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The formation of an apatite coating on carboxylated polyphosphazenes via a biomimetic process
Materials Letters 61 (2007) 3692 – 3695 www.elsevier.com/locate/matlet
The formation of an apatite coating on carboxylated polyphosphazenes via a biomimetic process Justin Lee Brown a , Lakshmi S. Nair b , Jared Bender c , Harry R. Allcock c , Cato T. Laurencin a,b,d,⁎ a
Department of Biomedical Engineering, University of Virginia, Charlottesville, USA Department of Orthopaedic Surgery, University of Virginia, Charlottesville, USA c Department of Chemistry, Pennsylvania State University, University Park, USA d Department of Chemical Engineering, University of Virginia, Charlottesville, USA b
Received 25 July 2006; accepted 15 December 2006 Available online 23 December 2006
Abstract The biomimetic deposition of an apatite coating on polyphosphazene was investigated. A polyphosphazene having carboxylatophenoxy side groups was synthesized as a candidate polymer with heterogeneous nucleation sites for apatite deposition. Poly[bis(carboxylatophenoxy) phosphazene] (PCPP) was synthesized by a two-step synthetic route involving the synthesis of poly(dichlorophosphazene) (PDP) followed by nucleophilic replacement of the chlorine atoms of PDP with carboxylatophenoxy groups. The surface of the carboxylated polyphosphazene induced the formation of a dense layer of apatite on the surface after incubation in simulated body fluid at 37 °C with an ionic concentration approximately the same as that of human blood plasma. The results suggested an early precipitation of octacalcium phosphate and calcium phosphate intermediates on the surface, both of which are known to promote osteoblast differentiation and proliferation. © 2006 Elsevier B.V. All rights reserved. Keywords: Biomimetic process; Polyphosphazene; Simulated body fluid; Hydroxyapatite
1. Introduction The bone binding biomaterials that preclude the formation of fibrous encapsulation in vivo are some of the most sought after materials for orthopaedic applications [1,2]. Kukubo et al. have demonstrated that the in vivo bone binding ability of biomaterials (metallic and polymeric) can be evaluated in vitro by their ability to form bone like apatite on their surface following incubation in simulated body fluid, which has the same ionic concentration as human blood plasma [1,2]. The ability of materials to form an apatite coating on a surface depends (a) on the specific surface chemistry of the material that induces heterogeneous nucleation of apatite and (b) on the presence of a high concentration of calcium ions in the surrounding fluid [3]. Studies have shown that negatively ⁎ Corresponding author. Department of Biomedical Engineering, 400 Ray C. Hunt Drive, Suite 330, University of Virginia, Charlottesville, VA 22903, USA. Tel.: +1 434 243 0250; fax: +1 434 243 0252. E-mail address: [email protected] (C.T. Laurencin). 0167-577X/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.matlet.2006.12.020
charged surfaces are highly favorable for the nucleation of apatite in the presence of simulated body fluid, which is a supersaturated ionic solution [4]. This has been attributed to the accumulation of calcium ions on negatively charged surfaces which in turn triggers the nucleation of apatite [4]. Therefore, several processes have been developed to form negatively charged metallic and polymeric surfaces to induce bone binding ability. These include acid or alkaline hydrolysis of metal surface, such as titanium, to form a titanium oxide gel layer capable of inducing apatite nucleation [5], surface hydrolysis of polymers such as poly(α-hydroxy esters) to form hydroxyl and carboxyl groups on the surface [6,7], coating of surfaces with self-assembled monolayers terminated with polar head groups such as hydroxyl, phosphoric and carboxylic acid [8–10]. In our laboratories we have developed a unique class of polymers – the polyphosphazenes – as potential polymeric biomaterials for orthopaedic applications. Polyphosphazenes are polymers with a phosphorous nitrogen backbone and with two organic side groups attached to each phosphorus atom [11]. The synthetic flexibility of polyphosphazenes allows the design
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(Phenomenex), and calibrated versus polystyrene standards (Polysciences). 2.1. Synthesis of poly[bis(carboxylatophenoxy)phosphazene] (PCPP)
Scheme 1. Synthesis of PCPP.
and development of polymers with functional groups for specific applications. Our previous studies using various biodegradable and non-degradable polyphosphazenes have demonstrated osteocompatibility of these polymers [12–14]. The objective of the present study was to evaluate the ability of a carboxylated polyphosphazene to nucleate and deposit an apatite layer on a surface using scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) as analytical tools. 2. Experimental Solution-state NMR spectra were obtained at 298 K using a Bruker WM-360 NMR spectrometer resonating at 360.23 MHz for 1H, 145.81 MHz for 31P, and 90.56 MHz for 13C. Molecular weights were estimated using a Hewlett-Packard HP 1090 gel permeation chromatograph equipped with an HP-1047A refractive index detector, two Phenogel 10 μ linear columns
Hexachlorocyclotriphosphazene (HCCTP) (Nippon Fine Chemicals Co., Japan) was purified by recrystallization from heptane and was sublimed at 55 °C (0.05 mm Hg). The macromolecular precursor, polydichlorophosphazene, (PDCP), was prepared by a ring opening polymerization of the HCCTP at 250 °C as described previously [11]. The nucleophilic substitution of the chlorine atoms of PDCP was performed as reported earlier [15–18]. The sodium salt of propyl 4-hydroxybenzoate was prepared by the addition of propyl 4-hydroxybenzoate (118.01 g, 0.655 mol) in THF (150 mL) to a stirred suspension of sodium hydride (25.80 g, 0.645 mol) in THF (1.25 L). The reaction mixture was stirred overnight at room temperature. A solution of poly(dichlorophosphazene) (15.0 g, 0.129 mol) in THF (1 L) was added drop wise to the sodium salt solution at room temperature. After complete addition of the poly (dichlorophosphazene) solution, the reaction mixture was refluxed for 48 h. The polymer solution was then cooled, and precipitated into aqueous acid (0.02 M HCl), dried, dissolved in a minimal amount of THF and reprecipitated into deionized H2O (once) and hexanes (twice). The resultant polymer (Poly[bis (propyl 4-hydroxybenzoate)phosphazene]) (Polymer 1) was dried under vacuum to give a white fibrous polymer. 1H NMR (CD2Cl2): δ 0.92 (t, 3H), 1.67 (m, 2H), 4.10 (br t, 2H), 6.63 (dd, 2H), 7.44 (dd, 2H); 13C NMR (CD2Cl2): δ 10.75, 22.55, 66.91, 120.60, 127.50, 131.30, 154.59, 165.56; 31P NMR (CD2Cl2): δ − 19.45; Mn = 2.42 × 105, Mw = 6.79 × 105, PDI = 2.8. Polymer 1 (15.0 g, 0.0372 mol) was dissolved in THF (1 L) and added drop wise to a stirred solution of potassium tertbutoxide (58.42 g, 0.521 mol) and water (5 mL) in THF
Fig. 1. SEM showing the surface morphology of PCPP films before and after incubation in r-SBF. (A) Before incubation; (B) after 1 day;(C) after 3 days; (D) after 5 days; (E) after 7 days; and (F) after 14 days.
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Table 1 Ca/P ratio of inorganic mineral deposit on PCPP at various time of incubation Time point
Ca/P ratio
(Ca + Mg)/P ratio
24 h 3 days 5 days 7 days 14 days
2.42 1.34 1.29 1.34 1.34
2.42 1.34 1.35 1.51 1.50
(750 mL). The reaction mixture was stirred at room temperature for 20 h, at which time the white precipitate was removed via filtration, dissolved in deionized water, and was purified by dialysis against deionized water (4 days). The polymer solution was removed from the dialysis membrane and concentrated to a viscous solution under reduced pressure which was precipitated into THF yielding polymer Poly[bis(potassium carboxylatophenoxy)phosphazene] (Polymer 2) as a brittle white solid. 1H NMR (D2O): δ 6.95 (br, 2H), 7.69 (br, 2H); 31P NMR (D2O): δ −18.2. Poly[bis(carboxylatophenoxy)phosphazene] (Polymer 3) was precipitated from a 4 wt.% solution of polymer 2 in deionized water through the slow addition of 1 M HCl. The white precipitate was removed via filtration, washed with copious amounts of deionized water, and dried under vacuum. 1 H NMR (d6-DMSO): δ 6.64 (d, 2H), 7.43 (s, 2H) 12.62 (br, 1H); 13C NMR (d6-DMSO): δ 119.70, 127.15, 130.55, 153.36, 166.04; 31P NMR (d6-DMSO): δ − 19.2 (Scheme 1).
SEM. The films were sputter coated with gold and imaged using a JEOL 840A scanning electron microscope. For EDS evaluation, uncoated samples were used and were analyzed with a Princeton Gamma-Tech X-ray Spectrometer attached to the JEOL 840A. All SEM images and energy dispersion spectrums were processed using Princeton Gamma-Tech's Spirit software package. The thin layer X-ray diffraction (XRD) characterization of the mineralized films was performed on a Siemens D5000 diffractometer using Cu radiation at 40 kV/30 mA. Full scans were run over the angular range of 10° to 50° with a step size of 0.03° and an exposure time of 22 h. 4. Results and discussion In the present study, the ability of the carboxylated polyphosphazene to induce heterogeneous nucleation and growth of apatite by incubation in simulated body fluid (r-SBF) was demonstrated. r-SBF was used in this study to closely mimic in vivo conditions because it has been reported that r-SBF has ion concentrations that are equal to or close to that of human blood plasma [19]. Fig. 1A shows the surface morphology of PCPP film cast from DMF solution before incubation in r-SBF. SEM reveals a relatively smooth surface of the polymer film. Fig. 1B–F show the SEM of PCPP films after incubation in r-SBF at 37 °C for various periods of time. It can be seen that after 24 h of incubation small spherical particles of apatite were formed on the surface of the film. Previous studies using self-assembled molecules have demonstrated the ability of carboxylic groups to induce apatite nucleation possibly by chelating calcium ions [20]. It is presumed that
2.2. PCPP film preparation The PCPP was dissolved in dimethylformamide (DMF) and the solution was poured into a Teflon lined Petri dish. The polymer film was allowed to dry under vacuum at 50 °C overnight. The brittle film obtained was immersed in water to increase the flexibility and circular matrices of (10 mm diameter) were bored using a cork borer. 2.3. Simulated body fluid preparation The revised simulated body fluid, r-SBF, was prepared as described previously by Oyane et al. [19]. Fresh solutions were prepared every week and stored at 4 °C. It has been reported that r-SBF is stable for up to 4 weeks at 5 °C [19]. 3. Incubation in simulated body fluid The apatite forming ability of PCPP was evaluated by incubating the polymer films in simulated body fluid as reported earlier [2]. The films were placed in 10 mL centrifuge tubes filled with 10 mL of r-SBF and placed in a water bath at 37.5 °C. The r-SBF was changed every 24 h. At predetermined times, the films (n = 3) were removed and gently washed with ultra pure water (5 times) and dried at room temperature for 24 h. 3.1. Surface characterization of PCPP films The surface structure of PCPP films before and after incubation in revised simulated body fluid was determined by
Fig. 2. Thin layer XRD pattern of mineral deposited on PCPP film: A. after 3 days of incubation and B. after 7 days of incubation.
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the initial stages of interaction between a carboxylated surface and supersaturated SBF involves the formation of –COOCa+ or (–COOH)2Ca which is followed by interaction with phosphate ions. Studies have also shown that the presence of surface carboxyl groups could significantly increase the adhesion of a deposited apatite layer to the surface. However, biomimetic studies using polymers with carboxyl side groups indicated the low reactivity of carboxyl groups in inducing apatite nucleation. In most cases carboxylated polymer surfaces showed an induction period of more than 24 h for apatite nucleation even after using 1.5× SBF, which has higher concentration of Ca2+ compared to SBF. (1× SBF has ion concentration close to human blood plasma and 1.5× SBF has 1.5 times higher ion concentration of that of 1× SBF). Therefore, pre-treating carboxylated polymer surfaces with Ca(OH)2 or CaCl2 has been investigated to decrease the induction period by presenting a high concentration of calcium ions for chelation with the carboxyl groups [21,22]. However, in the case of PCPP we have observed nucleation of apatite within 24 h using 1 × simulated body fluid without the treatment with Ca(OH)2 or CaCl2 (Fig. 1B). This may be due to the structure of PCPP which has two carboxyl groups per repeat unit that would facilitate the rapid chelation of calcium ions. Fig. 1C shows the SEM of PCPP surface after incubation in r-SBF for 3 days at 37 °C. By 3 days it can be seen that the surface of the PCPP film is almost covered with a layer of inorganic apatite. The density of the apatite layer increased with an increase in the incubation time. Thus, multilayers of apatite were observed after incubation of the film in r-SBF for 5, 7 and 14 days (Fig. 1D–F). The surface elemental composition of the formed apatite layer on PCPP surface was evaluated using EDS (Table 1). Since the polyphosphazene contains phosphorus atoms, the EDS was performed selectively on the deposited inorganic phase on PCPP film, particularly at earlier time points such as 1 and 3 days when the surface of the polymer film was not completely covered with the apatite layer. At 24 h, the spectrum showed strong calcium and phosphorous peaks along with small peaks that denoted potassium, chlorine and sulfur. The Ca/P ratio of the apatite layer after 24 h of incubation was found to be 2.42. The high Ca/P ratio can be attributed to carboxylate bound calcium that induces further apatite nucleation. After 3 days of incubation, the Ca/P ratio decreased to ∼1.3. This can be attributed to the formation of octacalcium phosphate Ca8(HPO4)2(PO4)4·5H2O [23,24]. It has been reported previously that octacalcium phosphate has a higher probability of precipitation from r-SBF at a pH of 7.4 [19]. The EDS spectra of the apatite formed after incubation for 5 days indicated the presence of magnesium on the surface. The (Ca + Mg)/P ratio of apatite on PCPP film incubated in r-SBF for 5 days was found to be ∼1.3 indicating the formation of octacalcium phosphate. The thin layer XRD pattern of the surface corroborated the formation of octacalcium phosphate after 5 days of incubation in SBF (Fig. 2). The sharp diffraction lines appeared at 2θ = 16.8° corresponding to the (−101) plane and 2θ = 25.5° corresponding to the (002) plane, both are characteristic of octacalcium phosphate crystal [25]. However, as reported earlier on biomimetic deposition on titanium substrates, the intensity of the diffraction line (−101) at 2θ = 16.8° is significantly higher than typical triclinic OCP crystals and has been attributed to crystal growth along those direction by the influence of substrate on crystal formation. The PCPP substrate did not show any significant peaks except two small peaks near 20–21°. The (Ca + Mg)/P ratio of apatite on PCPP surface after 7 and 14 days of incubation showed a value of 1.5 indicating that the composition of the mineral is approaching that of hydroxyapatite, possibly calcium-deficient apatite or tricalcium phosphate [26]. However, the XRD pattern obtained after 7 days of incubation in simulated body fluid was found to be not significantly different from day 5 indicating the crystalline composition to be more towards that of octacalcium phosphate.
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5. Conclusions The present study demonstrates the feasibility of carboxylated polyphosphazene films prepared to heterogeneously nucleate apatite mineral on the surface. The PCPP film showed nucleation of inorganic mineral by 24 h of incubation in r-SBF at 37 °C without any pretreatment with calcium ions. The density of the deposited layer significantly increased with time of incubation in simulated body fluid. After 5 days of incubation in simulated body fluid the crystalline composition of the mineral was found to be similar to that of octacalcium phosphate. Acknowledgements The authors acknowledge the financial support from NIH grant # EB004051. References [1] T. Kokubo, Biomaterials 12 (1991) 155. [2] T. Kokubo, H. Takadama, Biomaterials 27 (2006) 2907. [3] T. Kawai, C. Ohtsuki, M. Kamitakahara, T. Miyazaki, M. Tanihara, Y. Sakaguchi, S. Konagaya, Biomaterials 25 (2004) 4485. [4] P. Zhu, Y. Masuda, K. Koumoto, Biomaterials 25 (2004) 3915. [5] L. Jonasova, F.A. Muller, A. Helebrant, J. Strnad, P. Greil, Biomaterials 25 (2004) 1187. [6] A. Oyane, M. Uchida, C. Choong, J. Triffitt, J. Jones, A. Ito, Biomaterials 26 (2005) 2407. [7] W.L. Murphy, D.J. Mooney, J. Am. Chem. Soc. 124 (2002) 1910. [8] K. Sato, Y. Kumagai, J. Tanaka, J. Biomed. Mater. Res. 50 (2000) 16. [9] H. Lin, W.S. Seo, K. Kuwabara, K. Koumoto, J. Ceram. Soc. Jpn. 104 (1996) 291. [10] M. Tanahashi, T. Matsuda, J. Biomed. Mater. Res. 34 (1997) 305. [11] H.R. Allcock, Chemistry and Applications of Polyphosphazenes, WileyInterscience, Hoboken, NJ, 2003. [12] L.S. Nair, D.A. Lee, J.D. Bender, E.W. Barrett, Y.E. Greish, P.W. Brown, H.R. Allcock, C.T. Laurencin, J. Biomed. Mater. Res. 76A (2006) 206. [13] S. Sethuraman, L.S. Nair, S. El-Amin, R. Farrar, M.T. Ngyyen, A. Singh, H.R. Allcock, Y.E. Greish, P.W. Brown, C.T. Laurencin, J. Biomed. Mater. Res. 77A (2006) 679. [14] L.S. Nair, D.S. Katti, C.T. Laurencin, Adv. Drug Deliv. Rev. 55 (2003) 467. [15] Y.E. Greish, J.D. Bender, S. Lakshmi, P.W. Brown, H.R. Allcock, C.T. Laurencin, J. Biomed. Mater. Res. 77A (2006) 416. [16] C.S. Reed, K.S. TenHuisen, P.W. Brown, H.R. Allcock, Chem. Mater. 8 (1996) 440. [17] H.R. Allcock, S. Kwon, Macromolecules 22 (1989) 75. [18] H.R. Allcock, S. Kwon, U.S. Patent 5,053,451. [19] A. Oyane, H.M. Kim, T. Furuya, T. Kokubo, T. Miyazaki, T. Nakamura, J. Biomed. Mater. Res. Part A 65 (2003) 188. [20] M. Tanahashi, T. Matsuda, J. Biomed. Mater. Res. 34 (1997) 305. [21] T. Miyazaki, C. Ohtsuki, Y. Akioka, M. Tanihara, J. Nakao, Y. Sakaguchi, S. Konagaya, J. Mater. Sci., Mater. Med. 14 (2003) 569. [22] T. Kokuba, M. Hanakawa, M. Kawashita, M. Minoda, T. Beppu, T. Miyamoto, T. Nakamura, Biomaterials 25 (2004) 4485. [23] R. Rohanizadeh, R.Z. LeGeros, M. Harsono, A. Bendavid, J. Biomed. Mater. Res. A 72 (2005) 428. [24] X. Lu, Y. Leng, Biomaterials 26 (2005) 1097. [25] F. Barrere, P. Layrolle, C.A. Van Blitterswijk, K. De Groot, J. Mater. Sci., Mater. Med. 12 (2001) 529. [26] L. Grondahl, F. Cardona, K. Chiem, E. Wentrup-Byrne, T. Bostrom, J. Mater. Sci., Mater. Med. 14 (2003) 503.
The formation of an apatite coating on carboxylated polyphosphazenes via a biomimetic process Justin Lee Brown a , Lakshmi S. Nair b , Jared Bender c , Harry R. Allcock c , Cato T. Laurencin a,b,d,⁎ a
Department of Biomedical Engineering, University of Virginia, Charlottesville, USA Department of Orthopaedic Surgery, University of Virginia, Charlottesville, USA c Department of Chemistry, Pennsylvania State University, University Park, USA d Department of Chemical Engineering, University of Virginia, Charlottesville, USA b
Received 25 July 2006; accepted 15 December 2006 Available online 23 December 2006
Abstract The biomimetic deposition of an apatite coating on polyphosphazene was investigated. A polyphosphazene having carboxylatophenoxy side groups was synthesized as a candidate polymer with heterogeneous nucleation sites for apatite deposition. Poly[bis(carboxylatophenoxy) phosphazene] (PCPP) was synthesized by a two-step synthetic route involving the synthesis of poly(dichlorophosphazene) (PDP) followed by nucleophilic replacement of the chlorine atoms of PDP with carboxylatophenoxy groups. The surface of the carboxylated polyphosphazene induced the formation of a dense layer of apatite on the surface after incubation in simulated body fluid at 37 °C with an ionic concentration approximately the same as that of human blood plasma. The results suggested an early precipitation of octacalcium phosphate and calcium phosphate intermediates on the surface, both of which are known to promote osteoblast differentiation and proliferation. © 2006 Elsevier B.V. All rights reserved. Keywords: Biomimetic process; Polyphosphazene; Simulated body fluid; Hydroxyapatite
1. Introduction The bone binding biomaterials that preclude the formation of fibrous encapsulation in vivo are some of the most sought after materials for orthopaedic applications [1,2]. Kukubo et al. have demonstrated that the in vivo bone binding ability of biomaterials (metallic and polymeric) can be evaluated in vitro by their ability to form bone like apatite on their surface following incubation in simulated body fluid, which has the same ionic concentration as human blood plasma [1,2]. The ability of materials to form an apatite coating on a surface depends (a) on the specific surface chemistry of the material that induces heterogeneous nucleation of apatite and (b) on the presence of a high concentration of calcium ions in the surrounding fluid [3]. Studies have shown that negatively ⁎ Corresponding author. Department of Biomedical Engineering, 400 Ray C. Hunt Drive, Suite 330, University of Virginia, Charlottesville, VA 22903, USA. Tel.: +1 434 243 0250; fax: +1 434 243 0252. E-mail address: [email protected] (C.T. Laurencin). 0167-577X/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.matlet.2006.12.020
charged surfaces are highly favorable for the nucleation of apatite in the presence of simulated body fluid, which is a supersaturated ionic solution [4]. This has been attributed to the accumulation of calcium ions on negatively charged surfaces which in turn triggers the nucleation of apatite [4]. Therefore, several processes have been developed to form negatively charged metallic and polymeric surfaces to induce bone binding ability. These include acid or alkaline hydrolysis of metal surface, such as titanium, to form a titanium oxide gel layer capable of inducing apatite nucleation [5], surface hydrolysis of polymers such as poly(α-hydroxy esters) to form hydroxyl and carboxyl groups on the surface [6,7], coating of surfaces with self-assembled monolayers terminated with polar head groups such as hydroxyl, phosphoric and carboxylic acid [8–10]. In our laboratories we have developed a unique class of polymers – the polyphosphazenes – as potential polymeric biomaterials for orthopaedic applications. Polyphosphazenes are polymers with a phosphorous nitrogen backbone and with two organic side groups attached to each phosphorus atom [11]. The synthetic flexibility of polyphosphazenes allows the design
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(Phenomenex), and calibrated versus polystyrene standards (Polysciences). 2.1. Synthesis of poly[bis(carboxylatophenoxy)phosphazene] (PCPP)
Scheme 1. Synthesis of PCPP.
and development of polymers with functional groups for specific applications. Our previous studies using various biodegradable and non-degradable polyphosphazenes have demonstrated osteocompatibility of these polymers [12–14]. The objective of the present study was to evaluate the ability of a carboxylated polyphosphazene to nucleate and deposit an apatite layer on a surface using scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) as analytical tools. 2. Experimental Solution-state NMR spectra were obtained at 298 K using a Bruker WM-360 NMR spectrometer resonating at 360.23 MHz for 1H, 145.81 MHz for 31P, and 90.56 MHz for 13C. Molecular weights were estimated using a Hewlett-Packard HP 1090 gel permeation chromatograph equipped with an HP-1047A refractive index detector, two Phenogel 10 μ linear columns
Hexachlorocyclotriphosphazene (HCCTP) (Nippon Fine Chemicals Co., Japan) was purified by recrystallization from heptane and was sublimed at 55 °C (0.05 mm Hg). The macromolecular precursor, polydichlorophosphazene, (PDCP), was prepared by a ring opening polymerization of the HCCTP at 250 °C as described previously [11]. The nucleophilic substitution of the chlorine atoms of PDCP was performed as reported earlier [15–18]. The sodium salt of propyl 4-hydroxybenzoate was prepared by the addition of propyl 4-hydroxybenzoate (118.01 g, 0.655 mol) in THF (150 mL) to a stirred suspension of sodium hydride (25.80 g, 0.645 mol) in THF (1.25 L). The reaction mixture was stirred overnight at room temperature. A solution of poly(dichlorophosphazene) (15.0 g, 0.129 mol) in THF (1 L) was added drop wise to the sodium salt solution at room temperature. After complete addition of the poly (dichlorophosphazene) solution, the reaction mixture was refluxed for 48 h. The polymer solution was then cooled, and precipitated into aqueous acid (0.02 M HCl), dried, dissolved in a minimal amount of THF and reprecipitated into deionized H2O (once) and hexanes (twice). The resultant polymer (Poly[bis (propyl 4-hydroxybenzoate)phosphazene]) (Polymer 1) was dried under vacuum to give a white fibrous polymer. 1H NMR (CD2Cl2): δ 0.92 (t, 3H), 1.67 (m, 2H), 4.10 (br t, 2H), 6.63 (dd, 2H), 7.44 (dd, 2H); 13C NMR (CD2Cl2): δ 10.75, 22.55, 66.91, 120.60, 127.50, 131.30, 154.59, 165.56; 31P NMR (CD2Cl2): δ − 19.45; Mn = 2.42 × 105, Mw = 6.79 × 105, PDI = 2.8. Polymer 1 (15.0 g, 0.0372 mol) was dissolved in THF (1 L) and added drop wise to a stirred solution of potassium tertbutoxide (58.42 g, 0.521 mol) and water (5 mL) in THF
Fig. 1. SEM showing the surface morphology of PCPP films before and after incubation in r-SBF. (A) Before incubation; (B) after 1 day;(C) after 3 days; (D) after 5 days; (E) after 7 days; and (F) after 14 days.
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Table 1 Ca/P ratio of inorganic mineral deposit on PCPP at various time of incubation Time point
Ca/P ratio
(Ca + Mg)/P ratio
24 h 3 days 5 days 7 days 14 days
2.42 1.34 1.29 1.34 1.34
2.42 1.34 1.35 1.51 1.50
(750 mL). The reaction mixture was stirred at room temperature for 20 h, at which time the white precipitate was removed via filtration, dissolved in deionized water, and was purified by dialysis against deionized water (4 days). The polymer solution was removed from the dialysis membrane and concentrated to a viscous solution under reduced pressure which was precipitated into THF yielding polymer Poly[bis(potassium carboxylatophenoxy)phosphazene] (Polymer 2) as a brittle white solid. 1H NMR (D2O): δ 6.95 (br, 2H), 7.69 (br, 2H); 31P NMR (D2O): δ −18.2. Poly[bis(carboxylatophenoxy)phosphazene] (Polymer 3) was precipitated from a 4 wt.% solution of polymer 2 in deionized water through the slow addition of 1 M HCl. The white precipitate was removed via filtration, washed with copious amounts of deionized water, and dried under vacuum. 1 H NMR (d6-DMSO): δ 6.64 (d, 2H), 7.43 (s, 2H) 12.62 (br, 1H); 13C NMR (d6-DMSO): δ 119.70, 127.15, 130.55, 153.36, 166.04; 31P NMR (d6-DMSO): δ − 19.2 (Scheme 1).
SEM. The films were sputter coated with gold and imaged using a JEOL 840A scanning electron microscope. For EDS evaluation, uncoated samples were used and were analyzed with a Princeton Gamma-Tech X-ray Spectrometer attached to the JEOL 840A. All SEM images and energy dispersion spectrums were processed using Princeton Gamma-Tech's Spirit software package. The thin layer X-ray diffraction (XRD) characterization of the mineralized films was performed on a Siemens D5000 diffractometer using Cu radiation at 40 kV/30 mA. Full scans were run over the angular range of 10° to 50° with a step size of 0.03° and an exposure time of 22 h. 4. Results and discussion In the present study, the ability of the carboxylated polyphosphazene to induce heterogeneous nucleation and growth of apatite by incubation in simulated body fluid (r-SBF) was demonstrated. r-SBF was used in this study to closely mimic in vivo conditions because it has been reported that r-SBF has ion concentrations that are equal to or close to that of human blood plasma [19]. Fig. 1A shows the surface morphology of PCPP film cast from DMF solution before incubation in r-SBF. SEM reveals a relatively smooth surface of the polymer film. Fig. 1B–F show the SEM of PCPP films after incubation in r-SBF at 37 °C for various periods of time. It can be seen that after 24 h of incubation small spherical particles of apatite were formed on the surface of the film. Previous studies using self-assembled molecules have demonstrated the ability of carboxylic groups to induce apatite nucleation possibly by chelating calcium ions [20]. It is presumed that
2.2. PCPP film preparation The PCPP was dissolved in dimethylformamide (DMF) and the solution was poured into a Teflon lined Petri dish. The polymer film was allowed to dry under vacuum at 50 °C overnight. The brittle film obtained was immersed in water to increase the flexibility and circular matrices of (10 mm diameter) were bored using a cork borer. 2.3. Simulated body fluid preparation The revised simulated body fluid, r-SBF, was prepared as described previously by Oyane et al. [19]. Fresh solutions were prepared every week and stored at 4 °C. It has been reported that r-SBF is stable for up to 4 weeks at 5 °C [19]. 3. Incubation in simulated body fluid The apatite forming ability of PCPP was evaluated by incubating the polymer films in simulated body fluid as reported earlier [2]. The films were placed in 10 mL centrifuge tubes filled with 10 mL of r-SBF and placed in a water bath at 37.5 °C. The r-SBF was changed every 24 h. At predetermined times, the films (n = 3) were removed and gently washed with ultra pure water (5 times) and dried at room temperature for 24 h. 3.1. Surface characterization of PCPP films The surface structure of PCPP films before and after incubation in revised simulated body fluid was determined by
Fig. 2. Thin layer XRD pattern of mineral deposited on PCPP film: A. after 3 days of incubation and B. after 7 days of incubation.
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the initial stages of interaction between a carboxylated surface and supersaturated SBF involves the formation of –COOCa+ or (–COOH)2Ca which is followed by interaction with phosphate ions. Studies have also shown that the presence of surface carboxyl groups could significantly increase the adhesion of a deposited apatite layer to the surface. However, biomimetic studies using polymers with carboxyl side groups indicated the low reactivity of carboxyl groups in inducing apatite nucleation. In most cases carboxylated polymer surfaces showed an induction period of more than 24 h for apatite nucleation even after using 1.5× SBF, which has higher concentration of Ca2+ compared to SBF. (1× SBF has ion concentration close to human blood plasma and 1.5× SBF has 1.5 times higher ion concentration of that of 1× SBF). Therefore, pre-treating carboxylated polymer surfaces with Ca(OH)2 or CaCl2 has been investigated to decrease the induction period by presenting a high concentration of calcium ions for chelation with the carboxyl groups [21,22]. However, in the case of PCPP we have observed nucleation of apatite within 24 h using 1 × simulated body fluid without the treatment with Ca(OH)2 or CaCl2 (Fig. 1B). This may be due to the structure of PCPP which has two carboxyl groups per repeat unit that would facilitate the rapid chelation of calcium ions. Fig. 1C shows the SEM of PCPP surface after incubation in r-SBF for 3 days at 37 °C. By 3 days it can be seen that the surface of the PCPP film is almost covered with a layer of inorganic apatite. The density of the apatite layer increased with an increase in the incubation time. Thus, multilayers of apatite were observed after incubation of the film in r-SBF for 5, 7 and 14 days (Fig. 1D–F). The surface elemental composition of the formed apatite layer on PCPP surface was evaluated using EDS (Table 1). Since the polyphosphazene contains phosphorus atoms, the EDS was performed selectively on the deposited inorganic phase on PCPP film, particularly at earlier time points such as 1 and 3 days when the surface of the polymer film was not completely covered with the apatite layer. At 24 h, the spectrum showed strong calcium and phosphorous peaks along with small peaks that denoted potassium, chlorine and sulfur. The Ca/P ratio of the apatite layer after 24 h of incubation was found to be 2.42. The high Ca/P ratio can be attributed to carboxylate bound calcium that induces further apatite nucleation. After 3 days of incubation, the Ca/P ratio decreased to ∼1.3. This can be attributed to the formation of octacalcium phosphate Ca8(HPO4)2(PO4)4·5H2O [23,24]. It has been reported previously that octacalcium phosphate has a higher probability of precipitation from r-SBF at a pH of 7.4 [19]. The EDS spectra of the apatite formed after incubation for 5 days indicated the presence of magnesium on the surface. The (Ca + Mg)/P ratio of apatite on PCPP film incubated in r-SBF for 5 days was found to be ∼1.3 indicating the formation of octacalcium phosphate. The thin layer XRD pattern of the surface corroborated the formation of octacalcium phosphate after 5 days of incubation in SBF (Fig. 2). The sharp diffraction lines appeared at 2θ = 16.8° corresponding to the (−101) plane and 2θ = 25.5° corresponding to the (002) plane, both are characteristic of octacalcium phosphate crystal [25]. However, as reported earlier on biomimetic deposition on titanium substrates, the intensity of the diffraction line (−101) at 2θ = 16.8° is significantly higher than typical triclinic OCP crystals and has been attributed to crystal growth along those direction by the influence of substrate on crystal formation. The PCPP substrate did not show any significant peaks except two small peaks near 20–21°. The (Ca + Mg)/P ratio of apatite on PCPP surface after 7 and 14 days of incubation showed a value of 1.5 indicating that the composition of the mineral is approaching that of hydroxyapatite, possibly calcium-deficient apatite or tricalcium phosphate [26]. However, the XRD pattern obtained after 7 days of incubation in simulated body fluid was found to be not significantly different from day 5 indicating the crystalline composition to be more towards that of octacalcium phosphate.
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5. Conclusions The present study demonstrates the feasibility of carboxylated polyphosphazene films prepared to heterogeneously nucleate apatite mineral on the surface. The PCPP film showed nucleation of inorganic mineral by 24 h of incubation in r-SBF at 37 °C without any pretreatment with calcium ions. The density of the deposited layer significantly increased with time of incubation in simulated body fluid. After 5 days of incubation in simulated body fluid the crystalline composition of the mineral was found to be similar to that of octacalcium phosphate. Acknowledgements The authors acknowledge the financial support from NIH grant # EB004051. References [1] T. Kokubo, Biomaterials 12 (1991) 155. [2] T. Kokubo, H. Takadama, Biomaterials 27 (2006) 2907. [3] T. Kawai, C. Ohtsuki, M. Kamitakahara, T. Miyazaki, M. Tanihara, Y. Sakaguchi, S. Konagaya, Biomaterials 25 (2004) 4485. [4] P. Zhu, Y. Masuda, K. Koumoto, Biomaterials 25 (2004) 3915. [5] L. Jonasova, F.A. Muller, A. Helebrant, J. Strnad, P. Greil, Biomaterials 25 (2004) 1187. [6] A. Oyane, M. Uchida, C. Choong, J. Triffitt, J. Jones, A. Ito, Biomaterials 26 (2005) 2407. [7] W.L. Murphy, D.J. Mooney, J. Am. Chem. Soc. 124 (2002) 1910. [8] K. Sato, Y. Kumagai, J. Tanaka, J. Biomed. Mater. Res. 50 (2000) 16. [9] H. Lin, W.S. Seo, K. Kuwabara, K. Koumoto, J. Ceram. Soc. Jpn. 104 (1996) 291. [10] M. Tanahashi, T. Matsuda, J. Biomed. Mater. Res. 34 (1997) 305. [11] H.R. Allcock, Chemistry and Applications of Polyphosphazenes, WileyInterscience, Hoboken, NJ, 2003. [12] L.S. Nair, D.A. Lee, J.D. Bender, E.W. Barrett, Y.E. Greish, P.W. Brown, H.R. Allcock, C.T. Laurencin, J. Biomed. Mater. Res. 76A (2006) 206. [13] S. Sethuraman, L.S. Nair, S. El-Amin, R. Farrar, M.T. Ngyyen, A. Singh, H.R. Allcock, Y.E. Greish, P.W. Brown, C.T. Laurencin, J. Biomed. Mater. Res. 77A (2006) 679. [14] L.S. Nair, D.S. Katti, C.T. Laurencin, Adv. Drug Deliv. Rev. 55 (2003) 467. [15] Y.E. Greish, J.D. Bender, S. Lakshmi, P.W. Brown, H.R. Allcock, C.T. Laurencin, J. Biomed. Mater. Res. 77A (2006) 416. [16] C.S. Reed, K.S. TenHuisen, P.W. Brown, H.R. Allcock, Chem. Mater. 8 (1996) 440. [17] H.R. Allcock, S. Kwon, Macromolecules 22 (1989) 75. [18] H.R. Allcock, S. Kwon, U.S. Patent 5,053,451. [19] A. Oyane, H.M. Kim, T. Furuya, T. Kokubo, T. Miyazaki, T. Nakamura, J. Biomed. Mater. Res. Part A 65 (2003) 188. [20] M. Tanahashi, T. Matsuda, J. Biomed. Mater. Res. 34 (1997) 305. [21] T. Miyazaki, C. Ohtsuki, Y. Akioka, M. Tanihara, J. Nakao, Y. Sakaguchi, S. Konagaya, J. Mater. Sci., Mater. Med. 14 (2003) 569. [22] T. Kokuba, M. Hanakawa, M. Kawashita, M. Minoda, T. Beppu, T. Miyamoto, T. Nakamura, Biomaterials 25 (2004) 4485. [23] R. Rohanizadeh, R.Z. LeGeros, M. Harsono, A. Bendavid, J. Biomed. Mater. Res. A 72 (2005) 428. [24] X. Lu, Y. Leng, Biomaterials 26 (2005) 1097. [25] F. Barrere, P. Layrolle, C.A. Van Blitterswijk, K. De Groot, J. Mater. Sci., Mater. Med. 12 (2001) 529. [26] L. Grondahl, F. Cardona, K. Chiem, E. Wentrup-Byrne, T. Bostrom, J. Mater. Sci., Mater. Med. 14 (2003) 503.