Article pubs.acs.org/journal/abseba
Bioinspired Deposition-Conversion Synthesis of Tunable Calcium Phosphate Coatings on Polymeric Hydrogels Jacqueline L. Harding and Melissa D. Krebs* Department of Chemical and Biological Engineering, Colorado School of Mines, 1613 Illinois Street, Golden, Colorado 80401, United States S Supporting Information *
ABSTRACT: Inspired by natural mineralization processes, here we present the stepwise mineralization of hydrogels with synthetic control over the amount of deposited CaPi and selective tuning of the coating composition. Alternate immersion of the hydrogel at 3 min intervals in calcium salt (Ca2+) and inorganic phosphate (Pi) solutions under mild aqueous conditions results in the layer-by-layer deposition of a precursor CaPi polymorph, dicalcium phosphate dihydrate (DCPD, Ca/Pi 1.12 ± 0.07), as a surface coating. Successive immersion cycles were shown to linearly increase the amount of deposited Ca2+ and Pi ions over 20 cycles enabling direct control over the mineral coating density and crystal morphology. Conversion of the DCPD coating to apatite (CaPi 1.61 ± 0.02) is induced by aqueous hydrolysis at physiological temperature and pH (7.4, 37 °C, 5 days). After conversion, the apatite coating density was found to correlate with the amount of mineral initially deposited as DCPD, indicating this approach to mineralization imparts simultaneous synthetic control over the coating composition and density on the hydrogel substrate. Mineralized coatings were characterized by XRD, ATR-IR spectroscopy, SEM-EDX, and quantitative analysis of Ca2+ and Pi ions. Supplementation of the conversion solution with Ca2+, Pi, SBF, F−, or citrate ions results in apatite coatings exhibiting variations in chemical composition and morphology. In the presence of added Ca2+ ions and SBF, an increase in Ca2+ content of the coating is observed, and the resulting particles exhibit growth as plates and petal like clusters, respectively. Conversion with F− ions results in the formation of spherical F-apatite particles that exhibit clearly resolved peaks in the XRD pattern. Citrate ions were found to restrict the growth of apatite particles. The described deposition-conversion approach overcomes longstanding limitations in CaPi-based biomaterials as a versatile method for the predictable and tunable synthesis of CaPi coatings of preformed biopolymer substrates. KEYWORDS: bioinspired mineralization, calcium phosphate, hydrogel, composites, coatings
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INTRODUCTION
The deposition of CaPi coatings onto substrates for biomedical applications is a well established field and is the subject of numerous review articles.3,5,10,11,17,21−23 Robust titanium orthopedic implants employ high-temperature thermal spraying and vapor deposition approaches to produce uniform hydroxyapatite (HA) coatings.6,7 However, for the preparation of composite synthetic bone graft materials constructed from polymeric substrates, milder mineralization approaches are needed to prevent substrate degradation.17,24,25 As a result, aqueous mineralization mediums that are close to physiological pH and temperature conditions are preferred. The extended immersion of substrates in ion-rich solutions formulated as simulated body fluid or metastable calcium phosphate solutions have been extensively utilized as biomimetic approaches for
The deposition of bioactive calcium orthophosphate (CaPi) coatings onto orthopedic implants results in materials with improved capacities for biointegration and extended lifetimes of use.1−4 Early CaPi materials were titanium implants functionalized with a deposited hydroxyapatite coating,5−7 and now there are numerous commercial CaPi formulations available on the market as void fillers and synthetic grafting materials.8−10 Inspired by the composite nature of hard tissues, mineralized hydrogels are interesting materials to explore as synthetic bone grafts.11−13 Although hydrogels are well-established for their versatility as cellular growth templates,14 the regenerative potential of hydrogels to be used for osteoconduction has been shown to be enhanced with the addition of CaPi mineral phase.15 The composition, morphology, and resorbability of CaPi materials are known to be contributing factors to the efficacy of CaPi materials in regenerative applications.16−18 To date, the efficacy of synthetic graft materials has yet to rival the efficacy of natural bone grafts.19,20 © XXXX American Chemical Society
Special Issue: Tissue Engineering Received: May 5, 2017 Accepted: June 1, 2017 Published: June 1, 2017 A
DOI: 10.1021/acsbiomaterials.7b00280 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
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Figure 1. Mineralization of polymeric hydrogels. (A) Preparation of poly(vinyl alcohol) (PVA) hydrogel by physical cross-linking using a cyclic freezing/thawing approach. (B) Bioinspired mineralization of PVA hydrogel by alternate immersion in Ca2+ and Pi salt solutions under ambient conditions. Atomic layer deposition by repeat immersion cycles facilitates the layer-by-layer deposition of DCPD as an initial coating. Hydrolytic conversion of the coating to apatite is achieved by submersion of the DCPD-mineralized PVA hydrogel in aqueous Tris buffer solution at pH 7.4, 37 °C.
mineralization of materials.26−31 However, the lengthy reaction times, low deposition rates, and limited synthetic control over the resulting coatings obstruct practical application for the formulation of robust synthetic graft materials.21 Alternative wet chemical approaches to the in situ synthesis of apatite coatings have explored electrophoretic deposition, sol−gel chemistry, ionic diffusion, dip coating, and alternate soaking methods.21,23,32 In each instance, the approaches were effective in the deposition of CaPi polymorphs; however, synthetic control at the interface regarding chemical composition, particle morphology, and homogeneous dispersion of the resulting CaPi phase remain a critical barrier. Recently, we developed an approach to the preparation of composite hydrogels where the mineral phase incorporated within a hydrogel is selectively converted to biomimetic apatites by exposure to aqueous mediums under physiologically mimetic conditions.33 Inspired by natural apatite biosynthesis, a precursor CaPi polymorph, Dicalcium phosphate dihydrate (DCPD), encapsulated as a dispersion in the hydrogel matrix, is transformed to ionically substituted apatite by aqueous hydrolysis. Although the use of encapsulated CaPi materials was shown to promote cellular attachment on the surface of the hydrogels, ideally the formulation of synthetic graft materials would more closely resemble the composition of native hard tissues with the outermost surface being apatite. There is a need for the development of a mild wet chemistry approach for the rapid and selective synthesis of apatite coatings on polymeric hydrogels.23 An ideal approach to mineralization of hydrogels would provide a synthetic handle for the controlled deposition of coatings based on chemical composition, particle morphology, and thickness of the deposited CaPi layer. Previous studies have demonstrated that the surface properties of the substrate play a
significant role in directing the properties of the resulting coating. 34−36 In particular, surfaces functionalized with carboxyl, hydroxyl, amine, and phosphate groups are shown to promote nucleation of CaPi phases and direct particle growth on the surfaces of the substrate.12,34,37−40 The controlled deposition of surface coatings by atomic layer deposition (ALD) is a versatile method for producing uniform two component coatings initially utilized in the fabrication of semiconductors and more recently expanded to biomaterial applications.41,42 ALD is advantageous because the alternating exposure to the precursors in the gaseous or liquid state results in the layer by layer growth of coatings that is directed by the nature of the surface precursor interactions in a self-limiting way terminating when all reactive sites are occupied enabling coatings on large or complex substrates.43−45 The coating thickness is then modified based on the number of deposition cycles the substrate is subjected to. Enabling the formulation of materails with properties that can be tuned on an application specific basis. Traditionally, in the synthesis of CaP coatings apatite is the target product, and thus manipulation of the soak solutions to mimic the stoichiometric ratio of apatite at Ca/Pi 1.67, pH 7.4, and reaction temperatures of 37 °C are utilized.46−49 However, there are significant limitations to this approach particularly for ALD synthesis based on the slow rate of apatite nucleation and the preference for the depositon of biphasic compounds at a 1:1 ratio.41 Accordingly, we hypothesize that the deposition of the CaPi polymorph DCPD is favored for alternate immersion synthesis approaches based on the Ca/Pi of 1.0 and the established rapid precipitation in aqueous solutions at room temperature when the pH is adjusted between 4 and 6, eq 1. To achieve the desired apatite coatings, similar to biomineralization mechanisms, we propose the conversion of the depositied B
DOI: 10.1021/acsbiomaterials.7b00280 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
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mM K2HPO4, 0.1 M citric acid, 0.025 M NaF, or simulated body fluid (SBF). A 1x SBF solution was prepared with an ionic composition similar to that of human plasma in a 50 mM Tris buffer solution with added ionic species: 142 mM Na+, 5.0 mM K+, 1.5 mM Mg2+, 2.5 mM Ca2+, 103 mM Cl−, 2.7 mM HCO3, 1 mM HPO42−, 0.5 mM SO42−. The final solution was adjusted to pH 7.4 with 1 M HCl. Physical Methods for Characterization of CaPi Coatings. The CaPi phases deposited on PVA hydrogels were determined by powder X-ray diffraction (XRD) (Phillips X’pert) and Fourier Transform Infrared Spectroscopy (FTIR, Nexus 470 e.s.p.). Samples for analysis were prepared by evaporative dehydration under ambient conditions. Samples for XRD were analyzed over the 2 theta range of 10° to 60° with a step size of 0.02 degrees with Cu−K radiation (λ= 1.54060 Å). ATR-FTIR analysis of the CaPi coatings were investigated over the range of 550−4000 using an ATR accessory (Specac, Golden Gate) equipped with a germanium crystal. The mineral morphology and chemical composition of the CaPi -PVA hydrogels before and after hydrolysis were examined by scanning electron microscopy (Quanta F.E.I 600) equipped with energy-dispersive X-ray spectroscopy (SEMEDX). Samples were mounted on conductive tape and sputtered with gold. The accelerating voltage was set to 20 kV with a spot size of 4 units and visualized using both standard secondary electron and backscatter electron detectors. Line scans of the elemental composition ratios were acquired via EDX with a spot size of 4. Determination of Ca2+ and Pi Incorporation. Bulk PVA hydrogels prepared as previously described were transformed into 6 mm × 4 mm cylindrical punches that were subsequently coated with CaPi for 1−50 deposition cycles. For this study, the coated hydrogels from each sample set were split into two groups. Group 1 hydrogels were reserved for immediate analysis by dissolution of the coating in 0.5 mL of 1 M HCl and subsequently diluted to 4 mLwith DI water. Group 2 CaPi -PVA hydrogels were subjected to conversion treatment in 100 mM Tris buffer for 5 days at 37 °C, then dissolved for analysis as described for group 1 samples. The Ca/Pi value is determined by taking the total amount of Ca2+ detected and dividing it by the Pi content. Quantification of Phosphate. Colorimetric determination of phosphate concentration in the digestion solutions was determined by the formation of a phosphomolybdate complex and measurement by UV−vis spectroscopy at 390 nm. A 0.2 mL aliquot of the analyte solution was reacted with 1.6 mL of acetone-acid-molybdate (AAM) solution. The AAM solution was prepared by mixing 25 mL of 10 mM ammonium molybdate tetrahydrate with 25 mL H2SO4 and 50 mL of acetone. After reacting for 5 min, 0.16 mL of 1 M citric acid was added to the analyte−AAM solution. A standard curve between phosphate concentrations of 1 mM to 2.75 mM was prepared. Analyte solutions outside of this range were diluted with a 1:10 ratio. The resulting phosphate content supplied by the coating was correlated to mol mm−2. Quantification of Calcium. The Ca2+ content of the coatings was determined by compleximetric titration with EDTA. To prevent precipitation of CaPi in solution as the pH is shifted toward alkaline conditions, we utilized a back-titration approach. In brief, 1 mL of analyte was added to 10 mL of standardized 5 mM EDTA solution. The pH was subsequently increased to 10 by the addition of 1 mL of ammonium buffer, followed by the addition of 0.2 mL of eriochrome black T (EBT) indicator (0.87 mM EBT in ethanol). Standardized 5 mM MgCl2 solution was titrated into the EDTA solution until the end point was reached by turning the solution from blue to purple. The Ca2+ content of the analyte solution was determined by subtracting the amount of Mg2+ added from the amount of EDTA added (mol of EDTA − mol of Mgtitrant = mol of Caanalyte). Statistical Analysis. Statistical analysis for significance was computed using the student’s t test (n = 4) utilizing Microsoft Excel as a software platform.
DCPD coating to apatite by aqueous hydrolysis according to eq 2. Utilizing this approach, synthetic control over the ionic composition and morphology of the resulting apatite coatings can be imparted from a single versatile precursor. pH 2 − 6, 37 ° C
Ca 2 + + HPO4 2 − ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯→ CaHPO4 ·2H 2O
(1)
pH 7.4, 37 ° C
5CaHPO4 ·2H 2O ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯ → Ca5 − xH 2x 2+ 2− − Y:Ca , HPO4 , F , SBF, citrate
(PO4 )3 (OH)1 − Y (Y) + 2PO4 3 −
(2)
Here, we report a bioinspired approach to the two-step deposition-conversion of tunable apatite coatings on the surface of poly(vinyl alcohol) (PVA) hydrogels (Figure 1). An ALD approach to the deposition of an initial DCPD coating is described for the layer-by-layer growth of coatings on the surface of the hydrogel substrate. The effect of sequential immersion cycles is explored based on the composition determined by XRD, the morphology of the deposited crystals visualized by SEM, and the quantification of ionic deposition of Ca2+ and Pi with each immersion cycle. The transformation of the DCPD coating was induced by hydrolysis resulting from immersion of the DCPD-PVA materials in aqueous buffers under physiological mimetic conditions at pH 7.4 and 37 °C. Variations in the ionic composition and particle morphologies of the resulting apatite coatings were explored by the introduction of solution additives (Ca2+, Pi, F−, citrate ions) or the use of SBF as the hydrolysis medium. The CaPi coatings deposited on the hydrogel substrate are characterized for composition, morphology, and Ca/Pi ratio after initial deposition and conversion utilizing XRD, ATR-IR, SEM, and quantitative analysis of Ca2+ and Pi ionic content.
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EXPERIMENTAL SECTION
Preparation of Physically Cross-Linked PVA Hydrogels. Hydrogel substrates of 10 wt % PVA (30−50 kDa, Sigma) were prepared by dissolving 20 g of PVA in 180 g of DI water (treated at 18mΩ) under constant stirring at 75 °C. The resulting solution was aliquoted (5 mL) into circular polystyrene molds (35 mm diameter by 6 mm height). Physical cross-linking of the PVA solution to form the hydrogel was achieved by cyclic freezing and thawing of the PVA solution: the PVA was treated for 5 cycles of freezing at −80 °C for 24 h followed by thawing at 22 °C over a 6 h period. The resulting translucent hydrogels had an average height of 4 mm and were stored hydrated with DI water at room temperature until use. Deposition of DCPD Coating on PVA Hydrogels. PVA hydrogels were removed from molds and hydrated in DI water for 24 h prior to use. The hydrated PVA substrate was then immersed in 0.25 M CaNO3·4H2O (Sigma, 99%) at pH 6 for 3 min at 22 °C. Upon removal from the Ca2+ solution the Ca-PVA hydrogel was rinsed with DI water and subsequently immersed in 0.25 M K2HPO4 (Sigma, 98% +) at pH 7.5 for 3 min at 22 °C. A white precipitate was found to immediately form on the surface of the hydrogel substrate and was not removed upon rinsing in DI water. A single deposition cycle was characterized by immersion in both salt solutions with an intermediate and a final rinse. Deposition cycles on the substrate were repeated between 1 and 50 with Ca2+ and Pi salts refreshed every 10 cycles. Bioinspired Conversion of CaPi Coating to Apatite on PVA Hydrogels. The CaPi -PVA hydrogels were submerged in 60 mL of 100 mM Tris buffer (pH 7.4) for 5 days at 37 °C. The pH of the 100 mM Tris buffer (Trizma base, Sigma) reaction solution was monitored for the duration of the reaction and adjusted back to pH 7.4 as needed with 1 M NaOH (Sigma). Conversion with Solution Additives. In experiments examining the influence of ionic additives on the maturation product, 100 mM Tris buffer (pH 7.4) solutions were prepared with 5 mM CaNO3·4H2O, 5
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RESULTS AND DISCUSSION Deposition-Conversion Synthesis of Apatite Coatings on PVA Hydrogels. Physically cross-linked PVA hydrogels C
DOI: 10.1021/acsbiomaterials.7b00280 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
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Figure 2. Mineralization of PVA hydrogel by bioinspired deposition-conversion treatment. (A) SEM image at 100× magnification of CaPi/PVA hydrogel cross section after 20× deposition cycles. (B) Ionic quantification of Ca2+ and Pi ions deposited on the PVA hydrogels between 1 and 50 deposition cycles. (C) Top surface SEM images at 2000× magnification of initially deposited CaPi coatings (top row) and the conversion products (bottom row) after 1, 4, 12, and 20 deposition cycles. The corresponding Ca/Pi values are presented for each coating.
were prepared by cyclic freezing and thawing of the polymer solution.50 ALD method is used to deposit an encapsulating CaPi coating on the hydrogel surface by alternate immersion in calcium and phosphate salt solutions, as shown in Figure 1. A deposition cycle is complete after reaction with both salt solutions and final rinse is applied. Consecutive deposition cycles resulted in the layer-by-layer growth of CaPi particles on the surface of the substrate (Figure 2). As shown in Figure 2a, the mineral is deposited exclusively on the outer surface of the hydrogel as a coating and does not penetrate the interior of the hydrogel matrix. Quantitative determination of the Ca2+ and Pi ions incorporated with each deposition cycle indicate an increase in the amount of ionic incorporation up to 20 cycles (Figure 2b). Increasing the amount of immersion cycles out to 50 does not result in a significant increase in the amount of deposited Ca2+ or Pi ions. Thus, the ionic deposition reaches a maximum after 20 cycles. Analysis of the crystal growth based on deposition cycles on the hydrogel surface by SEM in Figure 2c revealed that an initial deposition cycle resulted in the deposition of small nucleating clusters which grew into welldefined crystalline platelike particles as the number of deposition cycles increased, with a corresponding increase in the density of particle surface coverage. The composition of the CaPi coating with each consecutive deposition cycle was investigated by XRD (Figure 3). The ATR-IR spectra (Figure S1) and the diffraction patterns of the initial CaPi-coated PVA substrate (Figure 3a) indicate the deposited mineral phase is DCPD. Peaks associated with DCPD are observed after a single deposition cycle. The
Figure 3. XRD patterns of (A) mineral initially deposited by alternate immersion and (B) mineral after conversion for 5 days in Tris buffer at pH 7.4, 37 °C, with 1, 4, 12, 20 deposition cycles applied.
presence of an additional broad diffraction peak at 20° on the XRD pattern is attributed to the PVA hydrogel. With each consecutive cycle, as the density of the coating thickness is increased, the appearance of the PVA diffraction peak diminishes after 4 cycles and is completely removed after 12 cycles. Notably, the appearance of the diffraction peak at 11.5 degrees frequently observed with DCPD was not initially D
DOI: 10.1021/acsbiomaterials.7b00280 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
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resulting diffraction pattern peaks identified as NaCl. Although the two-step approach developed in this work requires an initially rapid synthesis followed by a lengthy conversion and is more complex than existing methods of biomaterial mineralization, this method offers a synthetic handle that imparts superior control over the mineral density of the coating and predictable synthesis of tunable CaPi coatings. Thus, the controlled synthesis of an apatite coating under mild aqueous conditions on polymeric hydrogels is successfully demonstrated by a two-step deposition-conversion approach bioinspired of natural biomineralization processes. The hydroxyl groups of nonionic hydrogels including PVA are effective materials for promoting nucleation and growth of CaPi coatings by attracting precursor ions to the hydrophilic material surface.49 Previous research endeavors have explored the deposition of apatitic phases onto the surface of PVA by alternate immersion approaches, however these methods aimed to directly synthesize apatite on the material surface through tuning the stoichiometry of the salt solutions and reaction temperatures, resulting in coatings with low apatite depositions and difficulty in tuning the composition and morphology of the resulting particles.46,51 The method developed here differs significantly from previous approaches to hydrogel mineralization in that the mineral phase initially targeted by alternate immersion deposition synthesis is DCPD, not apatite. DCPD is an ideal apatite precursor because it is the polymorph that most readily forms under aqueous conditions, the Ca/Pi value of 1 promotes ALD, and it readily undergoes transformation to apatite when exposed to an aqueous environment under physiological conditions. Although the initial deposition of DCPD is rapid and can be achieved within a 1−3 h period depending on the number of deposition cycles applied, the transformation to apatite at pH 7.4 is a slow process that requires the extended immersion of the substrate over a 5 day period. By comparison, other approaches developed for hydrogel mineralization necessitate lengthy immersion times from days to weeks. The advantage of our approach in comparison to these more traditional one-step immersion methods is that through the use of an initial deposition step we can directly control the mineral content of the final coating, independent of the immersion duration, resulting in the formation of apatite. Conversion with Ionic Additives. Apatite formed by the aqueous hydrolysis of DCPD is subject to modifications resulting from the ionic composition of the conversion solution.52 This effect is analogous to the formation of biomineralized apatite, which is found to incorporate ions found in surrounding body fluids. This results in the formation of distinct hard tissue compositions characteristic of enamel, dentin, and bone minerals.53 Here we aim to investigate the ability to selectively tune the ionic composition of the resulting apatite coatings by examining the influence of ionic additives to the hydrolysis solutions. Conversion in the Presence of Excess Calcium and Phosphate. The formation of apatite coatings up to this point has relied on the reorganization of Ca2+ and Pi ions initially deposited on the surface as DCPD. Here we aim to investigate the influence of Ca2+ and Pi supplemented into the conversion solution on the transformation of DCPD to apatite particles. In Figure 4 the XRD patterns indicate the initial DCPD coating after 20 immersion cycles was successfully converted to apatite in the presence of either excess Ca 2+ or P i ions. Supplementation with Ca2+ and Pi solutions results in
evident as a coating on the surface. Further investigation of the coating by inducing a 90-degree rotation of the CaPi-coated hydrogel resulted in the appearance of the peak, shown in Figure S2 and greatly reduced the intensity of the remaining peaks. These results are suggestive of the oriented growth of minerals deposited on the surface of the hydrogel. Conversion of the DCPD coating to biologically relevant apatite was induced by aqueous hydrolysis according to eq 2. The DCPD/PVA hydrogel is immersed in Tris buffer maintained at pH 7.4 and 37 °C for 5 days. A transformation in the coating morphology is observed by SEM (Figure 2c) and confirmed as apatite by XRD patterns (Figure 3b) and ATRFTIR spectra (Figure S3). The diffraction patterns of conversion coatings between 1 and 20 cycles indicated the complete conversion of DCPD to apatite (Figure 2b). The diffraction pattern after 1 deposition cycle followed by conversion was dominated by the appearance of the PVA peak with trace detection of apatite diffraction. Increased deposition cycles resulted in the disappearance of the PVA peak and enhanced resolution of the peaks attributed to apatite. SEM images of apatite particle growth revealed the formation of fine bladed particles on the surface of the hydrogel. Apatite coating resulting from 1 deposition cycle lacked the formation of defined particle growth on the surface and the pore structure of the hydrogel substrate remained evident. Increased deposition cycles resulted in the formation of an apatite coating with increasing surface density and the formation of defined apatite particles on the surface. According to eq 2, the conversion of DCPD (CaHPO4) to apatite (Ca5(PO4)3OH) results in a change in the Ca/Pi value of the polymorphs from 1 to approximately 1.67 for stoichiometric apatites, where the change in Ca/Pi value results from the loss of Pi ions present in the initial DCPD polymorph. Consistent with this, we determined that for coatings with 20 deposition cycles, 101 ± 4% of Ca2+ ions and 77.9 ± 5% of Pi ions are retained from the initial DCPD coating as the apatite conversion product (Table 1). As a result, the apatite product was determined to have a Table 1. DCPD Coating Conversion to Apatite in the Presence of Ionic Additives additivea none Ca2+ Pi SBF F− Citrate SBF, Citrate
% Cab 101 117 100 115 102 85.3 91.7
± ± ± ± ± ± ±
4 4 5 2 2 4 4
% Pib 77.9 80.2 73.2 87.2 72.1 60.5 57.4
± ± ± ± ± ± ±
Ca/Pi 5 5 4 5 3 8 4
1.61 1.75 1.59 1.60 1.64 1.60 1.83
± ± ± ± ± ± ±
0.02 0.1 0.02 0.1 0.3 0.1 0.1
a Conversion in Tris buffer pH 7.4, 37 °C. b% ions deposited initially as DCPD.
Ca/Pi value of 1.61 ± 0.02. Apatite products formed by aqueous hydrolysis are observed to result in nonstoichiometric Ca/Pi values and furthermore are consistent with observations of Ca/Pi values found in naturally mineralized hard tissues. For comparison with established mineralization methodologies, we investigated the widely accepted approach to the mineralization of biomimetic apatite by immersion in SBF for 5 days at pH 7.4 and 37 °C. The resulting hydrogel retained the original transparency of the PVA hydrogel. Analysis of the SBFsoaked PVA hydrogel by XRD (Figure S4) indicated that apatite particles did not form on the hydrogel, with the E
DOI: 10.1021/acsbiomaterials.7b00280 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
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Figure 4. (A) SEM photomicrographs and (B) XRD spectra of conversion products when ionic additives (Ca2+, Pi, SBF, or F−) are present in the hydrolysis solution.
apatite when immersed in SBF. Observations of the resulting apatite particles by SEM (Figure 4b) reveal the formation of petal-like particles arranged in clusters on the surface of the hydrogel substrate, suggesting that ionic supplementation of the conversion solution enables control over the morphologies of the resulting apatite coatings. EDX spectra of these materials is presented in Figure S7. Quantitative analysis of the Ca2+ and Pi content of the coatings indicates a 15% and 10% increase, respectively, in ionic incorporation of the coatings compared to the conversion products in the absence of solution ionic supplements. As a result, the final Ca/Pi value of 1.60 + 0.1 was determined to correlate with additive-free reaction. Notably the incorporation of Ca2+ ions in SBF conversion solutions is equivalent to the amount incorporated in Ca2+ supplemented solutions, indicating there is a threshold for mineral growth as a surface coating. However, the corresponding increase in Pi ionic incorporation for SBF solutions and not Ca2+ ions despite solution availability in both instances is suggestive that Pi incorporation may also be concentration-dependent. Conversion in the Presence of Fluoride Ions. The importance of fluoride incorporation into the apatite lattice is well-documented for dental applications, where fluorinated apatite (F-apatite) improves resistance to the degradation of enamel that leads to dental carries.54 Here we demonstrate the direct synthesis of F-apatite coatings by the conversion of the DCPD coating in hydrolysis buffer supplemented with F− ions.55 DCPD hydrolysis with F− ions added to the conversion solution resulted in the formation of small mineral clusters deposited on the hydrogel surface determined to be F-apatite particles (Figure 4). The XRD pattern of the F-apatite particles indicated the formation of clearly resolved peaks that are consistent with the formation of crystalline hydroxyapatite particles synthesized at high temperatures. Elemental analysis of the coating by accompanying EDX spectroscopy confirmed the incorporation of F− into the apatite coating (Figure S8). Quantitative analysis of the F-apatite coatings demonstrated a Ca/Pi value of 1.64 ± 0.3, with the incorporation of Ca2+ remaining consistent at 102 ± 4% of the initial coating, but the Pi content decreased to 72.1 ± 3% of the initial coating. The
diffraction patterns that exhibit analogous peaks to the diffraction patterns observed for the conversion of DCPD to apatite in Tris buffer, indicating that the presence of ionic supplements in solution does not interfere with the hydrolysis of DCPD to apatite. Observation of the resulting apatite coatings by SEM (Figure 4) indicate that supplementation with Ca2+ or Pi ions influences the particle morphologies when compared to nonsupplemented buffer solution. In the presence of excess Ca2+ ions, the resulting apatite particles are formed as distinct platelike particles on the surface of the hydrogel. The apatite conversion product had a 17% increase in the amount of lattice Ca2+ ions compared to conversion without supplemental Ca2+. An increase in the Pi content of the apatite product was not observed despite the solution availability of up to 20% initially incorporated as DCPD, resulting in Ca-rich apatite coating with a Ca/Pi value of 1.75 ± 0.1. (Figure S6 shows EDX spectra of these materials.) Conversely, supplementation with Pi ions did not result in a corresponding increase in Pi content in the apatite product. Conversion to apatite in the presence of substituted Pi ions resulted in a decrease in the amount of incorporated Pi ions at 73%, while still utilizing 100% of the available Ca2+ ions. This resulted in the formation of Ca-deficient apatite with a Ca/Pi value of 1.59 ± 0.02 and is consistent with observations of apatite formed without ionic supplements in the hydrolysis solution. The observed particles deposited on the surface were irregularly shaped. Taken together, these results suggest that the conversion to apatite is limited by the availability of Ca2+ ions while the presence of excess Pi ions does not significantly influence the composition of the resulting apatite particles. Conversion in Simulated Body Fluid. Soaking materials in SBF, which contains both Ca2+ and Pi ions, is a classic approach to the deposition of biomimetic apatite coatings on the surfaces of hydrogels.26 To explore the effect of conversion to apatite when Ca2+ and Pi ions are both available as supplements in the hydrolysis solution, we utilized SBF as the hydrolysis solution. The diffraction pattern (Figure 4a) indicates the successful conversion of the DCPD coating to F
DOI: 10.1021/acsbiomaterials.7b00280 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
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lacked defined crystalline particle morphologies previously observed in the absence of citrate ions. Instead, the resulting CaPi coatings when citrate was added to either Tris or SBF exhibited restricted crystal growth with flattened surface morphologies and no distinguishable individual particles (Figure 5). The inclusion of citrate anions to the hydrolysis buffer resulted in the conversion of the coating to an apatite phase, however as shown by the diffaction patterns the resolution of characteristic peaks is reduced, Figure 5b. Further analysis of the resulting coatings by ATR-FTIR spectroscopy indicated the formation of apatite particles based on the presence of sharp peak at 1047 cm−1 attributed to the apatite P−O stretch. Additional peaks present between 1200 and 1600 cm−1 indicate the association of citrate ions with the apatite coating (Figure 5c). These peaks were not observed in apatite conversion products formed without citrate ions. Quantification of the apatite coating formed in the presence of citrate ions demonstrated a decrease in the amount of both incorporated Ca2+ and Pi ions (Table 1). Conversion in SBF with added citrate ions resulted in an increase in the amount of incorporated Ca2+ ions but an equivalent amount of Pi ions when compared to conversion in Tris buffer solutions. This resulted in the variation of the Ca/Pi values for each conversion product 1.60 ± 0.12 and 1.83 ± 0.10 for Tris and SBF conversions, respectively. Citrate molecules are found to direct the growth of apatite particles by restricting both the amount deposited and the three-dimensional particle growth, leading to flat particle morphologies without indication of individual particles. Similarly, in this work here the conversion of DCPD particles to apatite in the presence of citrate molecules resulted in the formation of apatite coatings with poorly defined particle morphologies and absorbed citrate molecules. The results of this study indicate that the morphologies of the resulting apatite coatings can be readily tuned by the incorporation of directing organic molecules, essential to the preparation of biomimetic apatite coatings.
formation of F-apatite results in a change in the morphology of the particles (Figure 4b). The F-apatite particles are spherical aggregates deposited on the surface of the hydrogel. These results indicate that in addition to the changing the composition of the final apatite product, the presence of F− in the conversion solution also modifies the morphology of the resulting particles. The conversion of DCPD in the presence of F− ions results in fluorination of the apatite lattice. The Ca/Pi ratio of F-apatite remained equal to unsubstituted apatite, which is consistent with the known substitution of backbone OH− ions, leaving the Ca2+ and Pi ions unaffected. The smaller ionic radius of F− ions compared to OH− ions is known to enhance crystallinity of the resulting apatite lattice by reducing the distortion along the lattice backbone.59 In accordance with this, diffraction patterns of F-apatite coatings in this study resulted in enhanced crystallinity with clearly resolved apatite peaks. In addition to improved crystallinity, F-apatite is known to be less soluble in comparison to its unsubstituted apatite counterpart.59 Conversion in the Presence of Citrate Ions. During apatite biosynthesis, small organic molecules and large proteins direct in vivo mineral growth.56,57 Citrate molecules account for 5.5% of the organic matter of bone apatite and are observed as absorbed species on the apatite.58 Through adsorption to the surface of the growing apatite mineral, the citrate molecules inhibit crystal growth perpendicular to the surface of the organic substrate.59 There is evidence to indicate that the incorporation of citrate anions results in a bridge between the growing CaPi crystals that ultimately facilitates the gradual transformation to mature bone crystals with thin platelike morphologies.60 Here, we investigated the influence of citrate ions added to the conversion solution on the resulting apatite particle morphologies (Figure 5a). Observation of the particle morphologies by SEM imaging indicated the resulting particles
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CONCLUSION In summary, we report a bioinspired method for the deposition of tunable apatite coatings onto polymeric hydrogels. Apatite coatings on the hydrogel surface are synthesized by the hydrolytic conversion of a DCPD coating initially deposited on the hydrogel substrate using a layer-by-layer growth approach. Controlled growth of deposited DCPD particles was achieved by the number of deposition cycles to which the substrate was exposed, with a linear rate of ionic deposition up to a maximum incorporation after 20 cycles. Hydrolytic conversion of the DCPD coating to apatite was successfully demonstrated by immersion of the DCPD hydrogels in aqueous buffer adjusted to mimic physiological conditions. Tuning the ionic composition of the conversion reaction solution selectively modified the chemical composition and particle morphologies of the apatite conversion coatings. Biomimetic apatite particles were successfully synthesized after immersion in SBF, which promoted the growth of well-defined particles coating the hydrogel surface. Also, fluorinated apatite coatings were prepared to mimic the composition of biosynthesized enamel. Lastly, the incorporation of citrate ions into the conversion buffer restricted the growth of apatite particles to the longitudinal directions across the substrate surface, analogous to the formation of platelike apatite particles evident in bone apatite. The described deposition-conversion approach overcomes longstanding limi-
Figure 5. (A, B) SEM photomicrographs of conversion products with citrate ions added to (A) Tris buffer and (B) SBF. (C) XRD patterns of CaPi coating conversion with citrate ions in Tris or SBF. (D) ATRIR spectra of CaPi-conversion coating in Tris buffer, Tris with citrate ions, SBF, and SBF with citrate ions. G
DOI: 10.1021/acsbiomaterials.7b00280 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
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ACS Biomaterials Science & Engineering
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tations in CaPi-based biomaterials as a versatile method for the predictable and tunable synthesis of CaPi coatings of preformed biopolymer substrates. Application-specific development of CaPi-coated biomaterials has far reaching implications in the development of implantable orthopedics with enhanced clinical activities as useful reconstructive materials.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.7b00280. XRD patterns of DCPD-PVA hydrogels with 90° of rotation applied to the PVA hydrogel substrate; XRD pattern of PVA hydrogel after extended immersion in SBF; ATR-IR figures of CaPi-PVA hydrogels comparing the initially deposited coating and after hydrolysis to apatite and comparing the conversion coating in the presence of ionic additives Ca2+, Pi, SBF, and F−; figure of EDX spectra of CaPi-PVA coatings initially deposited and after conversion in Tris and with F− and SBF additives (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Melissa D. Krebs: 0000-0002-5170-7116 Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We acknowledge financial support from the Colorado Office of Economic Development and International Trade, the Colorado School of Mines Foundation, and start-up funds from Colorado School of Mines, which supported this work.
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ABBREVIATIONS CaPi, calcium orthophosphate Pi, inorganic phosphate PVA, poly(vinyl) alcohol DCPD, dicalcium phosphate dihydrate OCP, octacalcium phosphate SBF, simulated body fluid SEM, scanning electron microscopy EDX, energy dispersive X-ray XRD, X-ray diffraction ATR-IR, attenuated total reflectance infrared spectroscopy
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REFERENCES
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DOI: 10.1021/acsbiomaterials.7b00280 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
