorporate dimethylaminohexadecyl methacrylate (DMAHDM) and 2-methacryloyloxyethyl phosphorylcholine (MPC) into rechargeable NACP composite, and (2) investigate mechanical properties, protein adsorption and biofilm response of composite, and the pH of biofilm medium.

Methods

MPC, DMAHDM and NACP were mixed into a resin of ethoxylated bisphenol A dimethacrylate (EBPADMA) and pyromellitic glycerol dimethacrylate (PMGDM). Protein adsorption was measured using a micro bicinchoninic acid method. A human saliva microcosm biofilm model was used to grow biofilms on composites. Colony-forming units (CFU), live/dead assay, metabolic activity, and biofilm culture medium pH were determined. The tests used n = 6.

Results

The composite with 3% MPC had protein adsorption an order of magnitude less than that of a commercial composite (p < 0.05). Control composites were fully covered by live bacteria. Live bacteria were reduced via MPC; 3% MPC + 3% DMAHDM had the least live bacteria (p < 0.05). The composite with 3% MPC + 3% DMAHDM inhibited biofilm growth and viability, reducing biofilm CFU by 3 log compared to commercial control composite (p < 0.05), while having a flexural strength similar to that of the commercial composite (p > 0.1). The composite containing 3% MPC + 3% DMAHDM with biofilm culture maintained a pH above 6.5, while the commercial composite had a cariogenic pH of 4.2 in biofilm culture medium.

Conclusions

The new protein-repellent and antibacterial NACP rechargeable composite substantially reduced biofilm growth, yielding a much higher pH than a commercial composite.

Clinical significance

This novel bioactive nanocomposite is promising to protect tooth structures from biofilm acids and caries. The method of using NACP, MPC and DMAHDM may be applicable to other dental materials to reduce plaque buildup and secondary caries.

Introductions

Tooth caries is a common biofilm-related oral disease . Tooth cavity restorations cost the United States annually $46 billion in 2005 . Because of their esthetics and direct-filling capability, resin composites are widely used to restore tooth cavities with bonding agents . Extensive efforts have improved the resin compositions, curing efficacy, and mechanical and physical properties of the composites . However, secondary caries still limits the longevity of composite restorations . More than half of all restorations fail in 10 years , and the replacement of failed restorations accounts for 50% to 70% of all restorations performed . Dental composites generally do not inhibit bacterial adhesion. On the contrary, previous studies showed that composites accumulated more biofilms and plaque than other restorative materials . Biofilm acids can decrease the local pH to a cariogenic range of 5.5–4, which could lead to tooth structure demineralization and secondary caries formation .

To combat caries, calcium phosphate (CaP) composites were developed that could release calcium (Ca) and phosphate (P) ions to remineralize tooth lesions . Re-incorporation of minerals into demineralized dentin could serve as sites for nucleation and remineralization . Composites with nanoparticles of amorphous calcium phosphate (NACP) were developed . NACP nanocomposite released Ca and P ions similar to traditional CaP composites , but with a 2-fold increase in strength for load-bearing restorations . NACP composite was “smart” and rapidly neutralized acids to protect tooth structures . In a human in situ model, NACP composite inhibited caries at the enamel-composite margins .

However, the Ca and P ion release from previous CaP composites lasted for only a couple of months and then diminished over time . Such short-term ion release is not suitable for dental applications where the restoration should last for many years. Recently, a rechargeable CaP composite 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 with increasing the recharge/re-release cycles . However, while CaP composites have remineralization and acid neutralizing capabilities, they do not have significant antibacterial activity.

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 . A quaternary ammonium dimethacrylate (QADM) was incorporated into a composite, yielding a strong antibiofilm effect . Recently, a new dimethylaminohexadecyl methacrylate (DMAHDM) was synthesized and incorporated into dental resin, which showed the strongest antibacterial effect among the antibacterial monomers tested . In addition, 2-metha-cryloyloxyethyl phosphorylcholine (MPC) was incorporated into resins to repel protein and bacterial attachment . However, to date, literature and patent searches revealed no report on rechargeable CaP dental composite that is also antibacterial.

Therefore, the objectives of this study were to develop the first rechargeable CaP dental composite with protein-repellent and antibacterial functions, and to investigate the effects of DMAHDM and MPC on mechanical properties, protein adsorption, dental plaque microcosm biofilm response and pH. It was hypothesized that: (1) Incorporating MPC and DMAHDM into the rechargeable NACP composite would still retain mechanical properties matching those of a commercial control composite; (2) Incorporating MPC and DMAHDM into rechargeable NACP composite would greatly decrease biofilm growth, viability and CFU; (3) Incorporating MPC and DMAHDM into rechargeable NACP composite would yield biofilm culture medium pH much higher than that of control.

Materials and methods

Fabrication of rechargeable NACP nanocomposite

Ethoxylated bisphenol A dimethacrylate (EBPADMA) (Sigma-Aldrich, St, Louis, MO) and pyromellitic glycerol dimethacrylate (PMGDM) (Esstech, Essington, PA) were mixed at a mass ratio of 1:1 to form the resin matrix . PMGDM was selected because it is an acidic adhesive monomer and can chelate with calcium and phosphate ions from the recharge solution to achieve recharge capability, follow recent studies on rechargeable CaP composite . This resin was rendered light-curable with 0.2% camphorquinone and 0.8% ethyl 4- N,N -dimethylaminobenzoate , and is referred to as the EBPM resin.

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 . As a co-filler for mechanical reinforcement, barium boroaluminosilicate glass particles with a median size of 1.4 μm (Caulk/Dentsply, Milford, DE) were silanized with 4% 3-methacryloxypropyltrimethoxysilane and 2% n -propylamine (mass fractions) . Mass fractions of 30% NACP and 35% glass particles were mixed with the EBPM resin to form a cohesive composite paste. The composite with EBPM resin containing NACP was shown previously to have long-term Ca and P ion release with recharge and re-release capability .

Development of protein-repellent, antibacterial and rechargeable NACP nanocomposite

The protein-repellent MPC was obtained commercially (Sigma-Aldrich) which was synthesized using a method reported previously . MPC is a methacrylate with a phospholipid polar group in the side chain . Antibacterial monomer DMAHDM was synthesized using a modified Menschutkin reaction where a tertiary amine group was reacted with an organo-halide . 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 ethanol in 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 .

MPC and DMAHDM were mixed with the EBPM resin, which was further mixed with NACP and glass fillers, at mass fractions listed in the following groups:

  • (1)

    Rechargeable NACP composite control: 35% EBPM resin matrix + 30% NACP and 35% glass fillers (referred to as “EBPM + NACP control”);

  • (2)

    Rechargeable NACP composite with MPC: 32% EBPM + 3% MPC + 30% NACP + 35% glass fillers (referred to as “EBPM + NACP + MPC”);

  • (3)

    Rechargeable NACP composite with MPC and 1.5% DMAHDM: 30.5% EBPM + 3% MPC + 1.5% DMAHDM + 30% NACP + 35% glass fillers (referred to as “EBPM + NACP + MPC + 1.5DMAHDM”);

  • (4)

    Rechargeable NACP composite with MPC and 3% DMAHDM: 29% EBPM + 3% MPC + 3% DMAHDM + 30% NACP + 35% glass fillers (referred to as “EBPM + NACP + MPC + 3DMAHDM”).

MPC mass fraction in the composite was 3% because a previous study showed that this yielded a strong protein-repellent property without compromising mechanical properties . The DMAHDM mass fractions in the composite were 0%, 1.5% and 3%, respectively, following previous studies . A commercial composite (Heliomolar, Ivoclar, Amherst, NY) also served as a control. Heliomolar contained 66.7% of nanofillers of 40–200 nm of silica and ytterbium-trifluoride with fluoride-release. According to the manufacturer, Heliomolar is indicated for Class I, II, III, IV and V restorations. Heliomolar is referred to as “commercial control”.

Mechanical testing

Each composite paste was placed into a mold of 2 × 2 × 25 mm and photo-cured (Triad 2000, Dentsply, York, PA) for 1 min on each open side of the specimen. The specimens were stored at 37 °C in water for 24 h. Flexural strength and elastic modulus of specimens were measured using a three-point flexural test with a 10 mm span at a crosshead-speed of 1 mm/min on a computer-controlled Universal Testing Machine (5500R, MTS, Cary, NC) . Flexural strength was calculated by: S = 3P max L/(2bh 2 ), where P max is the fracture load, L is span, b is specimen width and h is thickness. Elastic modulus was calculated by: E = (P/d) (L 3 /[4bh 3 ]), where load P divided by displacement d is the slope in the linear elastic region .

Characterization of protein adsorption

For protein adsorption and biofilm tests, each composite paste was filled into disk molds of 9 mm in diameter and 2 mm in thickness. They were light-cured and then immersed in distilled water at 37 °C for 24 h, as described above. The amount of protein adsorption on composite 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 composite 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. Tis totally 48 h of incubation, which was previously shown to form dental plaque microcosm biofilms on resins .

Live/dead staining of biofilms

Composite 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). The area of green staining (live bacteria) was computed using NIS Elements imaging software (Nikon, Kanagawa, Japan). The area fraction of live bacteria = green staining area/total area of the image. Six disks were evaluated for each group. Three randomly chosen fields of view were photographed from each disk, yielding a total of 18 images for each composite.

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 [43]. 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 composite disk .

pH of biofilm culture medium

Each composite 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 in section 2.5. Then, the disks with adherent biofilms were transferred to new 24-well plates with fresh culture 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 from 0 h to 24 h of culture, because the planktonic bacteria in the growth medium would affect the pH values. In this way, the measured pH was only related to the biofilm adherent on the composite disk, and there was no non-adherent bacteria contribution from the medium . The pH data were recorded once every hour from 24 h to 36 h of the incubation. Then no pH measurement was made at night for 12 h. The pH measurement was re-started the next morning once 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

Fabrication of rechargeable NACP nanocomposite

Ethoxylated bisphenol A dimethacrylate (EBPADMA) (Sigma-Aldrich, St, Louis, MO) and pyromellitic glycerol dimethacrylate (PMGDM) (Esstech, Essington, PA) were mixed at a mass ratio of 1:1 to form the resin matrix . PMGDM was selected because it is an acidic adhesive monomer and can chelate with calcium and phosphate ions from the recharge solution to achieve recharge capability, follow recent studies on rechargeable CaP composite . This resin was rendered light-curable with 0.2% camphorquinone and 0.8% ethyl 4- N,N -dimethylaminobenzoate , and is referred to as the EBPM resin.

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 . As a co-filler for mechanical reinforcement, barium boroaluminosilicate glass particles with a median size of 1.4 μm (Caulk/Dentsply, Milford, DE) were silanized with 4% 3-methacryloxypropyltrimethoxysilane and 2% n -propylamine (mass fractions) . Mass fractions of 30% NACP and 35% glass particles were mixed with the EBPM resin to form a cohesive composite paste. The composite with EBPM resin containing NACP was shown previously to have long-term Ca and P ion release with recharge and re-release capability .

Development of protein-repellent, antibacterial and rechargeable NACP nanocomposite

The protein-repellent MPC was obtained commercially (Sigma-Aldrich) which was synthesized using a method reported previously . MPC is a methacrylate with a phospholipid polar group in the side chain . Antibacterial monomer DMAHDM was synthesized using a modified Menschutkin reaction where a tertiary amine group was reacted with an organo-halide . 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 ethanol in 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 .

MPC and DMAHDM were mixed with the EBPM resin, which was further mixed with NACP and glass fillers, at mass fractions listed in the following groups:

  • (1)

    Rechargeable NACP composite control: 35% EBPM resin matrix + 30% NACP and 35% glass fillers (referred to as “EBPM + NACP control”);

  • (2)

    Rechargeable NACP composite with MPC: 32% EBPM + 3% MPC + 30% NACP + 35% glass fillers (referred to as “EBPM + NACP + MPC”);

  • (3)

    Rechargeable NACP composite with MPC and 1.5% DMAHDM: 30.5% EBPM + 3% MPC + 1.5% DMAHDM + 30% NACP + 35% glass fillers (referred to as “EBPM + NACP + MPC + 1.5DMAHDM”);

  • (4)

    Rechargeable NACP composite with MPC and 3% DMAHDM: 29% EBPM + 3% MPC + 3% DMAHDM + 30% NACP + 35% glass fillers (referred to as “EBPM + NACP + MPC + 3DMAHDM”).

MPC mass fraction in the composite was 3% because a previous study showed that this yielded a strong protein-repellent property without compromising mechanical properties . The DMAHDM mass fractions in the composite were 0%, 1.5% and 3%, respectively, following previous studies . A commercial composite (Heliomolar, Ivoclar, Amherst, NY) also served as a control. Heliomolar contained 66.7% of nanofillers of 40–200 nm of silica and ytterbium-trifluoride with fluoride-release. According to the manufacturer, Heliomolar is indicated for Class I, II, III, IV and V restorations. Heliomolar is referred to as “commercial control”.

Mechanical testing

Each composite paste was placed into a mold of 2 × 2 × 25 mm and photo-cured (Triad 2000, Dentsply, York, PA) for 1 min on each open side of the specimen. The specimens were stored at 37 °C in water for 24 h. Flexural strength and elastic modulus of specimens were measured using a three-point flexural test with a 10 mm span at a crosshead-speed of 1 mm/min on a computer-controlled Universal Testing Machine (5500R, MTS, Cary, NC) . Flexural strength was calculated by: S = 3P max L/(2bh 2 ), where P max is the fracture load, L is span, b is specimen width and h is thickness. Elastic modulus was calculated by: E = (P/d) (L 3 /[4bh 3 ]), where load P divided by displacement d is the slope in the linear elastic region .

Characterization of protein adsorption

For protein adsorption and biofilm tests, each composite paste was filled into disk molds of 9 mm in diameter and 2 mm in thickness. They were light-cured and then immersed in distilled water at 37 °C for 24 h, as described above. The amount of protein adsorption on composite 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 composite 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. Tis totally 48 h of incubation, which was previously shown to form dental plaque microcosm biofilms on resins .

Live/dead staining of biofilms

Composite 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). The area of green staining (live bacteria) was computed using NIS Elements imaging software (Nikon, Kanagawa, Japan). The area fraction of live bacteria = green staining area/total area of the image. Six disks were evaluated for each group. Three randomly chosen fields of view were photographed from each disk, yielding a total of 18 images for each composite.

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 [43]. 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 composite disk .

pH of biofilm culture medium

Each composite 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 in section 2.5. Then, the disks with adherent biofilms were transferred to new 24-well plates with fresh culture 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 from 0 h to 24 h of culture, because the planktonic bacteria in the growth medium would affect the pH values. In this way, the measured pH was only related to the biofilm adherent on the composite disk, and there was no non-adherent bacteria contribution from the medium . The pH data were recorded once every hour from 24 h to 36 h of the incubation. Then no pH measurement was made at night for 12 h. The pH measurement was re-started the next morning once 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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Jun 19, 2018 | Posted by in General Dentistry | Comments Off on Protein-repellent and antibacterial functions of a calcium phosphate rechargeable nanocomposite
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