Hydroxyapatite Mineralization on the Calcium Chloride Blended Polyurethane Nanofiber via Biomimetic Method
© Nirmala et al. 2010
Received: 18 June 2010
Accepted: 5 August 2010
Published: 19 August 2010
Polyurethane nanofibers containing calcium chloride (CaCl2) were prepared via an electrospinning technique for the biomedical applications. Polyurethane nanofibers with different concentration of CaCl2 were electrospun, and their bioactivity evaluation was conducted by incubating in biomimetic simulated body fluid (SBF) solution. The morphology, structure and thermal properties of the polyurethane/CaCl2 composite nanofibers were characterized by means of scanning electron microscopy (SEM), field-emission scanning electron microscopy, energy dispersive X-ray spectroscopy, X-ray diffraction, Fourier transform infrared spectroscopy and thermogravimetry. SEM images revealed that the CaCl2 salt incorporated homogeneously to form well-oriented nanofibers with smooth surface and uniform diameters along their lengths. The SBF incubation test confirmed the formation of apatite-like materials, exhibiting enhanced bioactive behavior of the polyurethane/CaCl2 composite nanofibers. This study demonstrated that the electrospun polyurethane containing CaCl2 composite nanofibers enhanced the in vitro bioactivity and supports the growth of apatite-like materials.
KeywordsPolyurethane Electrospinning Nanofibers Simulated body fluid Bioactivity
Electrospinning of biologically significant polymers has increased dramatically since the electrospun membranes were identified as a candidate for guided tissue regeneration applications [1–4]. In particular, polyurethane is a thermoplastic, biodegradable, biocompatible polymer with excellent mechanical and physical properties [5, 6]. Due to these merits, polyurethane nanofibers have been found to be a very promising material of interest. It has been already reported that the electrospun membranes have the potential to promote osteoblastic cell function and bone regeneration . Recently, the feasibility of incorporating non-electrospinnable inorganic nanoparticles into polymer solution to form composite nanofibers has attained electrospinning as an attractive technique in meeting some specific functional applications [8–12]. There were some reports on electrospun composite nanofibers, such as poly (ε-caprolactone)/tricalcium phosphate , hydroxyapatite/gelatin , silk/hydroxyapatite , poly-lactic acid/hydroxyapatite , poly (vinyl alcohol) coated poly (ε-caprolactone)  and triphasic hydroxyapatite/collagen/poly(ε-caprolactone) [10, 11] had been analyzed and explored for potential bone regeneration applications. However, the effect of calcium chloride (CaCl2) on the bioactivity analysis in polyurethane nanofibers has not been characterized so far. It is interesting to note that the use of CaCl2 salt is salient to entrap the cells and make them grow faster . Calcium facilitates the attachment of cells to scaffold and to one another. Calcium is widely used in all classic media, which is utilized in biomanufacturing and in the tissue culture applications. The bioactivity of these materials can be attributed to the formation of a biologically active bone-like carbonate containing apatite layer. Therefore, we have employed for the first time CaCl2 incorporation in the polyurethane nanofibers during electrospinning to enhance the bioactivity behavior of the composites.
In this study, in order to obtain the role of CaCl2, we synthesized polyurethane nanofiber mats with different concentrations of CaCl2 salt. The hydroxyapatite was coated by the biomimetic method on as-spun nanofibers by incubating in the simulated body fluid (SBF) solution for different timings to observe the formation of apatite-like materials. We performed extensive analysis to confirm the formation of apatite-like materials on the electrospun polyurethane/CaCl2 composite nanofibers, and we proposed a likely process for the mineralization of hydroxyapatite after incubating in the SBF solution.
Polyurethane (MW = 110,000) was purchased from Cardio Tech, Japan. 2-butanone (MEK) and NN-dimethylformamide (DMF) (analytical grade, Showa, Japan) were used as solvents without further purification. Polyurethane solution with 10 wt% was prepared by dissolving in MEK and DMF. Polyurethane with 0, 1, 1.5 and 2 wt% of CaCl2 (Sigma-Aldrich, USA) was used to prepare nanofiber mats. The polymer solution with CaCl2 was kept for 12 h to dissolve completely in the solvent. Adding more amount of CaCl2 in the solution did not dissolve properly, which resulted in the non-electrospinnability of the polymer solution. Therefore, we optimized the CaCl2 content (up to 2 wt%) in the polymer solution to obtain a complete mixing. A high voltage power supply (CPS-60 K02V1, Chungpa EMT, South Korea) of 22 kV to the syringe micro-tip was supplied to electrospin the nanofibers. The polymer solution was fed to the 5-ml syringe with a plastic micro-tip. The tip-to-collector distance was kept at 15 cm. During electrospinning, the drum was rotated at a constant speed by a DC motor to collect the developing nanofibers. All experiments were performed at room temperature.
The in vitro bioactivity analysis was performed by using SBF solution. The recipe for the preparation of SBF was adopted from the method reported elsewhere . The SBF solution used in the present study was five times the concentration than that of calcium and phosphate ions when compared to conventional SBF [18, 19]. The morphology of the as-spun and immersed polyurethane/CaCl2 composite nanofibers in SBF solution was observed by using scanning electron microscopy (SEM, S-7400, Hitachi, Japan). The SEM images were obtained for different durations (2 h, 4, 6 and 7 days) of incubation in SBF to monitor the mineralization of apatite-like materials on the nanofiber surfaces. The chemical composition of the polyurethane/CaCl2 composite nanofibers was analyzed by using energy dispersive X-ray (EDX) spectrometer attached to the field-emission scanning electron microscope (FE-SEM). Structural characterization was carried out by X-ray diffraction in a Rigaku X-ray diffractometer (XRD) operated with Cu-Kα radiation (λ = 1.540 Å). The bonding configurations of the samples were characterized by means of Fourier transform infrared (FTIR) spectroscopy. Thermogravimetric analysis (TGA, Perkin-Elmer, USA) was carried out for the electrospun mats under nitrogen ambient with a flow rate of 20 ml/min. The samples were heated from 30 to 600°C at a rate of 10°C/min, and the differential thermogravimetry graph was recorded.
Results and Discussion
The chemical composition of the as-spun nanofiber mats was analyzed by EDX spectrometer attached with FE-SEM. Figure 1a2–d2 show the EDX spectra of the polyurethane nanofibers containing different concentration of CaCl2 salt with 0, 1, 1.5 and 2 wt%. As shown in Figure 1a2, the signals of carbon and oxygen were only observed for pristine polyurethane nanofibers. The EDX spectra revealed the presence of Ca and Cl in polyurethane nanofibers containing CaCl2 salt. By adding CaCl2 salt in polyurethane nanofibers, their compositions were increased dramatically with increasing concentration as shown in Figure 1b2–d2. The successful blending of CaCl2 salt with polyurethane nanofibers was confirmed by the EDX spectra.
Polyurethane nanofibers containing different concentrations of CaCl2 were successfully produced by electrospinning technique. These as-spun nanofibers exhibited a smooth surface and uniform diameters without any beads along their lengths. Addition of CaCl2 salt resulted in the formation of mesh-like ultrafine nanofibers in between the main fibers. The formation of apatite-like materials was spontaneously commenced, and their size was monotonically increased with increasing CaCl2 concentration after incubation in SBF solution. Good attachment and fast proliferation of apatite-like materials was observed in SEM analysis after incubation in SBF solution. The EDX and XRD data clearly confirmed the presence of apatite-like materials in the polyurethane/CaCl2 composite nanofibers. The results of FTIR and TGA analyses were also revealed the formation of plenty of apatite-like materials on the surfaces of polyurethane/CaCl2 composite nanofibers. Because of good attachment and formation behaviors, there is a potential to utilize polyurethane/CaCl2 composite nanofibers scaffold for the bone regeneration applications.
This work was supported by the grant of the Korean Ministry of Education, Science and Technology (The Regional Core Research Program/Center for Healthcare Technology & Development, Chonbuk National University, Jeonju 561-756 Republic of Korea).
- Fujihara K, Kotaki M, Ramakrishan S: Biomaterials. 2005, 26: 4139. 10.1016/j.biomaterials.2004.09.014View Article
- Yang F, Both SK, Yang X, Walboomers XF, Jansen JA: Acta Biomater. 2009, 5: 3295. 10.1016/j.actbio.2009.05.023View Article
- Kim HW, Song JH, Kim HE: Adv Funct Mater. 2005, 15: 1988. 10.1002/adfm.200500116View Article
- Ramakrishna S, Mayer J, Wintermantel E, Leong KW: Comp Sci Tech. 2001, 61: 1189. 10.1016/S0266-3538(00)00241-4View Article
- Ojha U, Kulkarni P, Faust R: Polymer. 2009, 50: 3448. 10.1016/j.polymer.2009.05.025View Article
- Kidoaki S, Kwon IK, Matsuda T: J Biomed Mater Res B Appl Biomater. 2006, 76: 219.View Article
- Pham QP, Sharma U, Mikos AG: Tissue Eng. 2006, 12: 1197. 10.1089/ten.2006.12.1197View Article
- Sui G, Yang X, Mei F, Hu X, Chen G, Deng X, Ryu S: J Biomed Mater Res A. 2007, 82: 445.View Article
- Deng XL, Sui G, Zhao ML, Chen GQ, Yang XP: J Biomater Sci Polym. 2007, 18: 117. 10.1163/156856207779146123View Article
- Catledge SA, Clem WC, Shrikishen N, Chowdhury S, Stanishevsky AV, Koopman M, Vohra YK: Biomed Mater. 2007, 2: 142. 10.1088/1748-6041/2/2/013View Article
- Venugopal JR, Vadgama P, Kumar TSS, Ramakrishna S: Nanotechnology. 2007, 18: 1. 10.1088/0957-4484/18/5/055101View Article
- Zhang Y, Venugopal JR, Turki AE, Ramakrishna S, Su B, Lim CT: Biomaterials. 2008, 29: 4314. 10.1016/j.biomaterials.2008.07.038View Article
- Li C, Vepari C, Jin HJ, Kim H, Kaplan D: Biomaterials. 2006, 27: 3115. 10.1016/j.biomaterials.2006.01.022View Article
- Liao S, Wang W, Uo M, Ohkawa S, Akasaka T, Tamura K, Cui F, Watari F: Biomaterials. 2005, 26: 7564. 10.1016/j.biomaterials.2005.05.050View Article
- Li L, Li G, Wang Y, Jiang J: Appl Surf Sci. 2009, 255: 7734. 10.1016/j.apsusc.2009.04.154View Article
- Lee GM, Han BK, Kim JH, Palsson BO: Biotechnology Lett. 1992, 14: 891. 10.1007/BF01020624View Article
- Kong L, Gao Y, Lu G, Gong Y, Zhao N, Zhang X: European Polym J. 2006, 42: 3171. 10.1016/j.eurpolymj.2006.08.009View Article
- Kokubo T, Takadama H: Biomaterials. 2006, 27: 2907. 10.1016/j.biomaterials.2006.01.017View Article
- Kokubo T, Hanakawa M, Kawashita M, Minoda M, Beppu T, Miyamoto T, Nakamura T: Biomaterials. 2004, 25: 4485. 10.1016/j.biomaterials.2003.11.007View Article
- Murugan R, Ramakrishna S: Tissue Eng. 2006, 12: 435. 10.1089/ten.2006.12.435View Article
- Barakat NAM, Kanjwal MA, Sheikh FA, Kim HY: Polymer. 2009, 50: 4389. 10.1016/j.polymer.2009.07.005View Article
- Chen JL, Chu B, Hsiao BS: J Biomed Mater Res A. 2006, 79: 307.View Article
- Oyane A, Uchida M, Choong C, Triffitt J, Jones J, Ito A: Biomaterials. 2005, 26: 2407. 10.1016/j.biomaterials.2004.07.048View Article
- Sobhana SSL, Sundaraseelan J, Sekar S, Sastry TP, Mandal AB: J Nanopart Res. 2009, 11: 333. 10.1007/s11051-008-9385-0View Article
- Sastry TP, Sundaraseelan J, Swarnalatha K, Sobhana SSL, Makheswari MU, Sekar S, Mandal AB: Nanotechnology. 2008, 19: 245604. 10.1088/0957-4484/19/24/245604View Article
- Patlolla A, Collins G, Arinzeh TL: Acta Biomater. 2010, 6: 90. 10.1016/j.actbio.2009.07.028View Article
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.