Coating Electrospun Poly(ε-caprolactone) Fibers with Gelatin and

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Coating Electrospun Poly(ε-caprolactone) Fibers with Gelatin and Calcium Phosphate and Their Use as Biomimetic Scaffolds for Bone Tissue Engineering Xiaoran Li,†,‡ Jingwei Xie,† Xiaoyan Yuan,*,‡ and Younan Xia*,† Department of Biomedical Engineering, Washington UniVersity, St. Louis, Missouri 63130, and School of Materials Science and Engineering, Tianjin Key Laboratory of Composite and Functional Materials, Tianjin UniVersity, Tianjin 300072, P. R. China ReceiVed September 10, 2008. ReVised Manuscript ReceiVed October 11, 2008 Electrospinning was employed to fabricate fibrous scaffolds of poly(ε-caprolactone) in the form of nonwoven mats. The surfaces of the fibers were then coated with gelatin through layer-by-layer self-assembly, followed by functionalization with a uniform coating of bonelike calcium phosphate by mineralization in the 10 times concentrated simulated body fluid for 2 h. Transmission electron microscopy, water contact angle, and scanning electron microscopy measurements confirmed the presence of gelatin and calcium phosphate coating layers, and X-ray diffraction results suggested that the deposited mineral phase was a mixture of dicalcium phosphate dehydrate (a precursor to apatite) and apatite. It was also demonstrated that the incorporation of gelatin promoted nucleation and growth of calcium phosphate. The porous scaffolds could mimic the structure, composition, and biological function of bone extracellular matrix. It was found that the preosteoblastic MC3T3-E1 cells attached, spread, and proliferated well with a flat morphology on the mineralized scaffolds. The proliferation rate of the cells on the mineralized scaffolds was significantly higher (by 1.9-fold) than that on the pristine fibrous scaffolds after culture for 7 days. These results indicated that the hybrid system containing poly(ε-caprolactone), gelatin, and calcium phosphate could serve as a new class of biomimetic scaffolds for bone tissue engineering.

Introduction In recent years, synthetic scaffolds capable of replacing autologous or allogeneic bones have attracted broad interests.1 Besides the challenge of designing a scaffold that can mimic the structure and biological function of bone extracellular matrix (ECM), how to achieve a suitable bone-implant interface for the host is also a key issue in bone tissue engineering.2 Electrospun fibers have been studied as a class of promising scaffolds for tissue engineering, since they can mimic the nanoscale features of the ECM. The nonwoven, fibrous mats electrospun from biodegradable polyesters such as poly(lactic acid) (PLA),3 poly(lactic-co-glycolic acid) (PLGA),4 and poly(εcaprolactone) (PCL)5 have all been intensively investigated for bone tissue engineering due to their good mechanical properties and controllable degradation. However, most polyesters are unable to interact specifically with cells due to their relatively high hydrophobicity as compared to natural ECM and lack of functional groups for the attachment of biologically active molecules.6 The biological polymers which can render innate biological information guidance to cells are always introduced by making use of surface modification to the synthetic polymers. Various approaches have been developed for surface modification, including * To whom correspondence should be addressed. E-mail: xia@ biomed.wustl.edu (Y.X.); [email protected] (X.Y.). † Washington University. ‡ Tianjin University.

(1) Stevens, M. M. Mater. Today 2008, 11, 18–25. (2) Porter, A. E.; Patel, N.; Skepper, J. N.; Best, S. M.; Bonfield, W. Biomaterials 2004, 25, 3303–3314. (3) Badami, A. S.; Kreke, M. R.; Thompson, M. S.; Riffle, J. S.; Goldstein, A. S. Biomaterials 2006, 27, 596–606. (4) Xin, X.; Hussain, M.; Mao, J. J. Biomaterials 2007, 28, 316–325. (5) Li, W.; Tuli, R.; Okafor, C.; Derfoul, A.; Danielson, K. G.; Hall, D. J.; Tuan, R. S. Biomaterials 2005, 26, 599–609. (6) Croll, T. I.; O’Connor, A. J.; Stevens, G. W.; Cooper-White, J. J. Biomacromolecules 2004, 5, 463–473.

entrapment;7 cooperative electrostatic interactions;8 grafting via plasma,9 γ-ray irradiation,10 etching,11 or chemical reaction;12 among others. Layer-by-layer (LBL) deposition provides a simple means to generate polyelectrolyte multilayer coating on the surface with a wide variety of different biofunctional properties, especially for substrates with irregular shapes and inner structures where traditional methods are generally ineffective.13 Moreover, the multilayer surface coating formed by LBL deposition has been shown with unique characteristics of improving cytocompatibility to cells.14 To this end, Gao et al. studied electrospun poly(Llactic acid) (PLLA) scaffolds whose surfaces were coated with chitosan using the LBL method. It was found that the attachment, activity, and proliferation of human endothelial cells on the PLLA scaffolds covered by three or five bilayers of poly(styrene sulfonate) sodium salt (PSS)/chitosan (with chitosan as the outermost layer) were better than those with one bilayer of PSS/ chitosan or the control, pristine PLLA.15 Vodouheˆ et al. also reported that the viability of motoneurons on polyelectrolyte (7) Liu, Z.; Jiao, Y.; Zhang, Z.; Zhou, C. J. Biomed. Mater. Res. 2007, 83A, 1110–1116. (8) Smoukov, S. K.; Bishop, K. J. M.; Kowalczyk, B.; Kalsin, A. M.; Grzybowski, B. A. J. Am. Chem. Soc. 2007, 129, 15623–15630. (9) Ma, Z.; He, W.; Yong, T.; Ramakrishna, S. Tissue Eng. 2005, 11, 1149– 1158. (10) Shin, Y. M.; Kim, K.; Lim, Y. M.; Nho, Y. C.; Shin, H. Biomacromolecules 2008, 9, 1772–1781. (11) Nagai, M.; Hayakawa, T.; Makimura, M. J. Biomater. Appl. 2006, 21, 33–47. (12) (a) Edlund, U. E.; Ka¨llrot, M.; Albertsson, A. J. Am. Chem. Soc. 2005, 127, 8865–8871. (b) Zhu, A.; Zhang, M.; Wu, J.; Shen, J. Biomaterials 2002, 23, 4657–4665. (c) Yan, M.; Ren, J. J. Mater. Chem. 2005, 15, 523–527. (d) Gu, H.; Yang, Z.; Gao, J.; Chang, C. K.; Xu, B. J. Am. Chem. Soc. 2005, 127, 34–35. (13) Hammond, P. T. AdV. Mater. 2004, 16, 1271–1293. (14) Salloum, D. S.; Olenych, S. G.; Keller, T. C. S.; Schlenoff, J. B. Biomacromolecules 2005, 6, 161–167. (15) Zhu, Y.; Gao, C.; He, T.; Liu, X.; Shen, J. Biomacromolecules 2003, 4, 446–452.

10.1021/la802984a CCC: $40.75  2008 American Chemical Society Published on Web 11/16/2008

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multilayers was higher compared to polyelectrolyte monolayers.16 In bone tissue engineering, collagen (a main structural protein in bone ECM) is a well-known osteoconductive biomaterial. Polyelectrolyte films based on LBL deposition of collagen have been constructed. Human endothelial cells cultured on polyurethane covered by multilayers of collagen showed favorable adhesion, spreading, and proliferation.17 Therefore, the mutilayer coating formed by LBL deposition (with a biological polymer as the outermost layer) seems to offer many attractive advantages over the monolayer coating which can be prepared using many other methods such as simple dip coating. In addition to its ability to provide a more cell-friendly interface, many charged bioactive molecules can be easily incorporated into a multiplayer system without loss of activity during LBL self-assembly.18 With the desire to build an artificial analogue of native bone ECM, which is mainly composed of hydroxyapatite (HAp) dispersed within a fibrous collagen framework, many attempts have been made to produce collagen/calcium phosphate composites.19 Compared with pure polymers, the composite surface can provide a bioactive environment and lead to improvement of cell attachment, elevation of certain osteogenic biomarker expression levels, and better integration with the host tissue.20 The composite fibrous scaffolds can be generated from their blends using electrospinning21 or hybrid twin screw extrusion/ electrospinning techniques.22 For example, Venugopal et al. prepared PCL/HAp/gelatin (1:1:2), PCL/HAp (1:1), PCL/gelatin (1:2), and PCL fibrous scaffolds by electrospinning their blends or solutions. It was found that PCL/HAp/gelatin composite fibrous scaffolds not only showed highly flexible tensile property but also allowed osteoblasts to penetrate into the scaffolds.21 However, for the preparation of composite fibers via electrospinning, there was difficulty in dispersing hydrophilic ceramic powders in organic solvents, which could hamper the reduction of fiber size and homogeneous distribution of the inorganic components within the fibers.23 Biomimetic mineralization is an alternative route to the production of polymer/calcium phosphate composite in the simulated body fluid (SBF).24 By this means, bonelike apatite, which is very close to natural bone with low crystallinity and nanoscale sizes, can be formed on the surface.25 Further study showed that the growth behavior as well as the dimensions of crystals were also similar to biological apatite present in human bones.26 In the present study, we aim to demonstrate a hybrid fibrous scaffold that can closely mimic bone ECM and provide a friendly interface with the host. We achieve this goal by fabricating fibrous scaffolds from PCL by electrospinning, followed by surface modification with gelatin via a LBL method and deposition of calcium phosphate using a mild mineralization procedure. (16) Vodouheˆ, C.; Schmittbuhl, M.; Boulmedais, F.; Bagnard, D.; Vautier, D.; Schaaf, P.; Egles, C.; Voegel, J.; Ogier, J. Biomaterials 2005, 26, 545–554. (17) Zhu, Y.; Sun, Y. Colloids Surf., B 2004, 36, 49–55. (18) (a) Mu¨ller, K.; Quinn, J. F.; Johnston, A. P. R.; Becker, M.; Greiner, A.; Caruso, F. Chem. Mater. 2006, 18, 2397–2403. (b) Ma, L.; Zhou, J.; Gao, C.; Shen, J. J. Biomed. Mater. Res. 2007, 83B, 285–292. (19) Zou, C.; Weng, W.; Deng, X.; Cheng, K.; Liu, X.; Du, P.; Shen, G.; Han, G. Biomaterials 2005, 26, 5276–5284. (20) Yao, J.; Radin, S.; Leboy, P. S.; Ducheyne, P. Biomaterials 2005, 26, 1935–1943. (21) Venugopal, J. R.; Low, S.; Choon, A. T.; Kumar, A. B.; Ramakrishna, S. Artif. Organs 2008, 32, 388–397. (22) Erisken, C.; Kalyon, D. M.; Wang, H. Nanotechnology 2008, 19, 165302. (23) Kim, H.; Lee, H.; Knowles, J. C. J. Biomed. Mater. Res. 2006, 79A, 643–649. (24) Yuan, X.; Mak, A. F. T.; Li, J. J. Biomed. Mater. Res. 2001, 57, 140–150. (25) Abe, Y.; Kokubo, T.; Yamamuro, T. J. Mater. Sci.: Mater. Med. 1990, 1, 233–238. (26) Mu¨ller, F. A.; Mu¨ller, L.; Caillard, D.; Conforto, E. J. Cryst. Growth 2007, 304, 464–471.

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Figure 1. SEM images of electrospun PCL fibers (A) before and (B) after coating with 15 bilayers of alternating gelatin and PSS (with gelatin as the outermost layer).

Experimental Section Materials. Poly(ε-caprolactone) (PCL, Mn ) 42 500 g/mol), gelatin (type A, form porcine skin), poly(styrene sulfonate) sodium salt (PSS, Mw ) 7000 g/mol), dichloromethane (DCM), dimethylformaldehyde (DMF), acetic acid, and all the chemicals for preparation of the 10 times concentrated simulated body fluid (10SBF) were obtained from Sigma-Aldrich (St. Louis, MO). All chemicals were used as received. The water used in all experiments was purified by passing through a Millipore system. Fabrication of PCL Fibrous Scaffolds by Electrospinning. The electrospinning setup used in the present study was described in our previous publication.27 PCL solution at a concentration of 20% was obtained by dissolving it in a solvent mixture of DCM and DMF with a volume ratio of 80:20. The solution was loaded into a 5 mL plastic syringe with a 24-gauge needle attached and injected using a syringe pump at a flow rate of 0.5 mL/h. The collector was a piece of aluminum foil. The distance between the tip of needle and the collector was about 15 cm, and a voltage of 15 kV was applied. Deposition of Gelatin. The deposition of gelatin onto electrospun PCL fibers via LBL self-assembly was conducted as follows: electrospun PCL fibers were immersed in a 2 mg/mL gelatin solution in 20 mM acetic buffer (pH ) 4.0) and kept for 20 min. Thereafter, the fibers were thoroughly rinsed with water for 10 min and then immersed in a 3 mg/mL PSS solution in 20 mM acetic buffer (pH ) 4.0) for another 20 min. The sample was thoroughly rinsed with water for 10 min. The deposition cycle was repeated until the desired number of layers (or film thickness) was reached. The scaffolds (27) Li, D.; McCann, J. T.; Xia, Y. J. Am. Ceram. Soc. 2006, 89, 1861–1869.

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Langmuir, Vol. 24, No. 24, 2008 14147 The microstructures of gelatin-covered PCL scaffolds were studied by transmission electron microscopy (TEM, Hitachi H-7500). Water contact angles of PCL and gelatin-covered PCL scaffolds were measured with a contact angle meter (Rame´-Hart Inc.) at ambient temperature. In order to determine the crystallographic structure, we examined the gelatin-covered PCL scaffolds after mineralization in 10SBF for 8 h (to make sure there was a sufficient amount of mineral phase coating) by using a Rigaku Geigerflex D-MAX/A diffractometer with Cu KR radiation (50 kV, 50 mA). The scanning range was from 10° to 40° with a step size of 0.02°. Cell Culture. Mouse calvaria-derived, preosteoblastic cells (MC3T3-E1; ATCC CRL-2593) were cultured in alpha minimum essential medium (R-MEM, Invitrogen, Grand Island, NY), supplemented with 10% fetal bovine serum (FBS, Invitrogen) and 1% antibiotics (containing penicillin and streptomycin, Invitrogen). The medium was changed every other day, and the cultures were incubated at 37 °C in a humidified atmosphere containing 5% CO2. Both pristine and mineralized PCL fibrous scaffolds were cut into circular discs of 15 mm in diameter and placed in the wells of a 24-well plate (Corning). The samples were sterilized in 70% ethanol overnight and then washed with phosphate buffer saline (PBS) three times. Approximately 105 cells were seeded in each well and cultured in 300 µL of osteogenic medium containing FBS for different periods of time up to 7 days. The cell viability was measured using the 3-(4,5-dimethylthiazol)2,5-diphenyl tetrazolium bromide (MTT) assay, which is based on the mitochondrial conversion of tetrazolium salt. After 1, 3, and 7 days of incubation, the medium was removed, and 270 µL of fresh medium and 30 µL of MTT (5 mg/mL in PBS) (Invitrogen) were added to each well and incubated at 37 °C and 5% CO2 for 3 h. After removal of the medium, the converted dye was dissolved with isopropyl alcohol. The absorbance at a wavelength of 560 nm was measured using a microplate reader (Tecan). The cell morphology at day 7 was characterized by fluorescent microscopy. The cells on the samples were washed twice with PBS, fixed in 3.7% formaldehyde solution (Sigma-Aldrich) in PBS for 30 min at room temperature, and dyed with Alexa Fluor1 488 phalloidin (Invitrogen) and 4′-6-diamidino-2-phenylindole (DAPI, Invitrogen) for 1 h. The fluorescent images were taken using a QICAM Fast Cooled Mono 12-bit camera (Q Imaging, Burnaby, BC, Canada) attached to an Olympus microscope with Capture 2.90.1 (Olympus).

Results and Discussion Figure 2. TEM images of (A) PCL/(gelatin/PSS)15/gelatin fibers and (B) nanotubes obtained by dissolving the inner PCL fibers.

used in both mineralization and cell culture were nonwoven mats of PCL fibers whose surfaces had been coated with five bilayers of gelatin and PSS, with gelatin on the outermost surface (thereafter, they are referred to as gelatin-covered scaffolds). As a control, nonwoven mats of pristine PCL fibers were also used for mineralization and cell culture studies. Coating of Calcium Phosphate. We followed Tas and Bhaduri’s method to prepare the 10 times concentrated simulated body fluid (10SBF).28 A stock solution containing NaCl, KCl, CaCl2, MgCl2, and NaH2PO4 · H2O with a pH value of about 4.1 was prepared in advance. The stock solution could be kept at 4 °C for several weeks without precipitation. At the beginning of a coating process, NaHCO3 was added at room temperature under a stirring speed of 500 rpm, and the pH value rose to about 6.5. The electrospun PCL and gelatincovered PCL scaffolds were immersed in 10SBF hosted in a tightly capped plastic tube and kept at room temperature for 1-8 h. The 10SBF solution was changed every 2 h. After being removed from buffer solution, the samples were gently washed with water and then dried in air at room temperature. Characterization. The morphologies of the electrospun PCL fibers, the gelatin-covered PCL fibers, and the scaffolds after mineralization in 10SBF for different periods of time were examined by scanning electron microscopy (SEM, FEI Nova 200 NanoLab). (28) Tas, A. C.; Bhaduri, S. B. J. Mater. Res. 2004, 19, 2742–2749.

Preparation of PCL Fibers. In the present study, PCL was chosen as a model polymer due to its relatively slow degradation in ViVo, which is useful to bone tissue engineering.29 Figure 1A shows a typical SEM image of PCL fibers fabricated by electrospinning. The PCL fibers were smooth and free of beads, with an average diameter of 1.2 µm. The fibers were randomly oriented to form a porous scaffold. It has been reported that the fibrous scaffold could serve as a much better support for cell attachment and proliferation, in comparison with a solid film cast from the same polymer solution.30 Gelatin Deposition. In addition to the topographical structure, the biological activity is also critical to the performance of a scaffold. Decoration of synthetic polymers with natural polymers via surface modification has been developed to improve the biomaterial/cell interaction. In the present study, positively charged gelatin was deposited onto the electrospun PCL fiber surface in a LBL assembly manner using PSS as the negatively charged polyelectrolyte. This approach, which is free from gelatin cross-linking, can avoid the potential problem of cytotoxicity caused by the chemicals employed during the process of crosslinking gelatin. Gelatin derived from collagen has been exploited for a variety of biomedical applications due to its biocompatibility (29) Bo¨lgen, N.; Mencelog˘lu, Y. Z.; Acatay, K.; Vargel, |$$I˙.; Pis¸kin, E. J. Biomater. Sci., Polymer Ed. 2005, 16, 1537–1555. (30) Sombatmankhong, K.; Sanchavanakit, N.; Pavasant, P.; Supaphol, P. Polymer 2007, 48, 1419–1427.

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Figure 3. SEM images of electrospun PCL fibers after their surfaces had been coated with gelatin and then incubated in 10SBF for (A) 1, (B) 2, (C) 3, (D) 4, and (E) 5 h. (F) SEM image of electrospun PCL fibers which were directly incubated in 10SBF for 2 h without gelatin treatment. The scale bars in the insets are 2 µm.

and low cost.31 In addition, PSS, a component widely used in LBL assembly, exhibited good cytocompatibility and stability in culture medium.32 After coating, the scaffold still maintained the fibrous and porous structure (Figure 1B). Figure 2A shows a TEM image of a single PCL fiber after coating with 15 bilayers of gelatin and PSS, with gelatin on the outermost surface. In order to clearly resolve the coating thickness, we deposited 15 bilayers rather than 5 bilayers on the PCL fibers. A core/shell structure could be observed under TEM due to the density difference between PCL and the polyelectrolyte layer, with a corresponding average shell thickness of 76 nm. To further confirm the deposition of gelatin and PSS, the fibers were immersed in DCM, resulting in dissolution of PCL cores and (31) Sun, J.; Wu, S. Y.; Lin, F. Biomaterials 2005, 26, 3953–3960. (32) Diaspro, A.; Silvano, D.; Krol, S.; Cavalleri, O.; Gliozzi, A. Langmuir 2002, 18, 5047–5050.

formation of hollow fibers made of gelatin and PSS (Figure 2B). Water contact angle measurements provided additional evidence to support the LBL coating. The hydrophobic PCL scaffolds exhibited a high water contact angle of 117°, whereas the surface of gelatin-covered PCL fibers was completely hydrophilic with a water contact angle of almost zero. From these results, it is clear that gelatin had been deposited on the fiber surface via LBL assembly, and the amount of gelatin could be tailored easily by controlling the number of LBL deposition cycles. If needed, various bioactive agents capable of stimulating cell adhesion, proliferation, and differentiation, such as DNA and growth factors,18 could also be directly incorporated into the polyelectrolyte layers during LBL deposition. Biomimetic Coating. SBF has been widely used for biomimetic calcium phosphate coating on bioinert materials. This

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Figure 4. X-ray diffraction pattern taken from a scaffold of electrospun PCL fibers whose surfaces were coated with gelatin and then mineralized by incubation in 10SBF for 8 h.

Figure 5. Proliferation of MC3T3-E1 cells seeded on membranes of electrospun PCL fibers without and with surface functionalization (n ) 4). Bar represented means ( SD. A statistically significant difference was observed after 7 days of culture (Student’s t-test, *p < 0.05).

process is rather slow, and it normally takes up to several weeks.33 The in Vitro mineralization in 10SBF was developed as an effective and robust approach to calcium phosphate coating.28,34 Figure 3 (A-E) shows morphological changes of the gelatincovered PCL fibers after different periods of mineralization in 10SBF. After incubation for 1 h, a few tiny particles appeared on the surfaces of the fibers, showing a fast precipitation of calcium phosphate (Figure 3A). A prolonged incubation significantly changed the surface morphology. The calcium phosphate growth occurred preferentially along the longitudinal direction of the fibers. After 2 h of incubation, the scaffold was fully covered with nanotextured precipitates but maintained its porous and fibrous structure, indicating a homogeneous nucleation (Figure 3B). The deposition gradually grew to become globules after 3 and 4 h (Figure 3C and D). The fibers were completely wrapped by thick calcium phosphate layers after 5 h; meanwhile, the fibrous and porous structures disappeared (Figure 3E). Figure 3F shows an SEM image of a pristine PCL scaffold after incubation in 10SBF for 2 h, and it can be seen that the mineral (33) Zhang, R.; Ma, P. X. Macromol. Biosci. 2004, 4, 100–111. (34) Yang, F.; Wolke, J. G. C.; Jansen, J. A. Chem. Eng. J. 2008, 137, 154– 161.

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deposition over individual fibers was uneven and bare fibers inside the scaffold could still be easily found. This ineffectiveness in coating could be ascribed to the relatively inert surface of PCL, which lacks the ability to bind to calcium phosphate.34 Previous studies have reported that anionic groups such as -COO- on the surface of organic polymers could lead to enrichment of Ca2+, resulting in local supersaturation and nucleation of crystallites.35 Several methods have been employed to modify the surfaces of substrates toward activating the polymers, and typical examples include NaOH36 and plasma37 treatment. However, either toxic reagents (e.g., NaOH) or dedicated equipment (e.g., plasma cleaner) was required. In addition, for polymer fibers, which were not as strong as the bulk materials, the harsh reaction conditions such as NaOH and plasma treatment could possibly impound intrinsic mechanical and chemical properties, and even result in degradation and/or damage.38 In this work, the deposition of gelatin via the LBL technique followed by incubation in 10SBF successfully induced fast and uniform calcium phosphate coating on the surface of PCL fibers. The increased hydrophilicity and introduction of functional groups such as -COOH and -NH2 could be the possible reasons for the effective calcium phosphate deposition. Yao et al. have studied the in situ formation of nanohydroxyapatite on a chitosan-gelatin network.39 It was found that the carboxyl groups of gelatin, and carbonyl and amino groups of gelatin and chitosan played a crucial role in HAp formation. It has been reported that calcium phosphates in different forms such as HAp40 and octacalcium phosphate41 could grow on a gelatin matrix. The crystal structure of the mineral phase developed from the gelatin-covered PCL fibers after 8 h of incubation in 10SBF was determined by X-ray diffraction (XRD). As indicated in Figure 4, the characteristic peaks of dicalcium phosphate dehydrate (DCPD) appeared clearly in the diffraction pattern (labeled with /), and the peak labeled with a dot could be assigned to apatite. It can be concluded that the deposition consisted of a mixture of DCPC and apatite. It has been established that DCPD is a potential starting material for bone substitute.42 Cell Response. It is well-known that the porous structure of a scaffold is very important to bone tissue engineering. The pores favor the inward growth of bone, and the interconnective porous structure facilitates in Vitro nutrient/waste transportation and in ViVo vascularization.43 In this study, the gelatin-covered PCL fibers after mineralization for 2 h in 10SBF were selected as the scaffolds for cell culture because the calcium phosphate could effectively coat on the surfaces of individual fibers while leaving the inherent porous structures of the scaffolds unchanged. The in Vitro biocompatibility of the electrospun hybrid fibers was assessed in terms of the proliferation of MC3T3-E1 cells with pristine electrospun PCL fibers as a control. Figure 5 shows proliferation data of the cells seeded on pristine and mineralized PCL fibrous scaffolds. The mineralized scaffolds were composed of electropsun PCL fibers covered with five bilayers of gelatin/ (35) Huang, S.; Zhou, K.; Zhu, W.; Huang, B.; Li, Z. J. Appl. Polym. Sci. 2006, 101, 1842–1847. (36) Oyane, A.; Uchida, M.; Choong, C.; Triffitt, J.; Jones, J.; Ito, A. Biomaterials 2005, 26, 2407–2413. (37) Oyane, A.; Uchida, M.; Yokoyama, Y.; Choong, C.; Triffitt, J.; Ito, A. J. Biomed. Mater. Res. 2005, 75A, 138–145. (38) Ma, Z.; Kotaki, M.; Yong, T.; He, W.; Ramakrishna, S. Biomaterials 2005, 26, 2527–2536. (39) Li, J.; Chen, Y.; Yin, Y.; Yao, F.; Yao, K. Biomaterials 2007, 28, 781– 790. (40) Bigi, A.; Boanini, E.; Panzavolta, S.; Roveri, N.; Rubini, K. J. Biomed. Mater. Res. 2002, 59, 709–714. (41) Wen, H. B.; Moradian-Oldak, J.; Fincham, A. G. J. Dent. Res. 2000, 79, 1902–1906. (42) Tas, A. C.; Bhaduri, S. B. J. Am. Ceram. Soc. 2004, 87, 2195–2200. (43) Karageorgiou, V.; Kaplan, D. Biomaterials 2005, 26, 5474–5491.

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Figure 6. Fluorescence micrographs of MC3T3-E1 cells that were cultured for 7 days on (A, B) membranes of electrospun PCL fibers and (C, D) membranes of electrospun PCL fibers whose surfaces had been derivatized with gelatin and then calcium phosphate. The F-actin was stained with fluorescein isothiocyanate-phallodin (green color), while the cell nucleus was stained with 4′-6-diamidino-2-phenylindole (DAPI) (purple color).

PSS (with gelatin as the outermost layer), followed by a bonelike calcium phosphate coating. They showed more favorable adhesion and higher proliferation rate of MC3T3-E1cells. The ratios of cell growth on the mineralized fibers to pristine PCL fibers at day 3 and day 7 were 1.4-fold and 1.9-fold, respectively. The cell proliferation on the mineralized fibers was significantly (p < 0.05) higher compared with that on pristine PCL fibers after 7 days of cell culture. After culturing for 7 days, we also observed the cell morphology by fluorescent microscopy, as shown in Figure 6. The cells were observed to attach and spread well on both types of PCL scaffolds. However, the cells seemed to prefer expanding on the mineralized fibers. After 7 days of culture, the surface of the mineralized scaffold was covered with multilayers of cells, which was in good agreement with our proliferation result. It could thus be concluded that the PCL scaffolds functionalized with gelatin and calcium phosphate might offer a more favorable microenvironment for MC3T3 cell growth. In this study, the PCL/gelatin/ calcium phosphate fibrous scaffolds combine all the attractive features and unique properties of synthetic polymers, natural polymers, and mineral deposition: that is, PCL for the mechanical properties, and coatings of gelatin and calcium phosphate for the bioactivity and osteoconductivity, respectively. The fibrous matrix also exhibits a structure and components similar to those of bone ECM. All these attributes should make the nonwoven mats of

mineralized PCL fibers as a new class of promising scaffolds for bone tissue engineering.

Conclusion A class of hybrid scaffolds based on PCL, gelatin, and calcium phosphate was developed through surface modification on the electrospun PCL fibers. Gelatin was immobilized by LBL assembly, and calcium phosphate was deposited on the surface of gelatin-covered fibers by mineralization in 10SBF. We found that the presence of gelatin facilitated a homogeneous calcium phosphate coating. After 2 h of incubation, fibers were uniformly covered by a thin layer of mineral deposition. XRD results indicated that the composition of the deposited mineral was a mixture of dicalcium phosphate dehydrate (a precursor to apatite) and apatite. The hybrid scaffolds were then evaluated for the culture of MC3T3-E1 cells. Cell proliferation was significantly higher than that on pristine PCL scaffolds, which were used as a control. A multilayered film of cells was observed on the mineralized scaffolds after 7 days of culture. It can be concluded that the introduction of gelatin and calcium phosphate coatings was effective in enhancing the cytocompatiblity. The scaffold developed in this study can also accommodate the incorporation of drugs such as bone morphogenetic proteins (BMP) and other bioactive species in the fibers or the polyelectrolyte films to empower them with more functions. LA802984A