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Poly(ethylene oxide)-b-poly(3-sulfopropyl methacrylate) block copolymers for calcium phosphate mineralization and biofilm inhibition Tobias Mai, Ekaterina Rakhmatullina, Katrin Bleek, Susanne Boye, Jiayin Yuan, Antje Voelkel, Marlies Graewert, Zeinab Cheaib, Sigrun Eick, Albena Lederer, Adrian Lussi, Andreas Taubert, and Christina Günter Biomacromolecules, Just Accepted Manuscript • Publication Date (Web): 17 Sep 2014 Downloaded from http://pubs.acs.org on October 1, 2014

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Poly(ethylene oxide)-b-poly(3-sulfopropyl methacrylate) block copolymers for calcium phosphate mineralization and biofilm inhibition Tobias Mai1, Ekaterina Rakhmatullina2*†, Katrin Bleek1, Susanne Boye3, Jiayin Yuan4, Antje Völkel4, Marlies Gräwert4, Zeinab Cheaib2, Sigrun Eick2, Christina Günter5, Albena Lederer3, Adrian Lussi2, Andreas Taubert1* 1 Institute of Chemistry, University of Potsdam, D-14476 Potsdam, Germany. 2 Department of Preventive, Restorative and Pediatric Dentistry, University of Bern, CH-3010 Bern, Switzerland 3 Leibniz Institut für Polymerforschung, D-01005 Dresden, Germany 4 Max Planck Institute of Colloids and Interfaces, D-14476 Potsdam, Germany. 5 Institute of Earth and Environmental Sciences, University of Potsdam, D-14476 Potsdam, Germany

Poly(ethylene oxide) (PEO) has long been used as an additive in toothpaste, partly because it reduces biofilm formation on teeth. It does not, however, reduce the formation of dental calculus or support the remineralization of dental enamel or dentine. The present article describes the

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synthesis of new block copolymers on the basis of PEO and poly(3-sulfopropyl methacrylate) blocks using atom transfer radical polymerization. The polymers have very large molecular weights (over 106 g/mol) and are highly water soluble. They delay the precipitation of calcium phosphate from aqueous solution, but upon precipitation lead to relatively monodisperse hydroxyapatite (HAP) spheres. Moreover, the polymers inhibit the bacterial colonization of human enamel by Streptococcus gordonii, a pioneer bacterium in oral biofilm formation, in vitro. The formation of well-defined HAP spheres suggests that a polymer-induced liquid precursor phase could be involved in the precipitation process. Moreover, the inhibition of bacterial adhesion suggests that the polymers could be utilized in caries prevention.

Introduction Calcium-deficient carbonated hydroxyapatite (HAP) is the main constituent of enamel1. Although it is a hard tissue, dental enamel degrades over one’s lifetime and is, unlike bone, not naturally replaced by newly formed enamel. Dietary habits (consumption of dietary acids, sweets), quality of oral hygiene, and genetic susceptibilities are examples of factors that determine the risk of caries and dental erosion. Caries remains one of the most common diseases worldwide and is associated with bacterial processes that cause damage to hard dental tissues (enamel, dentine, cementum). Caries is caused by the localized, progressive demineralization of the dental hard tissues. The demineralization is induced by acidic metabolites of the adhered bacteria that, in turn, ferment dietary carbohydrates. The bacterial adhesion occurs via specific receptors located in the salivary pellicle layer and follows typical stages of biofilm formation. Thus, caries results from the interplay of three main factors: dietary carbohydrates, cariogenic bacteria on the dental surface and susceptibility of hard tooth tissues to demineralization.


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Contrary to caries, dental erosion is induced by intrinsic (gastric) or extrinsic (dietary) acids of non-bacterial origin2. Excessive consumption of acidic beverages is one of the common causes of dental erosion. Acid-induced loss of calcium from dental tissue results in demineralization and softening, making teeth more susceptible to further mechanical damage by abrasion (tooth brushing) or attrition3. Persistent regular demineralization of teeth leads to a pronounced loss of dental tissue. In the most severe clinical cases, enamel can be completely eliminated down to the dentine layer, resulting in a painful experience for the patient. Although the bacterial origin in the caries process differs significantly from the aetiology of the erosion, the susceptibility of teeth to demineralization and the efficiency of any subsequent remineralization remain important factors in the progression of caries and erosion. Accordingly, preventive measures are focused on i) diet modification, ii) combating caries-inducing microorganisms and their adhesion to dental surfaces, iii) increasing the resistance of dental tissues to demineralization, and iv) regular application of the effective remineralizing agents. The latter two strategies are often combined and support each other. Thus, fluoride (F-), and stannous (Sn2+)-containing compounds inhibit both caries4–8 and dental erosion9–11. Some food-approved polymers such as polyphosphates12, pyrophosphates12, carboxymethylcellulose12,13, pectin12,14, propylene glycol alginate14, and gum arabic14, also inhibit acid-induced HAP demineralization. Among these polymers, polyphosphates exhibit the highest reduction in HAP dissolution rate12,15, which could be partially related to a stronger polymer adsorption to the HAP surface due to chelation by phosphate groups16 and, therefore, a longer coating retention. Formation of the polymer coating is believed to provide a diffusion barrier for acids, thus inhibiting demineralization15. Other polymers comprised of anionic functional groups (carboxyl, phosphonate) have also been used for the HAP coatings15,17. Shimotoyodome et al.17 reported the


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targeted design and synthesis of a block copolymer possessing phosphate and carboxylic groups for adsorption to hydroxyapatite, and a poly(ethylene oxide) (PEO) block for the prevention of Streptococcus mutans adhesion. Hence, the anchoring ability of phosphate groups was combined with the known biopassivation property of PEO18. Although reduced bacterial adhesion was found in some of the copolymer coatings, the authors of this study did not test the effect of the copolymers on HAP dissolution. Clearly, new dual (or multi)-functional molecules that could inhibit both erosion progression and colonization of cariogenic bacteria on the tooth surface would make a tremendous contribution to the field of preventive dentistry. The current study describes the preparation of a series of polymers based on PEO and poly(potassium 3-sulfopropyl methacrylate) (PSPM). The PEO section of the macromolecule was selected for its biopassivation properties. The PSPM blocks were chosen because the monomer is commercially available, fairly cheap, and can, like poly(phosphates), potentially anchor the polymer to enamel surfaces. Consequently, this combination should result in a strongly bound bacteria-repellent polymer, which is potentially able to also prevent enamel dissolution by surface passivation through the sulfonate groups. Experimental Section Materials Poly(ethylene oxide) 4600 (PEO4600, nominal Mn = 4600 g/mol), poly(ethylene oxide monomethylether)

5000

(MPEO5000,

nominal

Mn

=

5000

g/mol),

N,N,N',N'-

tetramethylethylendiamine (TMEDA), 3-sulfopropylmethacrylate potassium salt (SPM), copper(I) chloride, triethylamine, ethylenediamine tetraacetic acid disodium salt (EDTA), and potassium peroxodisulfate (KPDS) were purchased from Sigma-Aldrich®. Poly(ethylene oxide) 100000 (PEO100k, nominal Mn = 100000 g/mol) was purchased from abcr®. Solvents were


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purchased at over 99% purity from abcr® or VWR International®. All chemicals were used as received except triethylamine and dichloromethane, which were dried using calcium hydride and chloroform, which, in turn, were dried using phosphorus pentoxide. Macroinitiator In a typical synthesis, 5 g of poly(ethylene oxide) (PEO4600) (1.08 mmol, 1 eq.) was dissolved in 10 mL of dry dichloromethane. The colourless solution was cooled to 0°C in an ice bath. With an argon-filled syringe, 384 µL of bromacetylbromide (4.40 mmol, 4 eq.) and 609 µL of dry triethylamine (4.4 mmol, 4 eq.) were added under stirring (400 rpm). A white solid immediately precipitated. After 2 min of additional stirring, the reaction mixture was poured into ice water. The organic phase was separated and dried with sodium sulfate. After filtration to remove sodium sulfate and evaporation of the solvent, the beige product was freeze dried from benzene yielding a light beige powder (4.78 g, 96%). The other macroinitiators (Figure 1) were synthesized accordingly, with the exception of MI3. In this case, 500 mL of chloroform and approximately 1 week of stirring at room temperature were required for full dissolution; the reason is the high molecular weight of the PEO100k precursor.

Figure 1. Macroinitiators used in the current study19. Polymerization Prior to polymerization, CuCl was purified and activated with glacial acetic acid and diethyl ether20.


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Diblock copolymer MPEO5000-b-PSPM and triblock copolymer PSPM-b-PEO4600-b-PSPM The diblock copolymer MPEO5000-b-PSPM was synthesized as follows: in an argon-flushed two-neck flask, 250 mg (0.05 mmol, 1 eq.) of MI1 and 6.158 g (25 mmol, 500 eq.) of 3sulfopropyl methacrylate potassium salt (SPM) were dissolved in 11 mL of water20. The solution was degassed, 10 µL (0.1 mmol, 2 eq.) of tetramethylethylendiamine (TMEDA) and 10 mg (0.1 mmol, 2 eq.) of CuCl were added, and the flask was sealed with a septum. After 10 min, the solution became very viscous and stirring was not possible anymore. After 30 min, the reaction was stopped by opening the flask to air and adding 2 mL of 0.1 M aqueous ethylenediaminetetraacetate disodium salt (EDTA). The turquoise solution was poured into 200 mL of MeOH, yielding a white precipitate. After dissolution in 5–10 mL of water and reprecipitation, the solid was freeze dried from water. A total of 5.43 g of product (84%) was obtained. The triblock copolymer PSPM-b-PEO4600-b-PSPM was synthesized via the same protocol, but using twice the amounts of CuCl and TMEDA (yield: 4.32 g (67%) after freeze drying from water). Triblock coplymer PSPM-b-PEO100k-b-PSPM The triblock copolymer based on the high molecular weight macroinitiator MI3 was made using a solution of 507 mg (0.005 mmol, 1 eq.) of MI3 in 15 mL of degassed water under argon. After complete dissolution (two days), 5 mL of degassed water was added and the solution was degassed again. Then, 2.464 g (10 mmol, 2000 eq.) of SPM was dissolved in 5 mL of degassed water, followed by the addition of 18 µL of TMEDA (0.2 mmol, 40 eq.) and 19 mg of CuCl (0.2 mmol, 40 eq.). Then, 5 mL of degassed water was added and the flask was closed. The reaction was stopped after 35 min. by opening the flask and adding 2 mL of 0.1 M aqueous EDTA. The solvent was evaporated to approximately 5 mL and precipitated in 100–200 mL


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MeOH, yielding a voluminous white precipitate. After dissolution in 5–10 mL of water and reprecipitation, the solid was freeze dried from water. A total of 1.8–2.4 g of product (63-80%) was obtained. PSPM homopolymer PSPM homopolymers were synthesized for comparison with the block copolymers. In total, 1.230 g (5 mmol, 200 eq.) of SPM was dissolved in 5 mL of water under argon and degassed for 5 min with argon. After the addition of 7.98 mg (0.025 mmol, 1 eq.) of potassium persulfate (KPDS), the flask was sealed with a septum and the solution was stirred for 90 min at 60°C. After opening the flask to air, the solution was allowed to cool to room temperature and then poured into 100 mL of MeOH. The white solid was dissolved in 3 mL of water and reprecipitated in methanol. After freeze drying from water, 847 mg (69%) of product was obtained.


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Figure 2. PSPM-based block polymers used in the present study. The labels (lowercase letters) on PSPM correspond to labels in the NMR spectra of the polymers shown in Figure 519. PSPM: FTIR (KBr, 298 K): 2960 cm-1 21, C–H asymmetric stretching vibration; 2897 cm-1 21, C–H symmetric stretching vibration; 1726 cm-1, C=O stretching vibration of saturated ester21; 1450 cm-1, C–H asymmetric deformation of CH321; 1485 cm-1, C–H symmetric deformation of CH321; 1190 cm-1, symmetric stretching vibration of SO321; 1041 cm-1, asymmetric stretching vibration of SO321. 1H-NMR (D2O, 298 K): 0.6–1.3 ppm (m); 1.5–2.3 ppm (m); 2.8–3.1 ppm (m); 3.2–3.4 ppm (m); 3.8-4.3 ppm (m). Elemental analysis (EA) experiment (calculated):


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C 31.9% (34.1%); H 4.9% (4.5%); S 12.2% (13.0%). Value dn/dc = 0.1183 mL/g. AF4 Mw = 1.320·106 g/mol, PDI = 1.12. MPEO5000-b-PSPM: FTIR (KBr, 298 K): 2960 cm-1 21, C–H asymmetric stretching vibration; 2897 cm-1 21 , C–H symmetric stretching vibration; 1726 cm-1, C=O stretching vibration of saturated ester21; 1450 cm-1, C–H asymmetric deformation of CH321 ; 1485 cm-1, C– H symmetric deformation of CH321 ; 1190 cm-1, symmetric stretching vibration of SO321; 1043 cm-1, asymmetric stretching vibration of SO321. 1H-NMR (D2O, 298 K): 0.6–1.3 ppm (m); 1.5– 2.3 ppm (m); 2.8-3.1 ppm (m); 3.2–3.4 ppm (m); 3.57-3.64 ppm (s); 3.8-4.3 ppm (m). EA experiment (calculated): C 32.6% (34.2%); H 5.0% (4.5%); S 12.0% (13.0%). Value dn/dc = 0.1091 mL/g. AF4 Mw = 1.895·106 g/mol, PDI = 1.11. PSPM-b-PEO4600-b-PSPM: FTIR (KBr, 298 K): 2960 cm-1 21 , C–H asymmetric stretching vibration; 2897 cm-1 21, C–H symmetric stretching vibration; 1725cm-1, C=O stretching vibration of saturated ester21; 1450 cm-1, C–H asymmetric deformation of CH321; 1485 cm-1, C–H symmetric deformation of CH321; 1190 cm-1, symmetric stretching vibration of SO321; 1043 cm-1, asymmetric stretching vibration of SO321. 1H-NMR (D2O, 298 K): 0.6–1.3 ppm (m); 1.5–2.3 ppm (m); 2.8–3.1 ppm (m); 3.2–3.4 ppm (m); 3.55-3.64 ppm (s); 3.8-4.3 ppm (m). EA experiment (calculated): C 32.6% (34.2%); H 5.0% (4.5%); S 12.0% (13.0%). Value dn/dc = 0.1091 mL/g. AF4 Mw = 3.255·106 g/mol, PDI = 1.01. PSPM-b-PEO100k-b-PSPM: FTIR (KBr, 298 K): 2960 cm-1 21, C–H asymmetric stretching vibration; 2897 cm-1 21 , C–H symmetric stretching vibration; 1727 cm-1, C=O stretching vibration of saturated ester21; 1450 cm-1, C–H asymmetric deformation of CH321 ; 1485 cm-1, C– H symmetric deformation of CH321; 1193 cm-1, symmetric stretching vibration of SO321; 1043 cm-1, asymmetric stretching vibration of SO321. 1H-NMR (D2O, 298 K): 0.6–1.3 ppm (m); 1.5–


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2.3 ppm (m); 2.8–3.1 ppm (m); 3.2–3.4 ppm (m); 3.51–3.70 ppm (s); 3.8–4.3 ppm (m). EA experiment (calculated): C 35.7% (34.7%); H 3.6% (4.6%); S 10.6% (12.7%). Value dn/dc = 0.1215 mL/g. AF4 Mw = 4.580·106 g/mol, PDI = 1.51 (contains a fraction of unreacted starter material with MW = 0.112·106 g/mol, PDI = 1.81). Turbidity measurments. Simulated body fluid (doubly concentrated, 2SBF) was prepared according to Kokubo et al.22–25(see supporting information for exact composition) and buffered with 100 mmol/L tris(hydroxymethyl)-aminomethane (TRIS). 2SBF was chosen because the calcium concentration is comparable to natural saliva26. Even though the phosphate concentration is only half that of natural saliva, 2SBF is thus a robust and established model system to evaluate the role of the present polymers on mineral formation and inhibition (see supporting information)23,24,26. The pH was adjusted to 7.42 with 1M HCl and the initial calcium concentration was measured via inductively coupled plasma optical emission spectroscopy (ICPOES, supporting information). In total, 10 mg of polymer was dissolved in 1 mL of the phosphate-containing 2SBF part. After complete dissolution of the polymer, 1 mL of the calcium-containing 2SBF part was added. Then, a 0.1 M CaCl2 solution was added in 20 µL and 100 µL steps (see supporting information). Between every addition, the mixture was stirred for 2 min. Once the calcium phosphate precipitated, the solution became turbid. The turbidity evolution was quantified with a Shimadzu UV mini 1240 at 746 nm. This wavelength was chosen because it is the location of a plateau in the absorption spectra that is easy to follow. The turbidity (absorption) was plotted vs. the total amount of calcium (initial calcium in 2SBF plus calcium added over the course of the titration). The resulting curves were fitted with a sigmoidal Boltzmann function. The Ca2+ concentration necessary for precipitation, [Ca]P, is represented by the center of the function x0 in the equation y = A1 + (A2 – A1)/(1 + e(x – x0)/dx), with A1 as the


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bottom value, A2 as the top value, x0 as the function center and dx as the steepness of the fit. All measurements were reproduced in triplicate. Figure 3 shows a representative dataset used for the determination of [Ca]P.

Figure 3. Representative titration curve of PSPM-b-PEO100k-b-PSPM27. Polymer-induced calcium phosphate dissolution. A total of 50 mg of polymer was dissolved in 10 mL of MiliporeWater® (S = 18.2 mΩ) and 100 mg of HAP was added. After shaking the suspensions for 3 days, the residual HAP powder was separated by centrifugation and 1 mL of the supernatant was taken to measure the Ca2+ concentration via ICP-OES. Control samples without polymer additives present in solution were prepared accordingly. Mineralization. Additional precipitation experiments were performed in two groups. For the first group of samples, 150 mg of polymer was dissolved in 15 mL of the calcium-containing part of 2SBF. This solution was then mixed with 15 mL of the phosphate-containing part of 2SBF. For the second group, 150 mg of polymer was dissolved in the phosphate-containing part and this solution was then mixed with the calcium-containing part. The mixing was performed by


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vortexing for 30 s and stirring for 3 min. at 500 rpm. Then 15 mL of a 0.1 M CaCl2 solution was added to all mixtures in three steps under stirring (500 rpm). After vortexing for 30 s, the mixtures were stirred for 5 days at room temperature. Precipitates were then isolated and purified by centrifugation (10000 rpm), washed five times with 30 mL of MiliporeWater® (S = 18.2 mΩ), and washed three times with ethanol. Scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDXS). SEM and EDXS were performed on a JEOL JSM-6510 microscope with an INCA-x-act detector. All samples were coated with carbon for 5 s using an EMITECH SC7620 sputter coater. For deposition on the sample holder, the samples were dispersed in 5 mL of ethanol. NMR spectroscopy. NMR spectra were recorded on a Bruker Avance 300 (300 MHz) spectrometer at room temperature. Infrared spectroscopy. FTIR spectra were recorded on a ThermoNicolet Nexus spectrometer using KBr pellets. Data were evaluated with the Omnic 6.2 software program. ATR-IR spectra were recorded on a Thermo Nicolet 6700 spectrometer with a diamond ATR crystal. Elemental analysis. EA was performed on a Vario EL III (elementar) element analyzer. Gel permeation chromatography. PEO macroinitiators: standard GPC with simultaneous UV and RI detection was performed in N-methyl-2-pyrrolidone (NMP + 0.5 wt% LiBr) at 70°C, at a flow rate of 0.8 mL/min, and using two 300 × 8 mm2 PSS-GRAM (7 µm particles) columns with porosities of 102 and 103 Å. Calibration was performed with PEO standards (PSS, Mainz, Germany). Block copolymers: GPC-MALLS with simultaneous UV, RI, and MALLS (multiangle laser light scattering, Wyatt Dawn Eos) detection was performed in 0.1 M aqueous NaNO3 at room temperature, flow rate of 1.0 mL/min, and using two 300 × 8 mm2 PSS-SUPREMA (10 µm particles) columns with porosities of 30 and 3000 Å. Solutions containing ~0.15 wt%


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polymer were filtered through 0.45 µm filters; the injected volume was 100 µL. Data were recorded and evaluated with the PSS-WinGPC Unichrom software package; the refractive index increment values used are listed in Table 1. Analytical ultracentrifugation. The determination of the partial specific volume of the samples was performed in a density oscillation tube DMA 5000 (Anton Paar, Graz, Austria). Sedimentation experiments were performed on an Optima XLI centrifuge (Beckman Coulter, Palo Alto, CA, USA) and Rayleigh interference optics at 25°C and a speed of 60,000 rpm. The sedimentation coefficient distributions were evaluated with the least squares g*(s) evaluation implemented in the software program SEDFIT (version 11.8, P. Schuck, 2009)28. Equilibrium experiments for every polymer sample were performed at 2500 rpm, 3500 rpm or 5000 rpm with different concentrations. Data were evaluated with the program MSTAR (K. Schilling, Nanolytics, Germany)29. All solutions were prepared with 0.5 M NaCl as a solvent to prevent non-ideal sedimentation due to charge effects. Static light scattering (SLS). SLS was performed with a CGS-3 Compact Goniometer possessing a He-Ne Laser (wavelength 632.8 nm) (ALV-7004). The samples were filtered with a 0.2 µm PVDF syringe filter and measured from 30° to 150° at different concentrations in 0.5 M NaCl. Data were analyzed with the software package ALV-Stat. The refractive index increments dn/dc of the polymers in solution were measured with an interferometric refractometer ScanRef (NFT Nanofilm Technologie GmbH, Göttingen, Germany). Inductively coupled plasma-optical emission spectroscopy. ICP-OES spectroscopy was performed on a Perkin Elmer Optima 5300 DV spectrometer by dilution with water. Preparation of human enamel specimens and calculation of the enamel surface area. Enamel specimens (n = 320) were prepared from caries-free (Leica, Zoom 2000, USA; 25×


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magnification) human premolar teeth extracted by dental practitioners in Switzerland (no water fluoridation, 250 ppm F- in table salt). Before the extraction, the patients were informed about the use of their teeth for research purposes and consent was obtained. Teeth crowns were separated from the roots using an Isomet Low Speed Saw (Buehler, Düsseldorf, Germany). The buccal sites were taken and further sectioned into cubic slabs. Every side of each enamel slab was polished on a Kuntch-Rotor polishing machine using carbide paper (30, 18, and 6 µm grain size) under water cooling. Samples were stored in a mineral solution (1.5 mmol/L CaCl2, 1.0 mmol/L KH2PO4, 50 mmol/L NaCl, pH 7.0). The calculation of the enamel surface area was performed using a light microscope (Leica M 420). Every side of each cubic enamel slab was imaged and recorded. Software program IM500 was used for the image analysis where the recorded enamel surface area was measured taking into account the magnification factor of the microscope. In every sample, the total surface area was further calculated as the sum of each side area. Prior to the experiment, the samples were disinfected with ethanol (70%) for 1 h, and washed in fresh portions of deionized water for another hour. Pool of sterile human saliva. Paraffin wax-stimulated whole saliva was collected from 25 healthy donors with no signs of gingivitis or active caries. Saliva collection was approved by the ethics committee of the University of Bern. The collected samples were pooled, and pooled saliva was centrifuged at 4000 g for 20 min at 4°C. The supernatant was taken and sterilized under UV radiation for 30 min. The prepared pool of sterile saliva was divided into small aliquots and stored at -80°C. Salivary aliquots were defrosted immediately before the experiment. In vitro bacterial adhesion assay. The prepared human enamel specimens were randomly selected and divided into a total of 16 groups (8 for each of two incubation times with ): 1) bare


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polished enamel (E, n = 20); 2) enamel coated by the in vitro salivary pellicle layer (P, n = 20); 3) enamel coated by the copolymer MPEO5000-b-PSPM (MPEO5000-b-PSPM, n = 20); 4) enamel coated by the copolymer PSPM-b-PEO4600-b-PSPM (PSPM-b-PEO4600-b-PSPM, n = 20); 5) enamel coated by the copolymer PSPM-b-PEO100k-b-PSPM (PSPM-b-PEO100k-bPSPM, n = 20); 6) in vitro salivary pellicle layer modified with the copolymer MPEO5000-bPSPM (MPEO5000-b-PSPM/P, n = 20), 7) in vitro salivary pellicle layer modified with the copolymer PSPM-b-PEO4600-b-PSPM (PSPM-b-PEO4600-b-PSPM/P, n = 20); 8) in vitro salivary pellicle layer modified with the copolymer PSPM-b-PEO100k-b-PSPM (PSPM-bPEO100k-b-PSPM/P, n = 20). To prepare polymer and polymer-modified pellicle coatings, enamel was incubated in the corresponding aqueous solutions (0.1 mg/mL) of the copolymers MPEO5000-b-PSPM, PSPM-b-PEO4600-b-PSPM, and PSPM-b-PEO100k-b-PSPM, or in the mixtures of the copolymer aqueous solutions (0.2 mg/mL) with pooled sterile human saliva (1:1, v:v). Incubation was carried out at room temperature for 2 h under sterile conditions. In vitro salivary pellicle layer was prepared by the incubation of the enamel samples in the pooled sterile human saliva for 2 h at room temperature under sterile conditions. S. gordonii ATCC-10558 was cultured on Trypticase soy agar (TSA, Oxoid, Basingstoke, UK) supplemented with 5% sheep blood in 5% CO2 at 37°C for 15 h. The bacterial suspension was prepared in Dulbecco's modified Eagle's medium (DMEM; Gibco, Invitrogen) having a targeted cell concentration of 5×108 cells/mL. All samples were simultaneously incubated in the bacterial suspension either for 30 or 120 min followed by a gentle three-step dip-and-rinse in DMEM to eliminate non-adhered cells. Afterwards, samples were ultrasonicated at 16 W for 30 s in 0.9% sterile NaCl solution (Bichsel, Interlaken, Switzerland), and subsequently vortexed (30 s) to detach adhered S. gordonii. Aliquots of this suspension were taken and diluted (×100) with


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DMEM followed by spreading over a TSA plate. The agar plates were incubated for 48 h at 37°C in 5% CO2. Bacterial colonies were counted using an Acolyte SuperCount Colony Counter (Synbiosis, Cambridge, UK). The number of adhered S. gordonii was expressed as the number (log10) of colony-forming units per enamel surface area (CFU/mm2). Statistical data analysis was performed using a nonparametric ANOVA model (F1_LD_F1)30 and pairwise Wilcoxon rank sum tests with Bonferroni–Holm corrections for multiple testing. The level of significance was set at 0.05. Atomic Force Microscopy (AFM). Freshly cleaved muscovite mica substrates (Bal-Tec AG, Liechtenstein) were incubated in the copolymer MPEO5000-b-PSPM aqueous solution (0.1 mg/mL), pooled sterile human saliva, or a mixture of copolymer MPEO5000-b-PSPM/saliva (1:1, v:v) for 2 h at room temperature. Afterwards, samples were dip-and-rinsed in three portions of fresh deionized water and left to dry at room temperature. Tapping-mode AFM imaging was performed using a PycoLE system (Molecular Imaging) and silicon nitride cantilevers (k = 42 N/m, 1 line/s). Images were recorded in height, amplitude and phase mode with a size of 512 × 512 pixels. Height images were flattened and plain adjusted. Height (topography) images were further analyzed using the Gwyddion software program. X-Ray Powder Diffraction (XRD). A Siemens D5005 X-ray diffractometer with Cu anode and vertical circle goniometer with a optional microfocus system was used. For comparison of the XRD patterns the database of the Intenational Center For Diffraction Data (ICDD version pdf2_46, 1996) was used. Asymmetric flow field-flow fraction (AF4). The measurements were performed on a regenerated cellulose membrane with a cut-off at 10 kDa and canal height of 490 µm using water with 0.2% sodium azide as eluent. For detection, an Agilent Technologies UV-detector, a DAWN


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EOS multi-angle scattering detector from Wyatt Technologies, and a RI-detector WGE Bures at 620 nm were used. A flow rate of 1 mL/min at 25°C was used with an exponential cross flow gradient from 3 to 0 mL/min for 20 min using a curve multiplier of 2. The sample size was 50 µL of a 2 mg/mL polymer solution. Results 1. Block copolymers The block copolymers were synthesized using atom transfer radical polymerization (ATRP) as described in the experimental section. The average molecular weights (number and weight averages, Mn and Mw, respectively) and the polydispersity index (PDI = Mw/Mn) of the block copolymers were determined by gel permeation chromatography with on-line light scattering detection (GPC-MALLS) and AF4. The results are summarized in Table 1. Regular SLS and analytical ultracentrifugation (AUC) experiments were also conducted to characterize PSPM. A Mw = 1.82·106 g/mol was obtained by SLS and a Mw = 1.90·106 g/mol was obtained using AUC. 1

H-NMR spectroscopy along with elemental analysis confirmed the formation of copolymers.

Figure 4 and S 6 (Supporting Information) show representative NMR and IR spectra of PSPM, MPEO5000-b-PSPM,

PSPM-b-PEO4600-b-PSPM,

and

PSPM-b-PEO100k-b-PSPM,

respectively. In the 1H-NMR spectra (Figure 4), the copolymers clearly show a PSPM-dominant spectrum. The signal at 3.6 ppm indicates that the PEO block is also present in the copolymers. The -CH2-CH2O- signals could not be observed in the corresponding IR spectra since they are dominated by the signals from the PSPM block, which has a much higher molecular weight in the block copolymer (Table 1).


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Figure 4. Representative NMR spectra of A) PSPM-b-PEO100k-b-PSPM, B) PSPM-bPEO4600-b-PSPM, C) MPEO5000-b-PSPM, D) PSPM. For signal assignments see experimental section and Figure 2. Table 1. Summary of analytical data. The length of the SPM blocks reported for the triblock polymers PSPM-b-PEO4600-b-PSPM and PSPM-b-PEO100k-b-PSPM is the medium length of one block and was calculated from AF4 data. GPC analysis of MI1, MI2 and MI3 was performed in NMP with PEO calibration. GPC-LS and AF4 measurements were performed in aqueous solution. The two peaks of the bimodal PSPM-b-PEO100k-b-PSPM were analyzed separately. The error in the dn/dc measurements was ±0.0008 mL/g for PSPM and PSPM-b-PEO100k-b-


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PSPM, and ±0.0073 mL/g for PSPM-b-PEO4600-b-PSPM. For MPEO5000-b-PSPM, the dn/dc of PSPM-b-PEO4600-b-PSPM was used. GPC Polymer

GPC-LS

AF4 Pn(SPM

Pn(PEO)

Mn PDI [kg/mol]

dn/dc Mn PDI [mL/g] [kg/mol]

PSPM

1902

1.24

0.1183

1320

1.12

5359

0

MPEO5000-bPSPM

2090

1.52

0.1091

1895

1.11

7673

110

MI1

Mn PDI [kg/mol]

5.000

1.05

PSPM-bPEO4600-b-PSPM MI2

1.33

3255

1.01

6598

100 100

700/4793 2.41 117.2

0.1091

1.13

PSPM-bPEO100k-b-PSPM MI3

110 3341

4.670

)

0.1215

112/4580 1.81/1. 9059 51

2.1

2658 2658

2. Calcium phosphate mineralization The first and foremost question in biomimetic mineralization is whether or not a polymer is able to inhibit or promote mineral nucleation and growth. We have therefore studied the effects of the polymers on calcium phosphate nucleation and growth. To that end we used a titration method with optical detection (see experimental section for details). In short, 2SBF was used as a model system. Figure 5 shows that the calcium concentrations at which the first precipitation occurs, [Ca]P, strongly depended on the chemical composition of the polymer additive. Without polymer, calcium phosphate immediately precipitated after mixing the calcium and the phosphate components of 2SBF. In the presence of all polymers, a significant delay of nucleation was observed. PSPM showed the least effective retention. Polymers containing a PEO block were more efficient in delaying the calcium phosphate precipitation. The polymer with the long


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PEO block (PSPM-b-PEO100k-b-PSPM) was the most effective additive with a retention efficiency of 211% compared to the polymer-free solution. In all cases, the experiments are highly reproducible and showed a standard deviation of below 7%. Clearly, if calcium phosphate is to be stabilized in a supersaturated solution, the PSPM-b-PEO100k-b-PSPM copolymer is the most attractive candidate among those studied here.

Figure 5. Precipitation concentrations determined from titration of 2SBF with CaCl2 solution for polymer-free and polymer-containing solutions. The higher the calcium concentration, the later precipitation occurs. Initial and end data of titration are shown in Table S 1 and S 227. If the precipitates described above are to be used for dental repair, they must have the right crystal phase, that is, HAP or any of its precursors31,32, because teeth are mainly composed of small HAP crystals1,33. We have therefore determined the crystal phases of all precipitates via XRD and the Ca/P ratios via EDXS. The blank samples showed a Ca/P ratio of 1.33, which is indicative of octacalcium phosphate (Ca8H2(PO4)6·5 H2O), a HAP precursor. The Ca/P ratio of


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the samples grown in the presence of the polymers was approximately 1.41, indicating a mixed phase of different calcium phosphates, possibly octacalcium phosphate 1.3331 and HAP 1.6731 or calcium deficiencies in the HAP crystal structure. Some EDX spectra also showed low amounts of sodium and sulfur; the presence of these elements in the precipitates is due to the fact that the 2SBF used for mineralization contains sodium chloride and sulfate. Table 2. Ca/P of precipitation products from the combination of 2SBF with different polymers Polymer

Ca/P

PSPM

1.41 ± 0.04

MPEO5000-b-PSPM 1.44 ± 0.05 PSPM-b-PEO4600-b1.42 ± 0.02 PSPM PSPM-b-PEO100k-b1.43 ± 0.02 PSPM PEO4600

1.44 ± 0.05

MPEO5000

1.11 ± 0.18

PEO100k

1.29 ± 0.06

blank

1.33 ± 0.10

The XRD patterns (Figure 6) of all samples exhibited broad reflections at 30–34 degrees 2Θ. The patterns were noisy and only exhibited low count rates (approximately 200). Both the noise and the broad reflections indicate rather poor order in the precipitates. In spite of this, all patterns can be assigned to HAP (ICDD number 03-0747, Ca10(PO4)6(OH)2). The absence of higher order reflections at higher angles indicates (consistent with EDXS) that the samples contain no chlorapatite or carbonated apatite, despite the fact that the 2SBF also contains chloride and hydrogen carbonate.


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Figure 6. XRD patterns and Miller indices34,35 of calcium phosphates precipitated with (A) MPEO5000-b-PSPM, (B) PSPM-b-PEO4600-b-PSPM, (C) PSPM-b-PEO100k-b-PSPM, (D) PSPM, (E) PEO4600, (F) MPEO5000, (G) PEO100k, (H) no polymer (control), and (I) a pattern of natural HAP (data taken from rruff database RRUFF ID: R050512)27,36. The small peaks of (A), (C), (D) and (F) at 14° and 28° are from the Si sample holder. Figure 7 shows representative scanning electron (SEM) microscopy images of control samples and samples grown with polymer additives. The precipitates grown in the absence of any polymer exhibited fairly uniform small particles that were highly aggregated, yielding porouslooking large structures. Addition of PEO4600 and MPEO5000 homopolymers led to more strongly aggregated particles. The polymers appeared to favor a less open aggregation than without PEO. In contrast, addition of PEO100k with its much larger molecular weight appeared to favor the formation small particles, but also showed a significant number of larger spheres with a broad size distribution and diameters up to ca. 5 µm. Overall, SEM showed that increasing PEO molecular weights led to more well-defined morphologies (spheres), but also to a broadening of the size distribution of the respective samples.


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In contrast to the control samples and the samples precipitated with the uncharged PEO additives, addition of the PSPM homopolymer, which is highly negatively charged, led to the formation of uniform spherical particles with a narrow size distribution. A similar observation can be made with samples grown with diblock and triblock copolymers. These samples all exclusively contained spherical particles, which appeared to be individual particles. Again, the size distribution was strongly affected by the polymer molecular weight. The most uniform size distribution was found in samples grown with the MPEO5000-b-PSPM diblock copolymer, while the broadest size distribution was found for samples grown with the high molecular weight PSPM-b-PEO100k-b-PSPM triblock coplymer.

Figure 7. SEM images of calcium phosphate precipitated without polymer (control sample) and polymer additives. (A) Control sample, (B) PEO4600, (C) MPEO5000, (D) PEO100k 1454 ± 1009 nm, (E) MPEO5000-b-PSPM 701 ± 199 nm with an inset at higher magnification, (F)


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PSPM-b-PEO4600-b-PSPM 1067 ± 209 nm, (G) PSPM-b-PEO100k-b-PSPM 1175 ± 470 nm, (H) PSPM 1154 ± 413 nm. Insets are the particle size distributions27 (x-scale 6 µm) of the samples shown in the respective SEM images37. Images without size distribution histograms are from samples where no size distributions could be determined, mostly due to very small particle sizes or intergrowth and undefined morphologies. The samples are quite sensitive to the electron beam and higher magnification images could not be obtained in sufficient quality. 3. Interaction of polymers with synthetic HAP We have shown above (Figure 5) that mainly the charged polymers delay the calcium phosphate nucleation and precipitation. If an application in dental repair is envisioned, not only the precipitation, but also the dissolution of HAP (“enamel”) induced by the polymers is of interest because suitable additives should not dissolve the enamel surface. We therefore investigated the capacity of the polymers for dissolution of calcium phosphate using synthetic HAP as an initial model. Figure 8 shows the results of the dissolution studies. Solutions containing the short PEO homopolymers dissolved approximately as much calcium as a blank sample (pure d.i. water), but solutions of the longer PEO100k homopolymer dissolved less calcium than lower molecular weight PEO. In contrast to the PEO homopolymers, the four SPM-based polymers show nearly the same dissolution ability irrespective of chemical composition or molecular weight. To further evaluate the differences between the charged polymers, their dissolution ability was also analyzed with respect to the degree of polymerization of the charged blocks (Figure 8B). This value is called κ and was calculated as detailed in the caption of Table 3.


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Table 3. Absolute data for calcium dissolution in the presence of different PEO and PSPM homo- and copolymers and the relative calcium dissolution per mL, κ = (nsample(Ca2+) – nblank(Ca2+))/n(monomer), of one monomer with respect to the dissolution in the absence of any polymer. Here, nsample is the amount of calcium released from the HAP powder in the presence of a polymer additive, nblank is the amount of calcium released from HAP in the absence of polymer, and n(monomer) is the amount of monomer units of PSPM in the solution. While values above zero represent higher calcium dissolution, values below 0 represent a stabilizing effect on the HAP surface (that is, less dissolution) compared to a blank sample (pure d.i. water). Values around 0 indicate that there is no effect on the dissolution of calcium. Calcium concentrations are in mg/mL.

Polymer

PEO4600

MPEO5000

PEO100k

PSPM

MPEO5000-bPSPM

0.2 mg/mL

1.0 mg/mL

3.0 mg/mL

5.0 mg/mL

[Ca2+]

[Ca2+]

[Ca2+]

[Ca2+] κ

0.051 ±0.000 0.048 ±0.000 0.053 ±0.005 0.044 ±0.001 0.047 ±0.001

κ -0.0058

-0.0227

0.0055

-0.2458

-0.1540

0.052 ±0.002 0.047 ±0.002 0.043 ±0.000 0.048 ±0.001 0.051 ±0.000

κ 0.0000

-0.0057

-0.0099

-0.0246

-0.0062

0.054 ±0.002 0.047 ±0.003 0.033 ±0.000 0.097 ±0.003 0.092 ±0.002

κ 0.0008

-0.0019

-0.070

0.0922

0.0822

0.052 ±0.000 0.051 ±0.000 0.028 ±0.001 0.116 ±0.002 0.118 ±0.016

0.0000

-0.0002

-0.0053

0.0787

0.0813


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0.2 mg/mL

1.0 mg/mL

3.0 mg/mL

5.0 mg/mL

[Ca2+]

[Ca2+]

[Ca2+]

[Ca2+] κ

PSPM-bPEO4600-bPSPM

0.044

PSPM-bPEO100k-bPSPM

0.042

±0.002

±0.000

κ -0.2462

-0.3154

0.049 ±0.001 0.053 ±0.002

κ -0.0185

0.0063

0.092 ±0.001 0.087 ±0.000

κ 0.0821

0.0736

0.126 ±0.008 0.107 ±0.001

0.0886

0.0694

Figure 8. (A) Absolute calcium dissolution from HAP in water vs. polymer composition. The dotted line at 0.052 ± 0.004 mg/mL Ca2+ represents blank samples without polymer. (B) Relative calcium dissolution (κ) vs. polymer composition. 4. Surface adhesion of Streptococcus gordonii Sixteen groups of samples (n = 20/group) were included and compared in the experiment. The following various preparations of enamel slabs were incubated either for 30 or 120 min with S. gordonii: bare human enamel slabs (control); human enamel slabs coated by the in vitro salivary pellicle (clinically relevant reference); bare enamel coated by the copolymers MPEO5000-b-


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PSPM, PSPM-b-PEO4600-b-PSPM, and PSPM-b-PEO100k-b-PSPM; and enamel slabs incubated in the saliva/polymer mixtures to form a polymer-modified salivary pellicle layer (MPEO5000-b-PSPM/P,

PSPM-b-PEO4600-b-PSPM/P,

PSPM-b-PEO100k-b-PSPM/P).

Copolymer-saliva mixtures were applied to mimic a possible copolymer application in a mouth rinse or toothpaste, where macromolecules would be potentially mixed with the salivary flow in the oral cavity. To compare the numbers of the adhered viable S. gordonii vs. surface type, the bacteria were detached from the substrates and colony forming units were counted. Figure 9 illustrates the CFU/mm2 in all tested surfaces after 30 and 120 min of incubation with S. gordonii cells (5×108 cells/mL). Significantly higher bacterial adhesion (p < 0.05) was detected in the in vitro salivary pellicle layer on the enamel surface (P, Figure 9) compared to all other tested surfaces at both incubation times, except enamel tissue alone after 30 min of incubation (Table S 4). There were no statistically significant differences (p > 0.05) between the number of S. gordonii that adhered to the bare enamel surface and enamel coated by the copolymers MPEO5000-b-PSPM, PSPM-b-PEO4600-b-PSPM and PSPM-b-PEO100k-b-PSPM (Enamel vs. MPEO5000-b-PSPM, PSPM-b-PEO4600-b-PSPM, PSPM-b-PEO100k-b-PSPM, Figure 9). However, modified salivary pellicles prepared from the polymer/saliva mixtures showed significantly less bacterial adherence (p0.05, Table S 4).

Figure 9. Number of adhered S. gordonii cells vs. surfaces: (enamel) clean polished human enamel; (pellicle) enamel coated by the in vitro salivary pellicle layer (2 h); (MPEO5000-bPSPM) enamel coated by the copolymer MPEO5000-b-PSPM; (PSPM-b-PEO4600-b-PSPM)


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Biomacromolecules

enamel coated by the copolymer PSPM-b-PEO4600-b-PSPM; (PSPM-b-PEO100k-b-PSPM) enamel surface coated by the copolymer PSPM-b-PEO100k-b-PSPM; (MPEO5000-b-PSPM/P) enamel incubated in the copolymer MPEO5000-b-PSPM-saliva mixture (1:1, v:v); (PSPM-bPEO4600-b-PSPM/P) enamel incubated in the copolymer PSPM-b-PEO4600-b-PSPM-saliva mixture (1:1, v:v); (PSPM-b-PEO100k-b-PSPM/P) enamel incubated in the copolymer PSPM-bPEO100k-b-PSPM-saliva mixture (1:1, v:v). The statistical analysis was conducted using a nonparametric ANOVA model and pairwise Wilcoxon rank sum tests. Results are presented as box plots with a dashed line in each box indicating the mean value from the analysis of 20 samples. A statistically significant difference was found between numbers of S. gordonii that adhered after 30 and 120 minutes of incubation time on the enamel surfaces coated by the non-modified and polymer-modified salivary pellicles (Table S 4). In contrast, no significant difference according to incubation time (p>0.05, Table S 4) was observed in the cultures interacting with bare enamel and enamel surfaces coated by the copolymers MPEO5000-b-PSPM and PSPM-bPEO4600-b-PSPM (Figure 9). In contrast, enamel specimens treated by the copolymer PSPM-bPEO100k-b-PSPM showed a significant difference (p200% compared to a control sample without polymer). We explain the strong effect of this particular polymer by the fact that it contains a very high fraction of highly charged sulfonate groups and a large PEO block. The very high number of sulfonate monomers provides a strong and rapid interaction with calcium ions in solution, similar to that observed by Gebauer et al.40, and also provides a good interaction with inorganic calcium phosphate surfaces41. The better stabilization compared to the block copolymers with lower molecular weights can also be attributed to the large PEO block, which, according to established models of steric stabilization, provides better stability than shorter PEO blocks42. Besides the significant delay in precipitation, we also observed a strong effect of the polymer on the particle morphology. While the control sample and the samples precipitated with PEO homopolymers yielded relatively poorly defined particles, the PSPM homopolymer and the block copolymers produced rather uniform spherical particles with moderately narrow size distributions (ca. 20% in the most uniform samples, Figure 7). Both the delayed precipitation with the polyelectrolytes and the spherical shape of the final particles suggest a strong stabilizing effect for early states of nucleation. Possibly, the spherical shape also indicates that the particles form via an amorphous intermediate or a polymer-induced liquid precursor (PILP)43–45. An additional possibility, but this will have to be verified in the future, is an interaction of the PEO with surface -OH groups of the precipitate46,47. In the current case, -OH groups may originate from either phosphate ions or water in and on the inorganic particles. In any case, EDXS and XRD provided strong indications of either a transition through an amorphous or PILP precursor (Figure 6 and Table 2). This is because the XRD patterns are noisy


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and the reflections are very broad, indicating the final products are poorly ordered. This may originate from incomplete transformation of either an amorphous or PILP phase to HAP. The absence of a strong amorphous halo in the XRD patterns suggests that, possibly, the particles form through a PILP phase. This will again need to be confirmed in the future. EDXS found in all cases a Ca/P ratio between ca. 1.3 and 1.5 (Table 2). This is far from stoichiometric HAP, but is between HAP (1.67) and other calcium phosphate species that are HAP precursors (ACP 1, DCPD 1, OCP 1.33)31,48. Thus EDXS neither supports nor contradicts a model where the final spheres form through an amorphous precursor, but XRD possibly favors a PILP pathway. A important aspect is the fact that the particles precipitated with PSPM-b-PEO100k-b-PSPM had a very broad size distribution. We currently explain this observation with the fact that this polymer has a bimodal size distribution and both individual size distributions have a PDI that is significantly larger than the PDIs of the other polymers. Thus the combination of these two factors likely is responsible for the very broad size distribution of the respective precipitates. Besides the mineralization, we have also investigated the effect of the polymers on HAP dissolution. Generally, the PEO homopolymers show no or even a decreasing HAP dissolution efficiency in aqueous media (Figure 8). The strongest reduction in the dissolution efficiency vs. a polymer-free control experiment was observed with PEO100k, the largest PEO homopolymer investigated in the present study. Possibly, this is due to the fact that this large PEO species more strongly interacts with the HAP surfaces because there are more possibilities for hydrogen bonding between the polymer and the particle surface, similar to results by Rubio and Kitchener46on silica and by Shen47 on montmorillonite. By analogy to these studies, the interaction between PEO and HAP could be realized by partially protonated phosphate groups in HAP and the oxygen lone electron pairs in PEO. This interaction is more pronounced at higher


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Biomacromolecules

molecular weights. A higher molecular weight PEO could thus interact more strongly with the HAP surface and thus reduce the dissolution rate. In contrast, treatment of HAP powders with PSPM-based polymers led to a significant removal of calcium from HAP, indicating that these polymers do attack the HAP surface and remove at least calcium ions, but possibly also phosphate ions, from the solid HAP particles (Figure 8). Likely, the negatively charged SPM groups interact with partially protonated phosphate groups via hydrogen bonding like PEO, but they are also able to chelate calcium and thus more efficiently attack the HAP surface. This explanation is consistent with recent simulations on the dissolution of HAP49,50 and on the interaction of phosphorylated peptides with calcium phosphate51,52. All studies show that the efficient complexation of calcium is a key step in calcium phosphate dissolution. Besides the mineralization and HAP dissolution, we have also studied the effect of the polymers on biofilm formation on human enamel to evaluate the potential of these copolymers for application in dental care, specifically for the inhibition of biofilm formation. Formation of the oral biofilm typically follows several steps53,54. In the beginning (after tooth brushing, for example), salivary (glyco)proteins and lipids adhere on the enamel surface, forming a semipermeable membrane (the acquired salivary pellicle layer). This layer is permanently present and renewed on the surface of teeth. It contains important receptors and binding sites for oral bacteria54. Subsequent bacterial adhesion to the pellicle layer involves certain bacterial species, which are known as early or primary colonizers. These species are specific strains such as Actinomyces naeslundii, Streptococcus mitis, Streptococcus oralis, or S. Gordonii55, which provide optimal conditions for the secondary colonizers, i.e. Porphyromonas gingivalis, Streptococcus mutans, Fusobacterium nucleatum, or Veillonella atypical56,57. The biofilm


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provides nutrition networks, communication among bacteria, quorum-sensing, and protection against external factors (e.g. antimicrobial agents, oxygen deficiency)56. Therefore, as biofilm formation on teeth critically depends on the presence or absence of early colonizers, the prevention of the initial colonization step would be an attractive option for the prevention of oral diseases. Moreover, the investigation of early colonizers allows for an understanding of the molecular interactions between bacterial cells and dental surfaces. In this study, S. gordonii was used for evaluating biofilm formation on the copolymer-modified enamel and salivary pellicle layers. The adhesion of a bacterial cell to a substrate is driven by the interplay of specific and nonspecific interactions. Lifshitz-van der Waals, hydrogen bonding, acid-base, and electrostatic interactions are examples of non-specific contributions, whereas receptor-ligand interactions refer to specific ones. Van der Mei et al.58 showed that the specific binding interactions between a bacterial cell and the salivary pellicle film are stronger than the non-specific interactions between the same bacterial cell and a BSA-coated surface. Additionally, specific interactions strengthen over more extended periods of time compared to non-specific ones58. S. gordonii specifically binds to the salivary components in the pellicle layer. The alpha-amylase-binding protein A of S. gordonii interacts with salivary amylase59, while the antigen I/II family provides binding to agglutinin and proline-rich proteins present in the salivary pellicle57. Non-specific interactions of S. gordonii with the enamel and pellicle layer could potentially be driven by Ca2+bridging60, electrostatic61, hydrophobic62 or van der Waals interactions. To acquire first-hand knowledge about specific and non-specific contributions to the interaction of S. gordonii with polymer-modified enamel and pellicle, 30 and 120 min of incubation time with bacterial cells were applied in the experiments. Clearly, more extended incubation times, varied cell


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concentrations and molecular biological methods would be required for a complete study of cellsurface interactions. It is well known that molecules containing phosphate, phosphonate, sulfate, and sulfonate groups interact with HAP surfaces17,63–66. Indeed, we have shown above that the polymers strongly interact with calcium phosphate surfaces. Thus, the enhanced dissolution of calcium was found in the presence of charged sulfonate groups of copolymers as well as a modified surface morphology of the salivary pellicle after the addition of copolymers (Figures 8 and 10). In contrast, PEO interacts much less with calcium phosphate, but provides anti-fouling properties67,68, and surface hydrophilization17. The polymer-induced change of the pellicle morphology at the enamel surface could directly affect bacterial colonization. These effects have been studied using S. gordonii as a model. Because human enamel tissue consists mainly of calcium-deficient HAP and water1,69 (97% by weight), non-specific interactions were expected to mainly contribute to the adhesion of S. gordonii on bare enamel as well as on enamel coated with PSPM-b-PEO4600-b-PSPM, MPEO5000-b-PSPM, and PSPM-b-PEO100k-b-PSPM. Indeed, the fact that we observed no effect of incubation time on bacterial adhesion to bare enamel slabs, to enamel coated with MPEO5000-b-PSPM, and to enamel coated with PSPM-b-PEO4600-b-PSPM copolymers (Table S 4) indicates the prevalence of non-specific interactions. After 120 min of incubation, these surfaces were only slightly more colonized by S. gordonii (1.03–1.04 times more CFU/mm2) than after 30 min of incubation. Bare enamel surfaces as well as enamel coated by the copolymers were equally colonized by the bacterial cells after each particular incubation time (Table S 4, Figure 9). Apparently, the addition of polymer to the enamel surfaces does not lead to biopassivation.


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Biomacromolecules

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Once enamel tissue is coated by the salivary pellicle layer, significantly greater bacterial adhesion is detected after 120 min of incubation (P, Figure 9, Table S 4) compared to the noncoated clean enamel surface. First, only non-specific interactions take place on a clean enamel surface, whereas non-specific and specific interactions operate together on the salivary pellicle layer, leading to stronger adhesion of S. gordonii to the pellicle-coated enamel at a longer exposure time. The observed tendency correlates well with data from the literature58,60,70. The addition of the copolymers into the saliva resulted in the formation of mixed copolymerpellicle layers, which are less attractive for the interactions with S. gordonii compared to the native salivary pellicle (P vs. MPEO5000-b-PSPM/P, PSPM-b-PEO4600-b-PSPM/P, PSPM-bPEO100k-b-PSPM/P; Table S 4, Figure 9) at both incubation times. Apparently, the presence of synthetic macromolecules affects specific interactions between salivary adhesins and S. gordonii cells. There are several possible reasons for this observation: i) synthetic macromolecules compete with the salivary proteins for the same adsorption sites on the enamel surface, thus disturbing the regular protein arrangement and stabilization within the pellicle layer; ii) copolymer chains interact with the salivary adhesins/proteins and inactivate their function or disable their contact with bacterial anchors; iii) the copolymers hydrophilize the pellicle layer and thus hamper tight cell-substrate contact. Obviously two or more of these factors may jointly contribute to the observed effects. Most likely, not only specific interactions are altered upon addition of the copolymers but also non-specific interactions between copolymer coatings and bacterial cells are affected by the addition of salivary proteins. After 30 min of incubation, there were no statistically significant differences between the interaction of the bacteria with the polymer coatings alone versus the polymer-modified salivary pellicles (MPEO5000-b-PSPM vs. MPEO5000-b-PSPM/P, PSPM-b-


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Biomacromolecules

PEO4600-b-PSPM vs. PSPM-b-PEO4600-b-PSPM/P, PSPM-b-PEO100k-b-PSPM vs. PSPM-bPEO100k-b-PSPM/P, Figure 9); However, a tendency for slightly reduced bacterial adhesion can be noticed in the case of pellicles modified by the copolymers MPEO5000-b-PSPM and PSPMb-PEO4600-b-PSPM (MPEO5000-b-PSPM vs. MPEO5000-b-PSPM/P, PSPM-b-PEO4600-bPSPM vs. PSPM-b-PEO4600-b-PSPM/P, Figure 9). It cannot be excluded that the salivary proteins somewhat hinder the non-specific interactions between S. gordonii and the polymer chains by reducing the availability of macromolecules for direct contact with bacterial cells. On the other hand, salivary proteins should provide additional adhesion sites for stronger58 specific interactions, but the former require more extended periods of time. Indeed, statistically significant differences were found in the number of bacterial cells that attached to copolymermodified pellicles after 120 min of incubation compared to a short 30 min contact period (Table S 4). Note, no effect of the copolymer type (MPEO5000-b-PSPM vs. PSPM-b-PEO4600-b-PSPM vs. PSPM-b-PEO100k-b-PSPM) was found on the adhesion of S. gordonii to tested surfaces. The salivary pellicle modified by the copolymer PSPM-b-PEO100k-b-PSPM (PSPM-b-PEO100k-bPSPM/P, Figure 9) was more effective for adhesion of S. gordonii over a short-term period (30 min) than the pellicle layers modified by the copolymer MPEO5000-b-PSPM (MPEO5000-bPSPM/P, p

Poly(ethylene oxide) - American Chemical Society

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