ARTICLE pubs.acs.org/Biomac
Polymer Brush Controlled Bioinspired Calcium Phosphate Mineralization and Bone Cell Growth Ruben L€obbicke,† Munish Chanana,‡,^ Helmut Schlaad,§ Christine Pilz-Allen,|| Christina G€unter,# Helmuth M€ohwald,‡ and Andreas Taubert*,†,§ †
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Institute of Chemistry, University of Potsdam, Karl-Liebknecht-Str. 24-25, D-14476 Golm, Germany Departments of ‡Interfaces, §Colloid Chemistry, and Biomaterials, Max Planck Institute of Colloids and Interfaces, Research Campus Golm, D-14424 Potsdam, Germany ^ Departamento de Química Física, Universidade de Vigo, Campus Universitario, E-36310 Vigo, Spain # Institute of Earth and Environmental Sciences, University of Potsdam, Karl-Liebknecht-Str. 24-25, D-14476 Golm, Germany ABSTRACT: Polymer brushes on thiol-modified gold surfaces were synthesized by using terminal thiol groups for the surfaceinitiated free radical polymerization of methacrylic acid and dimethylaminoethyl methacrylate, respectively. Atomic force microscopy shows that the resulting poly(methacrylic acid) (PMAA) and poly(dimethylaminoethyl methacrylate) (PDMAEMA) brushes are homogeneous. Contact angle measurements show that the brushes are pH-responsive and can reversibly be protonated and deprotonated. Mineralization of the brushes with calcium phosphate at different pH yields homogeneously mineralized surfaces, and preosteoblastic cells proliferate on both the nonmineralized and mineralized surfaces. The number of living cells on the mineralized hybrid surfaces is ca. 3 times (PDMAEMA) and 10 times (PMAA) higher than on the corresponding nonmineralized brushes.
’ INTRODUCTION Bioinspired calcium phosphate hybrid materials have attracted tremendous interest; this is mainly due to the fact that these hybrids have a wide range of very useful properties and potential applications, most prominently in the biomedical field.15 There are numerous studies on the effects of water-soluble or amphiphilic polymers on calcium phosphate mineralization from bulk aqueous solution,611 but surface-controlled mineralization has only recently gained some attention.6,1221 However, in nature (including implantology, etc.), mineralization processes typically do not occur in bulk solution but in gel phases or at interfaces at roughly ambient conditions.22 There is thus a need for understanding the effects of interfaces on calcium phosphate nucleation, growth, and structure formation. Zhang et al. suggest that calcium phosphate formation at monolayers of low molecular weight surfactants such as arachidic acid proceeds via an amorphous intermediate, which later transforms into crystalline hydroxyapatite (HAP).23 A more recent cryo-electron microscopy study indeed demonstrates that initially amorphous prenucleation clusters form on an arachidic acid surface; these clusters later transform into amorphous calcium phosphate and carbonated apatite.24 Block copolymer monolayers at the airwater interface have also been studied. Both polyanionic and polycationic blocks have a distinct effect on calcium phosphate mineralization. In all cases, high charge densities favor the formation of well-controlled, uniformly mineralized polymer monolayers.1517 r 2011 American Chemical Society
Besides monolayers at the airwater interface, also the effect of polymer-modified solid surfaces on calcium phosphate mineralization has been investigated. Ngankam et al.19 and Ball et al.18 demonstrated that polymer multilayers influence nucleation and growth of calcium phosphate. Multilayers of poly(sodium 4-styrenesulfonate) (PSS) and poly(allylamine hydrochloride) (PAH) on silica influence the crystal phase selection.19 In contrast, multilayers of poly(L-glutamic acid) or poly(L-aspartic acid) and poly(L-lysine) affect the prenucleation induction times.18 Other studies demonstrated that polyelectrolyte coatings strongly modify calcium phosphate deposition on titanium and nickeltitanium alloy surfaces.13,14,25 Self-assembled monolayers (SAMs) also control nucleation, growth, crystal orientation, crystal sizes and shapes, and crystal phases.2631 This has been assigned to lattice matching/mismatching effects between the well-organized SAMs and the crystal lattice of the growing inorganic. For example, Aizenberg and co-workers used micropatterned SAMs to study calcium carbonate crystallization.32 They found specific dependencies of crystal size, crystallographic orientation, and morphology on the surface chemistry of the SAMs. Besides crystalline inorganics, also amorphous minerals such as silica precipitate on functional SAMs. For example, Tahir et al. used nitrilotriacetic acid (NTA) Received: July 18, 2011 Revised: August 16, 2011 Published: August 18, 2011 3753
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Biomacromolecules Scheme 1. Experimental Approach for SAM Preparation, Polymerization, and Mineralization with Calcium Phosphate
terminated alkanethiol SAMs on gold to show that silica only precipitates on silicatein-modified surfaces.33 The current study uses an approach combining SAMs and polymers. We have previously studied calcium phosphate mineralization on polymer monolayers at the airwater interface.1517 The goal of the current study is to evaluate whether or not it is possible to transfer this concept to solid surfaces (or a solid/ liquid interface, respectively). If successful, this could have implications for biomedical engineering and related areas, as the efficient mineralization of solid surfaces is a key requirement, for example, in implantology. The approach is based on 1,8octanedithiol, which forms a stable, dense, and uniform monolayer on gold,3436 and the subsequent thiyl radical-initiated photopolymerization37 of methacrylic acid (MAA) or dimethylaminoethyl methacrylate (DMAEMA). The resulting brushes can be mineralized with calcium phosphate (Scheme 1).
’ EXPERIMENTAL SECTION Materials. Hydrogen peroxide (35%, stabilized), sulfuric acid (96%), sodium dihydrogen phosphate dihydrate (g98%), and sodium acetate (g99%, anhydrous) were purchased from Roth, ethanol (abs, g99.9%) from VWR, tris(hydroxymethyl)aminomethane (TRIS, 99.8%) and Triton-X-100 (laboratory grade) from Sigma-Aldrich, hydrochloric acid (37%), calcium chloride (anhydrous, granular), methacrylic acid, and dimethylaminoethyl methacrylate (98%, stabilized with hydroquinone monomethyl ether) from Merck, 1,8-octanedithiol (98%) from Alfa Aesar, 40 ,6-diamidin-2-phenylindole (DAPI) from Roche, and Phalloidin-FITC from Invitrogen. DMAEMA was distilled prior to use. All other chemicals were used as received.
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Wafer Preparation. Silicon wafers of 25 20 mm were washed with a mixture of ammonia and hydrogen peroxide (3:1 v/v) at 80 C, a mixture of sulfuric acid and hydrogen peroxide (1:1 v/v), and deionized water, followed by drying in a nitrogen flow. The wafers were coated with 3 nm of chromium and 150 nm of gold by thermal vapor deposition in an Edwards FL400 coating system at p e 5 106 mbar. The deposition rate was about 1 Å/s. This method produces flat Au surfaces with a (111) orientation. The freshly prepared wafers were stored in pure ethanol under nitrogen in the dark. SAM Preparation. SAMs were prepared by immersion of a plasmacleaned Au(111) substrate with a contact angle of 20 in 1 mM ethanolic solutions of 1,8-octanedithiol for 24 h at room temperature. The vials were covered with aluminum foil to minimize light exposure. Thiol Surface-Initiated Photopolymerization. Gold wafers with freshly prepared SAMs were deposited in a 50 mL Schlenk flask. 10 mL of an ∼4 wt % aqueous solution of the monomer (MAA or DMAEMA) were added. The flask was sealed with a septum and Parafilm, and the mixture was degassed at least three times to remove traces of oxygen and ozone (ozone is a major cause for SAM instability)38 and subsequently flushed with argon. The flasks were then exposed to UV light at room temperature, using a Heraeus TQ 150 immersion lamp (150 W, Hg medium pressure). The light passes through the borosilicate glass of the Schlenk vessel, and the glass removes high-energy UV light (λ < ∼300 nm). This is important because only low temperature and low-energy UV light prevent thiol desorption from the gold substrate. After 24 h, the resulting polymer-modified Au wafers were rinsed extensively with Milli-Q water (0.055 μS/cm) to remove the remaining monomer and loose polymer material. (Occasionally, MAA polymerization led to a gel-like material, which could be removed via rinsing, leaving the attached polymer film behind.) The samples were dried at 65 C for 2 h in a nitrogen flow. Mineralization on Polymer-Modified Wafers. A buffer solution was prepared by mixing TRIS (20 mmol) and 100 mL of water. While stirring moderately, the pH was adjusted to 8.5 via addition of HCl (37%). The PMAA-modified Au wafers were placed in the solution vertically, and after 30 min, 2 mL of a 2 mM CaCl2 solution were added, yielding a 40 μmol solution of CaCl2. After another 30 min, 2 mL of a 2 mM of NaH2PO4 solution was added. Subsequently, the beaker was sealed with Parafilm, and the mixture was stirred for 24 h. The wafers were cleaned by rinsing several times with water and dried under air flow. For mineralization of the PDMAEMA-modified wafers, an acetate buffer was prepared by mixing sodium acetate (20 mmol) and 100 mL of deionized water, and the pH was adjusted to 5.5 with acetic acid under stirring. Mineralization was as described for PMAA-modified wafers, but the sequence of ion addition was reversed. Contact Angle Measurements. Contact angle measurements were performed on a Kr€uss G10 with a CCD camera. The system was aligned with a sapphire ball of 4 mm radius. Every value is the mean value of at least five single measurements. In all measurements Milli-Q water was used. Polyelectrolyte protonation and deprotonation were done with 1 M HCl and NaOH, respectively. Rinsing with Milli-Q was essential after NaOH treatment; without rinsing the drop irregularly strayed across the whole sample. Ellipsometry. Ellipsometry measurements were made with an Elli 2000 Brewster angle microscope (BAM, NFT Nanofilm Technology, G€ottingen, Germany) with a solid-state laser (50 mW, λ = 532 nm, experimental light intensity 23%). The machine utilizes a polarizer compensatorsampleanalyzer (PCSA) setup39 where the compensator (C) is fixed at 45. The angular region was between 50 and 75 (multiangle ellipsometry). For all measurements a nulling ellipsometry scheme was adopted. Within this scheme, ψ and Δ are obtained by minimizing the amount of light reaching the detector by rotating the polarizer (P) and analyzer (A). Data (ψ and Δ) were analyzed with a 3754
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Biomacromolecules fitting algorithm with wavelength λ, refractive indices, and thickness as adjustable parameters. The substrate was gold (nD = 0.4757, k = 2.492, h = 150 nm). Data used for calculation were nD = 1.503, k = 0.21 (1,8-DT) and nD = 1.58, k = 0.08 (polymer). IR Spectroscopy. IR spectra were recorded as difference spectra on a Bruker Equinox 55/S with a built-in DTGS-detector. A plasma-cleaned gold wafer was used as a reference. To avoid water vapor and CO2 bands, the instrument was continuously flushed with nitrogen. Raman Spectroscopy. Raman spectroscopy was performed on a CRM300 confocal Raman microscope (WITEC, Germany) with a Nikon objective and a linearly polarized excitation laser (diode pumped laser, λ = 532 nm, CrystaLaser). The Raman light was detected with a CCD camera (DV401-BV, Andor) with a grating spectrometer (600 L/ mm, UHTS 300, WITec). UVvis Spectroscopy. UVvis spectra were acquired on a Cary 50 UVvis spectrophotometer. 3 mL of DMSOformazan solution in a polystyrene (PS) cuvette were used for each measurement, and a DMSO-filled PS cuvette was used as reference. Absorption was recorded at 570 and 630 nm. Scanning Electron Microscopy. SEM measurements were performed on a JEOL JSM 6510 SEM with tungsten hairpin filament (15 kV) and an Everhart-Thornley secondary electron detector. For EDXS experiments an Oxford INCAx-act SN detector with a resolution of 135 eV at 5.9 keV was used. Atomic Force Microscopy. AFM images were collected in noncontact mode using a Veeco Dimension 3100 and tips with a resonance frequency of 285 kHz and a spring constant of 42 N/m. Fluorescence Microscopy. FM images were collected with a Leica DM RXA 2 fluorescence microscope with integrated filters for FITC (I 3 filter) and DAPI (N2.1). Cell Culture Experiments. Preosteoblastic Cellline MC3T3-E1 (a generous gift from Ludwig-Boltzmann-Instiute in Vienna) was used to investigate the biocompability of the wafers. The wafers were sterilized in 70% ethanolic solution and placed in a cell culture dish. 6 104 cells/cm2 per dish surface were seeded. The culture medium was α-modified Eagle’s medium (α-MEM, Sigma) with 4.5 g/L glucose (Sigma), 10% fetal calf serum (FCS) (PAA), 10 μg/mL gentamicin (Sigma), and 50 μg/mL ascorbic acid (Sigma). The cells were cultured for 72 h at 37 C and 5% CO2.40,41 Cell numbers were determined with a Casy Model TT (Casy Technolgies, Sch€arfe Systems).42 This instrument works on a combination of resistance measurement and pulse area analysis. The cells were detached with Pronase/EDTA solution (25 mg of Pronase and 100 mg of EDTA in 250 mL of PBS). PBS was added to the cell/Pronase suspension and then centrifuged at 650g for 10 min. The supernatant was removed, and the cells were redispersed in 1 mL of α-MEM. 100 μL of the resulting suspension were transferred to an Eppendorf vessel containing 900 μL of α-MEM. Subsequently, 10 mL of Casitron solution and 50 μL of cell suspension from the Eppendorf tube were mixed and shaken. Cell numbers of the resulting liquid were determined with a CASY 1 cell counter. Cell Viability Test (MTT Assay). MTT-solution (reagent A) was made by mixing 5 mg/mL of M 5655 MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide) with PBS buffer (without Ca2+ and Mg2+) and was filtered sterile. MTT solvent (reagent B) was made by mixing 99.4 mL of DMSO, 0.6 mL of glacial acetic acid, and 10 g of sodium dodecyl sulfate. For MTT testing, the cell-coated samples were transferred into a new aseptic 3 cm well. A solution of 250 μL of reagent A and 2250 μL of DMEM were added and incubated for 4 h at 37 C. Afterward, the liquid was removed, and 2500 μL of reagent B were added. After 5 min at room temperature, followed by 5 min of gently shaking, the supernatant was analyzed via UV/vis spectroscopy at 570 nm. Reference wavelength was 630 nm. Results are averages of at least three separate measurements.43
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Figure 1. AFM image of a C8-DT SAM after annealing. Surface roughness is 0.65 nm.
Immunofluorescence. Fluorescein isothiocyanate (FITC)-conjugated phalloidin solution was obtained by mixing 1 μL of FITCphalloidin stock solution (Invitrogen, Alexa Fluor 488 phalloidin c = 4 104 M in 1.5 mL of methanol, M = 1252.4 g/mol) and 99 μL of PBS, yielding a 4 106 M FITC-phalloidin solution. Excitation was done at 495 nm, and the emission maximum was at 513 nm. DAPIPBS solution was obtained by mixing 20 μL of DAPI (Roche Diagnostics) 1 mg/mL in Milli-Q water with 10 mL of PBS, yielding a final concentration of 2 μg/mL. DAPI excitation wavelength was 355 360 nm, and the fluorescence maximum was at 450 nm. Substrates carrying the attached cells were washed with PBS and fixed with 4% of paraformaldehyde (Sigma) in PBS for 20 min at 4 C. After three washing cycles with PBS cells were lysed with 0.1% Triton X-100 (Sigma) in PBS for 10 min at 4 C. After three more washing steps 100 μL of FITC-conjugated phalloidin solution was added, and the samples were incubated for 1 h at 4 C in the dark. After 3 7 min washing with PBS, the DAPIPBS solution was added, and after 10 min of incubation at 37 C in darkness, the samples were cleaned by rinsing with PBS.
’ RESULTS AND DISCUSSION SAMs of 1,8-Octanedithiol on Gold Surfaces. Selfassembled monolayers (SAMs) of 1,8-octanedithiol (C8-DT) on gold were characterized via ellipsometry, FTIR spectroscopy, atomic force microscopy (AFM), and contact angle (CA) measurements. Advancing contact angle (ACA) measurements of C8-DT SAMs show a CA of 81 ( 3 and a hysteresis of up to 20. The receding contact angle (RCA) is 68 ( 5. The latter is in good agreement with literature (68 ( 4, hysteresis 5),44 and indeed, annealing of the SAM at 90 C for 2 h also yields an ACA of 70 ( 2 and a hysteresis on the order of 5. Annealing thus has a significant effect on the homogeneity and roughness of the SAM. FTIR spectroscopy shows the formation of a covalent gold sulfur bond through a broad band at 500 cm1 which matches with the CS stretch mode. Peaks at 1251 and 1459 cm1 correspond to methyl symmetric and antisymmetric bending modes.45 Figure 1 shows a representative AFM height image of an annealed SAM. AFM confirms the homogeneity of the SAM and also shows that the SAMs have a small average surface roughness 3755
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Figure 2. Contact angles of PMAA and PDMAEMA brushes: (A) PMAA brush after extensively washing with water, (B) after treatment with aqueous NaOH and rinsing with water, and (C) after treatment with aqueous HCl. (D) PDMAEMA brush after extensively washing with water, (E) after treatment with aqueous HCl and rinsing with water, and (F) after treatment with aqueous NaOH.
of ca. 0.7 nm. AFM also reveals that there are essentially no defects in the SAM. AFM therefore confirms CA measurements in that in both cases evidence for a very homogeneous SAM is found. Ellipsometry finds a SAM thickness of 1233 Å. Molecular mechanics calculations predict a length of 11.8 Å for C8-DT.34 Assuming a tilt angle of 30, the SAM should be 10.2 Å thick.46 Ellipsometry therefore suggests that the SAM is actually a multilayer, which may arise from multilayer formation due to the interlayer linking chemistry between the thiol head groups.34,47 This is, however, less important for the polymerization of the brushes (see below) and has therefore not been further investigated. Nevertheless, as evidenced by AFM, the film is defect-free, and is therefore a suitable starting surface for polymerization (Scheme 1). Polymer Brushes by Surface-Initiated Photopolymerization. Photopolymerization of methacrylic acid (MAA) is an established technique.4851 By using light with wavelengths of λ > 300 nm at ambient temperature under argon (see Experimental Section for details), the detachment of the dithiol SAM during photopolymerization can be avoided, and thiyl radicals are only generated on the surface. There should be no radical transfer to monomers in solution; therefore, no polymerization occurs in bulk solution.37 Consequently, polymer brushes can be synthesized directly from the SAM (Scheme 1). Figure 2 shows representative contact angle measurements of the resulting brushes. The CA of the as-prepared PMAA brush surface is 44 ( 3. The CA decreases to 15 ( 4 if the surface is treated with aqueous NaOH. It must be noted at this point that the experimental setup only allows CA measurements of 8 and higher. Occasionally, however, the CAs of the surfaces treated with NaOH were lower than 8. It is thus likely that the real CA may be a few degrees less than 15. Upon treatment with aqueous HCl the contact angle increases again to 41 ( 3. CA measurements thus clearly show the presence of a pH responsive polymer brush on the surface. Correspondingly, the CA of the PDMAEMA surfaces is 69 ( 3, which is in very good agreement with reported values of 70 for DMAEMA polymers prepared by atom transfer radical polymerization (ATRP).52 Analogous to the PMAA surfaces, the PDMAEMA surfaces respond to changes in pH. The CA can be adjusted by treatment with aqueous HCl to 13 ( 3, which can be reversed by treating the surface with aqueous NaOH.
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Figure 3 shows representative AFM data of the PMAA brush. The films are smooth with no defects and have a surface roughness of ∼2.2 nm. All AFM images also show protrusions with a height of ca. 40 nm or less. Their origin is unknown at the moment. Further measurements on a scratch reveal a polymer film thickness of 71 nm, which is in good agreement with ellipsometry (7080 nm). It must, however, be stated at this point that the fitting of the ellipsometry data was done with a layer model and that the protrusions could so far not be accounted for adequately. Nevertheless, AFM height measurements on numerous scratches clearly show that the thickness estimation from ellipsometry is reasonable. Figure 4 shows the corresponding AFM data obtained from the PDMAEMA brushes. The PDMAEMA films have a roughness of ∼1.2 nm, which is similar to the PMAA brushes described above. In contrast to the PMAA brushes, the PDMAEMA surfaces do not exhibit the protrusions shown in Figure 3. Scratch depth profiles reveal a polymer thickness of around 90 nm, which is about 20 nm thicker than the PMAA films. In summary, AFM, CA, and ellipsometry show that in both cases (PMAA and PDMAEMA brushes) homogeneous surfaces have been generated. Calcium Phosphate Mineralization on Polymer Brushes. Charged polymer surfaces are much more efficient nucleation and crystallization templates than noncharged surfaces and lead to much more homogeneous mineral films.15,16,18,19,23,53 We have therefore performed the mineralization of both brushes (PMAA and PDMAEMA) under conditions where they are charged. Mineralization of the PMAA brushes was done at pH 8.5 (negatively charged brush, addition of calcium followed by addition of phosphate) and mineralization of PDMAEMA brushes at pH 5.5 (positively charged brush, addition of phosphate followed by addition of calcium). Already visual inspection of the films suggests significant differences. While mineralization of the PMAA brush results in a white film after 24 h, the PDMAEMA surface does macroscopically not appear mineralized. Figure 5 shows representative scanning electron microscopy (SEM) images of a mineralized PMAA and PDMAEMA brushes. Both types of brushes are mineralized, although the morphology of the films is quite different. Low-magnification SEM images of the PMAA films show a dense, although not complete, coverage of the polymer brush. Higher magnification shows a fine, porouslooking network of small particles. Focusing on different areas shows that also the parts that initially appear nonmineralized are at least coated with a thin mineral layer. In contrast to the PMAA brushes, mineralization of the PDMAEMA brushes leads to a much less dense mineral coating, but the high contrast of the features again suggests that some mineralization has occurred. The observed features are larger and less dense than those observed with the PMAA brushes, but also here, SEM shows that the polymer brush is densely coated in some areas and much less in interstitial areas. Table 1 shows quantitative energy-dispersive X-ray spectroscopy (EDXS) data of mineralized PDMAEMA and PMAA brushes. The calcium and phosphorus signals are much higher in the case of the PMAA brush, indicating a much higher degree of mineralization. Moreover, the Ca/P ratio (atom %) of these samples is 1.56, which is less than in pure, stoichiometric hydroxyapatite (HAP, 1.67).54 EDXS of mineralized PDMAEMA brushes was difficult because of a poor signal-to-noise ratio. This indicates that the degree of mineralization of the PDMAEMA brushes is much lower than in the case of PMAA, which is consistent with recent studies on the mineralization of PDMAEMA-based 3756
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Figure 3. (A) AFM height image (size 5 5 μm2, z-range 10 nm) of an as-prepared PMAA brush. (B, C) AFM height measurements (image size 30 12 μm2, z-range 250 nm) on a scratch in the polymer film with corresponding height profile. Line in panel B indicates the location of the height profile.
Figure 4. (A) AFM height image (size 5 5 μm2, z-range 20 nm) of an as-prepared PDMAEMA brush. (B, C) AFM height measurements (image size 15 6 μm2, z-range 400 nm) on a scratch in the polymer film with corresponding height profile. Line in panel B indicates the location of the height profile.
block copolymer at the airwater interface 16,17 and with SEM data of the current materials. Here, the Ca/P ratio (atom %) is 1.02, which is, for example, close to the Ca/P ratio in brushite (1.0). Figure 6 shows representative Raman spectra of the mineralized brushes. Spectra of the mineralized PMAA brushes show intense bands at 430, 590, 963, and 1046 cm1, which can be assigned to PO43 stretching vibrations ν2, ν4, ν1, and ν3 of HAP.55,56 The sharp band at 522 cm1 may originate from HPO42,57 indicating the presence of some protonated phosphate species in the material. Alternatively, this band may also originate from a disulfidealkyl vibration from 1,8-DT.58 A broad band at 2934 cm1 can be assigned to CH vibrations of the PMAA polymer brush. Finally, the band at 3573 cm1 can be assigned to polymer vibrations. The Raman spectra of mineralized PDMAEMA brushes exhibit a lower signal-to-noise ratio, and the signals are less intense. This is
consistent with EDXS (Table 1) and again suggests that the degree of mineralization of the PDMAEMA brushes is much lower. Although less intense, the spectra correspond to those of the mineralized PMAA films. Bands at 963 and 1050 cm1 can again be assigned to PO43 stretching vibrations ν1 and ν3 of HAP.55,56 The sharp band at 522 cm1 may again originate from HPO42 or disulfidealkyl vibrations.57,58 A broad band at 2934 cm1 can be assigned to CH vibrations of the PDMAEMA polymer brush. The band at 3573 cm1 can be assigned to polymer vibrations. Interestingly, the characteristic HPO band of brushite (dicalcium phosphate dihydrate, DCPD, at 872 cm1)55,56 is not observed in either sample, indicating that DCPD, a low-pH modification of calcium phosphate (which could be expected on the PDMAEMA brushes), is not present in significant amounts. Finally, it must be noted that bands between 1040 and 1080 cm1 may also be assigned to antisymmetric stretch vibrations of 3757
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Figure 5. Low- (left) and high-magnification SEM images (center) and EDX spectra (right) of mineralized brushes. (A, B, C) are mineralized PMAA brushes and (D, E, F) are mineralized PDMAEMA brushes. Scale bars are 200 μm (A, D) and 5 μm (B, E).
Table 1. Results of EDXS Measurementsa PMAA brush element
PDMAEMA brush
wt %
atom %
wt %
atom %
Si
9.77 ( 1.8
10.28 ( 2.0
19.13 ( 11.8
19.00 ( 5.7
P
9.52 ( 3.2
9.50 ( 2.3
3.30 ( 2.0
3.41 ( 3.0
19.65 ( 8.6
14.84 ( 3.8
1.56 ( 0.8
1.11 ( 0.2
O
32.29 ( 7.5
62.37 ( 0.7
33.92 ( 11.7
62.33 ( 0.5
Au
41.62 ( 4.4
7.44 ( 1.4
41.49 ( 1.4
7.71 ( 0.3
Ca
a
Ca/P ratio = 1.56 and 1.02 for mineralized PMAA brushes and mineralized PDMAEMA brushes, respectively. Carbon signals are due to the thiol and polymer present in the samples, oxygen signals are due to the phosphate, the polymer (methacrylate and methacrylic acid, respectively), and the oxidized silicon surface, and the silicon signals are due to the silicon wafers used as supports. Nitrogen of the PDMAEMA brush was below the detection limit.
Figure 6. Raman spectra of PMAA and PDMAEMA brushes after mineralization with calcium phosphate.
octacalcium phosphate (OCP).59 It may therefore be possible that some fraction of OCP is present in the samples along with HAP. Cell Viability Test and Cell Screening. To evaluate whether or not our surfaces are interesting for bone cell growth (and therefore for modification of e.g. implant surfaces), we have performed cell viability tests with preosteoblasts via MTT test. In reference experiments cells were cultivated in a blank PS Petri dish. After 24 h cells were counted, and the extinction was measured via MTT test. The fitted logarithmic correlation (y = 0.0213 ln(x) 0.1826) was used to calculate cell numbers (x) from the measured extinction (y) of the real experiment. Figure 7 shows the results of the cell viability tests vs blank Petri dishes (Nunclon Surface, Nunc) and unmodified gold substrates. The different surfaces evoke different responses in the cell cultures. In comparison to the standard PS Petri dish, pure gold is not a suitable substrate for adhesion of osteoblasts, and the number of cells is 1 order of magnitude lower than on the reference Petri dish. The cell viability on both polymer brushes is higher than on the pure gold, but the viability on the PDMAEMA brush is ca. 3 times higher than on the PMAA brush. Moreover, cell viabilities on the mineralized brushes are significantly higher than on the pure polymer brushes. Figure 7 therefore clearly shows
that the mineralization of the polymer brushes leads to surfaces that are attractive to osteoblasts, regardless of the chemical nature of the polymer brush. This is interesting, in particular because the degree of mineralization of the PDMAEMA brush is much lower than of the PMAA brush. In summary, the current study focuses on PMAA and PDMAEMA brushes for calcium phosphate mineralization. IR spectroscopy, AFM, and CA measurements (Figures 2, 3, and 4) show that rather uniform polymer brushes can be grown from surfaces via photochemically initiated polymerization,37 even though in the case of the PMAA brushes protrusions are visible in AFM. As the nature of these is currently unclear, it is not straightforward to discuss their role in mineralization. Mineralization yields surfaces with quite different levels of inorganic deposits, depending on the chemical nature of the polymer brush. SEM, EDXS, and IR and Raman spectroscopy (Figures 5, 6, and Table 1) suggest that the PMAA brushes exhibit a much higher mineralization level than the PDMAEMA brushes. This is consistent with previous reports, indicating that nucleation and growth on anionic surfaces is favored over cationic surfaces.15,19,53 Finally, cell viability tests (Figure 7) show that (i) the cationic PDMAEMA brush is a somewhat better substrate for cell growth than the PMAA 3758
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Given the low pH during mineralization, the formation of OCP and/or traces of DCPD is likely6,54 although Raman spectroscopy cannot unambiguously rule in or out the presence of either phase. We therefore speculate that the local pH in the polymer films is higher than in the bulk solution, consistent with an earlier publication suggesting a pH gradient in a polymer gel.60 Further work is however necessary to confirm local pH gradients or changes within the films.
Figure 7. Extinction from MTT assay vs surface type. Numbers above the columns are numbers of living cells/cm2 calculated from extinction data after 3 days of incubation. Inset is a representative fluorescence micrograph of cells attached to a PMAA surface. Error bars in the three columns on the right-hand side of the graph are too small to see.
brush and (ii) that the mineralized brushes exhibit cell viabilities on the order of the control substrate. This suggests that mineralization of polymer brushes is a simple yet effective method for improving the cellsurface contact. Figure 7 clearly shows that the calcium phosphate deposit on the surfaces is a key step in improving osteoblast adhesion. In accordance with previous work,1517 the current data suggest that the degree of mineralization of the PDMAEMA brush is much lower than that of the PMAA brush. In spite of this, the cell viabilities of both mineralized brushes are roughly identical. This suggest that polycation/calcium phosphate hybrid materials could indeed also be interesting biomaterials, consistent with an earlier report on poly(ethylene imine)/calcium phosphate hybrid materials.60 It must be noted here that the characterization of such tiny amounts of inorganic material is a marvelous challenge; it is thus not straightforward to clearly identify the calcium phosphate phase on the brushes. From Raman spectroscopy, we nevertheless conclude that in both cases (PMAA and PDMAEMA brushes) predominantly HAP forms. EDXS suggests that the HAP phase is nonstoichiometric, similar to a study by Bertoni et al., who have shown that increasing amounts of poly(acrylic acid) reduce the Ca/P ratios from 1.67 (pure HAP) to 1.56.61 A similar effect can also be postulated for the current situation, where locally (on the surface where the mineralization occurs) the PMAA concentration is very high, leading to a low Ca/P ratio of 1.56 (calcium-deficient HAP). Alternatively, it may also be possible that the calcium phosphate mineral layer is a mixture of two phases, similar to an earlier study by Shkilnyy et al.60 For example, Raman spectroscopy suggests that OCP or a HPO42-containing calcium phosphate species is also present on the surface. Surprisingly, Raman spectroscopy does not provide evidence of DCPD formation on the PDMAEMA brushes, although the experimental conditions (pH ≈ 5.5) should at least partly favor DCPD formation. Nevertheless, the Ca/P ratio obtained from EDXS (1.02) suggests that the precipitate is calcium-deficient compared to the samples grown on PMAA. This suggests that on the PDMAEMA brushes a mixture of different calcium phosphates with different Ca/P ratios is present, some of which may contain the HPO42 ion, as suggested by Raman spectroscopy.
’ CONCLUSION Functional soft surfaces and interfaces are major factors controlling key processes of (biological and pathological) mineralization such as nucleation, growth, growth kinetics, chemical composition, crystal shape, crystal organization, crystal phase, and crystal size of, for example, calcium phosphate.6,11,14,15,1821,23 The current study shows that (1) surface-initiated polymerization leads to uniform and pH-responsive surfaces, (2) not only polyanionic but also polycationic surfaces (mineralized or not) are interesting substrates for osteoblast cultivation, and (3) the mineralization with calcium phosphate greatly enhances the cell adhesion and growth. The study therefore suggests that surfaceinitiated polymerization/mineralization is a promising approach for the modification of surfaces in contact with biological hard tissue. ’ AUTHOR INFORMATION Corresponding Author
*Tel ++49 (0)331 977 5773, e-mail
[email protected].
’ ACKNOWLEDGMENT We thank A. Heilig, G. Wienskol, and J. Hartmann for help with AFM, Raman spectroscopy, and SEM/EDX, respectively. The Ludwig-Boltzmann-Institute, Vienna/Austria, is acknowledged for the donation of the preosteoblasts. The Max Planck Institute of Colloids & Interfaces and the University of Potsdam are thanked for financial support. ’ REFERENCES (1) Du, C.; Moradian-Oldak, J. Biomed. Mater. 2006, 1, R10. (2) Moradian-Oldak, J.; Fan, Y. In Biomimetic and Bioinspired Nanomaterials; Kumar, C. S. S. R., Ed.; Wiley: Weinheim, 2010; p 41. (3) Ishizaki, T.; Teshima, K.; Lee, S. H.; Masuda, Y.; Saito, N.; Takai, O. In Biomimetic and Bioinspired Nanomaterials; Kumar, C. S. S. R., Ed.; Wiley: Weinheim, 2010; p 303. (4) Gupta, H. S. In Biomimetic and Bioinspired Nanomaterials; Kumar, C. S. S. R., Ed.; Wiley: Weinheim, 2010; p 511. (5) Wischerhoff, E.; Badi, N.; Lutz, J. F.; Laschewsky, A. Soft Matter 2010, 6, 705. (6) Schweizer, S.; Taubert, A. Macromol. Biosci. 2007, 7, 1085. (7) Mann, S.; Comptom, R. G.; Davies, S. G.; Evans, J. Biomineralization: Principles and Concepts in Bioorganic Material Chemistry; Oxford University Press: Oxford, 2002. (8) Gorna, K.; Munoz-Espi, R.; Gr€ohn, F.; Wegner, G. Macromol. Biosci. 2007, 7, 163. (9) Shkilnyy, A.; Friedrich, A.; Tiersch, B.; Sch€ one, S.; Fechner, M.; K€otz, J.; Schl€apfer, C. W.; Taubert, A. Langmuir 2008, 24, 2102. (10) Shkilnyy, A.; Sch€one, S.; Rumplasch, C.; Uhlmann, A.; Hedderich, A.; Taubert, A. Colloid Polym. Sci. 2011, 289, 881. (11) Shkilnyy, A.; Brandt, J.; Mantion, A.; Paris, O.; Schlaad, H.; Taubert, A. Chem. Mater. 2009, 21, 1572. (12) Huang, Z.; Newcomb, C. J.; Bringas, P.; Stupp, S. I.; Snead, M. L. Biomaterials 2010, 31, 9202. 3759
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