Graphical abstract
Abstract
Objective
In this study we demonstrate that inorganic polyphosphate (polyP) exhibits a dual protective effect on teeth: it elicits a strong antibacterial effect against the cariogenic bacterium Streptococcus mutans and, in form of amorphous calcium polyP microparticles (size of 100–400 nm), it efficiently reseals cracks/fissures in the tooth enamel and dentin.
Methods
Three different formulations of amorphous polyP microparticles (Ca-polyP, Zn-polyP and Sr-polyP) were prepared.
Results
Among the different polyP microparticles tested, the Ca-polyP microparticles, as a component of a newly developed formulation of a dentifrice, turned out to be most effective in inhibiting growth of S. mutans . Further studies have shown that it is mainly the soluble polyP, which has a strong antibacterial activity, either given as sodium salt of polyP or formed by partial disintegration of the microparticles via the alkaline phosphatase present in the oropharyngeal cavity. In addition, we demonstrate that the developed toothpaste containing incorporated amorphous polyP microparticles, efficiently reduces dental biofilm formation.
Significance
From our results we conclude that polyP microparticles, if added to toothpaste in an amorphous state, might be beneficial not only for restoring tooth damages but also because they provide a suitable depot of functionally/antibacterially active soluble polyP.
1
Introduction
The development of tooth is an intricate process and the result of a cumulative and reiterative molecular signaling between dental epithelium and dental mesenchyme . While dental mesenchyme differentiates into the pulp and the dentin segment of the complex tooth organ, the epithelial tissues give rise to the enamel layer producing the dental enamel . Both inorganic matrices, enamel and dentin, are composed primarily of calcium and phosphate linked together by physico-chemical interactions . The overall chemical composition of tooth basically consists of hydroxyapatite (HA) crystals, Ca 10 (PO 4 ) 6 (OH) 2 , supplemented with minor components, e.g ., carbon, magnesium, sodium, and fluoride. These latter constituents are biologically important . While enamel is built from ribbon-like carbonatoapatite crystals, measuring 60–70 nm in width and 25–30 nm in thickness, dentin is formed of plate-like crystallites, with 2–5 nm in thickness and 60 nm in length . Mature enamel is an acellular zone that does not regenerate by itself, in contrast to other biomineralized tissues such as bone and dentin .
Consequently, the enamel is a biologically inert biomaterial, while dentin, likewise a biomaterial, is regeneratively active. However, appositional Ca-phosphate deposits can be formed on both components of teeth if suitable externally supplied precursors are provided. It is proven that phosphate and fluoride ions can be integrated into living enamel and dentin . In addition, amorphous, non-crystalline, but not (or only slightly) crystalline solids can be integrated into the crystalline bone or tooth matrix . Learning from nature and asking for the source of phosphate, required for the synthesis of bone and tooth, it became overt that the substrate of choice is inorganic polyphosphate (polyP), a bio-polymer synthesized by cells in humans, in particular blood platelets . PolyP is a polymer of three to hundreds of orthophosphate residues that are linked together by high-energy phosphoanhydride bonds, like in ATP . Like prokaryotes, eukaryotes, including humans, store polyP together with Ca 2+ in 100–200 nm large acidocalcisomes, where the polymer surely remains in the amorphous state . Recently, we succeeded to fabricate amorphous polyP microparticles, as well as nanoparticles by adding Ca 2+ in an over-stoichiometric ratio to soluble Na-polyP (based on phosphate units) . The amorphous calcium polyP (Ca-polyP) particles formed were found to be capable of stimulating bone formation both in vitro and in vivo . PolyP is a decisive inducer of the gene encoding the alkaline phosphatase (ALP) . In addition, this polymer, as Na-polyP or as polyP particles, is prone to enzymatic hydrolysis by ALP . Experimental evidence suggests that in mammalian cells polyP is taken up, as microparticles or nanoparticles, by clathrin-mediated/receptor-mediated endocytosis .
The ALP has been identified in dental pellicle, a protein film that is formed on the surface enamel by binding of glycoproteins present in the saliva . Dental pellicle is formed in seconds after tooth cleaning and by that protects the tooth from metabolic acids, produced by oral microorganisms, as well as from extrinsic stains . Among the powerful agents, displacing stain from teeth, are Na-pyrophosphate and Na-tripolyphosphate, which act both as dentifrice abrasives and as antibacterial agents against Streptococcus mutans . S. mutans have a central role in the etiology of dental caries since they are strong acid producers; by that, these bacteria cause an acidic environment with the (potential) consequence of creating cavities. The synthesis of polyP by bacteria in the dental pellicle has been experimentally demonstrated and reported to be associated with the appearance of electron-lucent “holes”, resembling acidocalcisomes .
Very recently we demonstrated that dentifrice, supplemented with polyP microparticles, can sustainably reseal cracks in both tooth enamel and dentin . In the present study we highlight the dual effect of polyP, first to elicit strong antibacterial effect against the caries- and cavity-inducing bacterium S. mutans , and, second to reseal dental cracks/fissures. In addition, we present a formulation of a dentifrice containing polyP compounds/particles in a functionally active amorphous state.
2
Materials and methods
2.1
Materials
Sodium polyphosphate (Na-polyP) with an average chain length of ≈40 phosphate units was obtained from Chemische Fabrik Budenheim (Budenheim, Germany).
2.2
Preparation of amorphous Ca-polyP microparticles
Based on our previously outlined strategy we formulated solid salts between Ca 2+ and polyP (Ca-polyP) that are amorphous. The standard Ca-polyP particles have a size range between 100 nm and 400 nm and can be produced with a definite size range by selecting a tuned alteration of the Ca:P starting molar ratio. A higher ratio of Ca:P (>2) will result in a smaller size of the particles, compared to larger particles that are formed at lower Ca:P ratios (<2).
In the present study Ca-polyP microparticles (Ca-polyP-MP) were prepared following the standard procedure . In this procedure the weight concentration ratio between Ca and P was set to ≈2. For this formulation, 2.8 g of CaCl 2 ·2H 2 O (#223506; Sigma–Aldrich, Taufkirchen; Germany) were dissolved in 50 ml ethanol solution (96%) and added drop-wise to 1 g of Na-polyP, dissolved in 50 ml distilled water at room temperature. The suspension formed was kept at pH 6 (using 1 N NaOH) and stirred for 5 h. The microparticles formed were collected by filtration (Nalgene Filter Units [pore size 0.45 μm]; Cole-Parmer, Kehl/Rhein; Germany) and washed three times with ethanol. Subsequently, the particles were dried at 60 °C and sieved through a sieve shaker AS 200 (mesh size 100 μm; Retsch GmbH, Haan; Germany). The resulting microparticles are termed “Ca-polyP-MP”. The particles, for which Ca 2+ has been replaced by zinc (Zn 2+ ) [“Zn-polyP-MP”] or strontium (Sr 2+ ) cations [“Sr-polyP-MP”], were prepared accordingly, by replacing CaCl 2 ·2H 2 O with 2.74 g of ZnCl 2 (anhydrous zinc chloride, #793523; Sigma) or 3.19 g of SrCl 2 (hexahydrate; #107865, Merck, Darmstadt; Germany), respectively.
2.3
Tooth samples
We used molar and premolar human teeth as sample material for treatment with the experimental dentifrice. They were provided by the Institute of Functional and Clinical Anatomy, University Medical Center of the Johannes Gutenberg University, Mainz, Germany, following the ethical guidelines of the University Medical Center Mainz. Prior to the experiments, the specimens were cleaned from organic material by incubation in 4% sodium hypochlorite solution for 4 h. Subsequently, the samples were thoroughly rinsed with distilled water then air dried.
2.4
Electron microscopy
For high-resolution scanning electron microscopic (SEM) analysis a HITACHI SU 8000 (Hitachi High-Technologies Europe GmbH, Krefeld, Germany), equipped with a low voltage (<1 kV) near-surface organic surfaces detector , was used.
The teeth samples, after washing for 3-times in phosphate buffered saline (PBS), were inspected with an ESEM XL-30 machine (Philips, Eindhoven; The Netherlands). The teeth were dehydrated in ethanol, freeze-dried, mounted onto specimen holders and finally sputtered with gold in an argon atmosphere as described .
2.5
Digital light microscopy
Digital light microscopy was performed with a VHX-600 Digital Microscope (Keyence, Neu-Isenburg; Germany) equipped with a VH-Z100 zoom lens.
2.6
Energy dispersive X-ray spectroscopy
For EDX spectroscopy an EDAX Genesis System was used that was attached to a scanning electron microscope (Nova 600 Nanolab; FEI, Eindhoven, The Netherlands), operating at 10 kV with a collection time of 30–45 s.
2.7
Fourier transformed infrared spectroscopy
Fourier transformed infrared spectroscopic (FTIR) analyses were performed with micro-milled powder in an ATR (attenuated total reflectance)-FTIR spectroscope/Varian 660-IR spectrometer (Agilent, Santa Clara, CA), fitted with a Golden Gate ATR unit (Specac, Orpington; UK), as outlined .
2.8
Composition of toothpaste
For the preparation of 20 g of the water based toothpaste formulation, used here, the following ingredients were mixed: 4 g of diatomaceous earth (pretreated with 100 mM Na-orthophosphate solution—final concentration of 20% [w/w]; Health Leads UK Ltd., Horeb; UK), 2 g of xylitol (10% [w/w]; Natur Total B.V., Apeldoorn; The Netherlands), 100 mg of xanthan (0.5% [w/w]; Special Ingredients Ltd., Foxwood Industrial Park, Chesterfield; UK), 100 mg of κ-carrageenan (0.5% [w/w]; Special Ingredients Ltd.) and 100 mg of ι-carrageenan (0.5% [w/w]; Special Ingredients Ltd.). Then, 10 g of glycerol (50% [w/w]; Dragonspice Naturwaren, Reutlingen; Germany) were added and the mixture was manually stirred until homogeneity. Afterwards, 3.1 ml bidistilled water were added and the mixture was stirred until reaching again homogeneity. The mixture was then heated to 70 °C for 5 min using a tempered water bath, mixed again, and subsequently allowed to cool down to ambient room temperature. Where indicted either 600 mg of Na-polyP per 20 g of dentifrice (3% [w/w] final concentration), or 1% [w/w] of the respective microparticles were added to the toothpaste.
2.9
Treatment of the teeth
Brushing of the respective tooth was performed with an electric toothbrush (Braun Oral-B PRO 6000; Procter & Gamble, Cincinnati, OH) at 8000 rpm and 100 g force for 3 min at room temperature . An amount of ≈0.2 g of dentifrice was distributed on top of the respective enamel and dentin surface both with the herein described dentifrice, free of or containing 1% [w/w] “Ca-polyP-MP” together with 50 μg/ml of Na-polyP. Routinely the specimens were brushed twice a day for 5 min each.
During the brushing periods (usually 3 h) the teeth remained in a humid chamber.
2.10
Streptococcus mutans growth and testing for inhibition
S. mutans (DSM No. 20523) was obtained from the DSMZ-German Resource Centre for Biological Material (Braunschweig; Germany). The S. mutans was cultivated as described on 5% defibrinated sheep blood agar (Becton-Dickinson, Le Pont-de-Claix; France). Cultivation was performed in an incubator (5% CO 2 ).
Testing for antibacterial activity was performed applying the paper disc assay as described before . Sterile paper discs (Whatman 3MM; Fisher Scientific, Schwerte; Germany) with a diameter of 5 mm were placed onto the Petri dishes (94 × 16 mm), containing the culture agar. Overnight cultures of S. mutans (density of ≈1.2 OD 600nm ) were grown; after dilution (1:3) with LB medium (Luria/Miller; #X968.2; Roth, Karlsruhe; Germany), giving an OD 600nm of ≈0.4, 350 μl suspension were evenly placed onto the plates with a Drigalski spatula. Where indicated, the filter paper discs were placed onto the plated bacteria. Then, the mixture of polyP samples (the respective amounts are mentioned in the text [7 μg, 20 μg or 70 μg]) and the dentifrice sample, either the commercial dentifrice or the new formulation (7 mg) were pipetted onto the filter papers. After an incubation period of 18 h or 26 h at 36 °C the agar samples were inspected. A clear visual resolution between the dense bacterial colony regions and the bacterial growth inhibition zones around the filter discs was observed.
2.11
In vitro incubation of teeth with Streptococcus mutans
In order to determine the effect of polyP on the extent of biofilm formation by S. mutans the human teeth were submersed into an aqueous solution of 20% [w/w] grade-1 Nestle/Nido skimmed/goat milk powder (Nestle/Bielevon Group GmbH, Bielefeld; Germany) and 5% d -(+)-glucose (#G8270; Sigma). Incubation was performed for 1 h while shaking at 37 °C. Then the samples were washed three-times in PBS and finally incubated in an overnight culture of S. mutans at an initial OD 600nm of 0.3. After 2 d of incubation, biofilm formation was assessed by scanning electron microscopy. The bacteria load applied to the teeth with OD 600nm of 0.3 surely exceeded the normal density existing in human individuals . But this level of bacterial colonization was chosen on purpose to ensure a reliable effect of the polyP sample towards the bacteria.
2.11.1
Statistical analysis
The data had been collected in Microsoft Excel and the analyses were performed with the SPSS Statistics for Windows, Version 19.0 . The analysis of variance (ANOVA) was performed, followed by the Tukey’s post-hoc test. In all the above tests, P < 0.05 was accepted as indicating significance.
2
Materials and methods
2.1
Materials
Sodium polyphosphate (Na-polyP) with an average chain length of ≈40 phosphate units was obtained from Chemische Fabrik Budenheim (Budenheim, Germany).
2.2
Preparation of amorphous Ca-polyP microparticles
Based on our previously outlined strategy we formulated solid salts between Ca 2+ and polyP (Ca-polyP) that are amorphous. The standard Ca-polyP particles have a size range between 100 nm and 400 nm and can be produced with a definite size range by selecting a tuned alteration of the Ca:P starting molar ratio. A higher ratio of Ca:P (>2) will result in a smaller size of the particles, compared to larger particles that are formed at lower Ca:P ratios (<2).
In the present study Ca-polyP microparticles (Ca-polyP-MP) were prepared following the standard procedure . In this procedure the weight concentration ratio between Ca and P was set to ≈2. For this formulation, 2.8 g of CaCl 2 ·2H 2 O (#223506; Sigma–Aldrich, Taufkirchen; Germany) were dissolved in 50 ml ethanol solution (96%) and added drop-wise to 1 g of Na-polyP, dissolved in 50 ml distilled water at room temperature. The suspension formed was kept at pH 6 (using 1 N NaOH) and stirred for 5 h. The microparticles formed were collected by filtration (Nalgene Filter Units [pore size 0.45 μm]; Cole-Parmer, Kehl/Rhein; Germany) and washed three times with ethanol. Subsequently, the particles were dried at 60 °C and sieved through a sieve shaker AS 200 (mesh size 100 μm; Retsch GmbH, Haan; Germany). The resulting microparticles are termed “Ca-polyP-MP”. The particles, for which Ca 2+ has been replaced by zinc (Zn 2+ ) [“Zn-polyP-MP”] or strontium (Sr 2+ ) cations [“Sr-polyP-MP”], were prepared accordingly, by replacing CaCl 2 ·2H 2 O with 2.74 g of ZnCl 2 (anhydrous zinc chloride, #793523; Sigma) or 3.19 g of SrCl 2 (hexahydrate; #107865, Merck, Darmstadt; Germany), respectively.
2.3
Tooth samples
We used molar and premolar human teeth as sample material for treatment with the experimental dentifrice. They were provided by the Institute of Functional and Clinical Anatomy, University Medical Center of the Johannes Gutenberg University, Mainz, Germany, following the ethical guidelines of the University Medical Center Mainz. Prior to the experiments, the specimens were cleaned from organic material by incubation in 4% sodium hypochlorite solution for 4 h. Subsequently, the samples were thoroughly rinsed with distilled water then air dried.
2.4
Electron microscopy
For high-resolution scanning electron microscopic (SEM) analysis a HITACHI SU 8000 (Hitachi High-Technologies Europe GmbH, Krefeld, Germany), equipped with a low voltage (<1 kV) near-surface organic surfaces detector , was used.
The teeth samples, after washing for 3-times in phosphate buffered saline (PBS), were inspected with an ESEM XL-30 machine (Philips, Eindhoven; The Netherlands). The teeth were dehydrated in ethanol, freeze-dried, mounted onto specimen holders and finally sputtered with gold in an argon atmosphere as described .
2.5
Digital light microscopy
Digital light microscopy was performed with a VHX-600 Digital Microscope (Keyence, Neu-Isenburg; Germany) equipped with a VH-Z100 zoom lens.
2.6
Energy dispersive X-ray spectroscopy
For EDX spectroscopy an EDAX Genesis System was used that was attached to a scanning electron microscope (Nova 600 Nanolab; FEI, Eindhoven, The Netherlands), operating at 10 kV with a collection time of 30–45 s.
2.7
Fourier transformed infrared spectroscopy
Fourier transformed infrared spectroscopic (FTIR) analyses were performed with micro-milled powder in an ATR (attenuated total reflectance)-FTIR spectroscope/Varian 660-IR spectrometer (Agilent, Santa Clara, CA), fitted with a Golden Gate ATR unit (Specac, Orpington; UK), as outlined .
2.8
Composition of toothpaste
For the preparation of 20 g of the water based toothpaste formulation, used here, the following ingredients were mixed: 4 g of diatomaceous earth (pretreated with 100 mM Na-orthophosphate solution—final concentration of 20% [w/w]; Health Leads UK Ltd., Horeb; UK), 2 g of xylitol (10% [w/w]; Natur Total B.V., Apeldoorn; The Netherlands), 100 mg of xanthan (0.5% [w/w]; Special Ingredients Ltd., Foxwood Industrial Park, Chesterfield; UK), 100 mg of κ-carrageenan (0.5% [w/w]; Special Ingredients Ltd.) and 100 mg of ι-carrageenan (0.5% [w/w]; Special Ingredients Ltd.). Then, 10 g of glycerol (50% [w/w]; Dragonspice Naturwaren, Reutlingen; Germany) were added and the mixture was manually stirred until homogeneity. Afterwards, 3.1 ml bidistilled water were added and the mixture was stirred until reaching again homogeneity. The mixture was then heated to 70 °C for 5 min using a tempered water bath, mixed again, and subsequently allowed to cool down to ambient room temperature. Where indicted either 600 mg of Na-polyP per 20 g of dentifrice (3% [w/w] final concentration), or 1% [w/w] of the respective microparticles were added to the toothpaste.
2.9
Treatment of the teeth
Brushing of the respective tooth was performed with an electric toothbrush (Braun Oral-B PRO 6000; Procter & Gamble, Cincinnati, OH) at 8000 rpm and 100 g force for 3 min at room temperature . An amount of ≈0.2 g of dentifrice was distributed on top of the respective enamel and dentin surface both with the herein described dentifrice, free of or containing 1% [w/w] “Ca-polyP-MP” together with 50 μg/ml of Na-polyP. Routinely the specimens were brushed twice a day for 5 min each.
During the brushing periods (usually 3 h) the teeth remained in a humid chamber.
2.10
Streptococcus mutans growth and testing for inhibition
S. mutans (DSM No. 20523) was obtained from the DSMZ-German Resource Centre for Biological Material (Braunschweig; Germany). The S. mutans was cultivated as described on 5% defibrinated sheep blood agar (Becton-Dickinson, Le Pont-de-Claix; France). Cultivation was performed in an incubator (5% CO 2 ).
Testing for antibacterial activity was performed applying the paper disc assay as described before . Sterile paper discs (Whatman 3MM; Fisher Scientific, Schwerte; Germany) with a diameter of 5 mm were placed onto the Petri dishes (94 × 16 mm), containing the culture agar. Overnight cultures of S. mutans (density of ≈1.2 OD 600nm ) were grown; after dilution (1:3) with LB medium (Luria/Miller; #X968.2; Roth, Karlsruhe; Germany), giving an OD 600nm of ≈0.4, 350 μl suspension were evenly placed onto the plates with a Drigalski spatula. Where indicated, the filter paper discs were placed onto the plated bacteria. Then, the mixture of polyP samples (the respective amounts are mentioned in the text [7 μg, 20 μg or 70 μg]) and the dentifrice sample, either the commercial dentifrice or the new formulation (7 mg) were pipetted onto the filter papers. After an incubation period of 18 h or 26 h at 36 °C the agar samples were inspected. A clear visual resolution between the dense bacterial colony regions and the bacterial growth inhibition zones around the filter discs was observed.
2.11
In vitro incubation of teeth with Streptococcus mutans
In order to determine the effect of polyP on the extent of biofilm formation by S. mutans the human teeth were submersed into an aqueous solution of 20% [w/w] grade-1 Nestle/Nido skimmed/goat milk powder (Nestle/Bielevon Group GmbH, Bielefeld; Germany) and 5% d -(+)-glucose (#G8270; Sigma). Incubation was performed for 1 h while shaking at 37 °C. Then the samples were washed three-times in PBS and finally incubated in an overnight culture of S. mutans at an initial OD 600nm of 0.3. After 2 d of incubation, biofilm formation was assessed by scanning electron microscopy. The bacteria load applied to the teeth with OD 600nm of 0.3 surely exceeded the normal density existing in human individuals . But this level of bacterial colonization was chosen on purpose to ensure a reliable effect of the polyP sample towards the bacteria.
2.11.1
Statistical analysis
The data had been collected in Microsoft Excel and the analyses were performed with the SPSS Statistics for Windows, Version 19.0 . The analysis of variance (ANOVA) was performed, followed by the Tukey’s post-hoc test. In all the above tests, P < 0.05 was accepted as indicating significance.
3
Results
3.1
Microparticle preparations of Ca-polyP, Zn-polyP and Sr-polyP
High resolution SEM analysis was applied to characterize the three different microparticle compositions. The “Ca-polyP-MP”, having an average size of 285 ± 95 nm, are close to spherical ( Fig. 1 A and C), while the “Sr-polyP-MP” measure in the average sizes of 170 ± 65 nm (to be published). In contrast to these particles the “Zn-polyP-MP” show a more brick-like morphology ( Fig. 1 B and D); the size of the cuboid-shaped particles measures 1–6 μm.
The EDX spectra for the microparticles show that the “Ca-polyP-MP” consist predominantly of Ca, P and O with a minor component of Na ( Fig. 1 E). The measured Ca to P atomic ratio was determined to be 0.84 ± 0.08 (quantitative EDX). With respect to “Sr-polyP-MP” the dominant signals were recorded for Sr, P and O; Na was negligible. The atomic ratio for Sr to P was determined to be 0.44 ± 0.03. The EDX spectrum for “Zn-polyP-MP” shows the major Zn and P signals with an atomic ratio for Zn to P 0.39 ± 0.03 ( Fig. 1 F). As described previously for “Ca-polyP-MP”, also the “Sr-polyP-MP” and the “Zn-polyP-MP” were found to be in an amorphous state , as revealed by X-ray diffraction analysis.
The FTIR spectra obtained from the polyP samples show the peaks at ∼3400 cm −1 and 1627 cm −1 that can be mainly assigned to the OH stretching and bending vibrations of absorbed water ( Fig. 2 ). The band near 1261 cm −1 in the spectrum for Na-polyP is designated to ν as (PO 2 ) − . The weak band at 1160 cm −1 originates from ν s (PO 2 ) − . The absorption bands close to 1083 cm −1 and 999 cm −1 are assigned to ν as (PO 3 ) 2– and to ν s (PO 3 ) 2– , respectively. The absorption band near 864 cm −1 is ν as (P O P) and the partially split band centered around 763 cm −1 is characteristic to ν s (P O P). In the spectrum for “Ca-polyP-MP” the signals are 1240 cm −1 ν as (PO 2 ) − , 1100 cm −1 ν s (PO 2 ) − , 1043 cm −1 ν as (PO 3 ) 2– , 991 cm −1 ν s (PO 3 ) 2– , 897 cm −1 ν as (P O P) and 723 cm −1 ν s (P O P). For “Zn-polyP-MP” they are 1256 cm −1 ν as (PO 2 ) − , 1134 cm −1 ν s (PO 2 ) − , 1087 cm −1 ν as (PO 3 ) 2– , 1010 cm −1 ν s (PO 3 ) 2– , 869 cm −1 ν as (P O P) and 780 cm −1 ν s (P O P). Consequently, some recorded shifts of the peaks in the spectra between Na-polyP and both the “Ca-polyP-MP” and the “Zn-polyP-MP” particles reflect the different interaction strengths of the respective cations and the anionic polymer.
