In Situ Growth Kinetics of Hydroxyapatite on Electrospun Poly(dl

In the aminolysis and gelatin grafting process, the amount of amino groups ... or cortical bone,(5) and the homogeneous distribution of bioceramic par...
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In Situ Growth Kinetics of Hydroxyapatite on Electrospun Poly(DL-lactide) Fibers with Gelatin Grafted Wenguo Cui, Xiaohong Li,* Jiangang Chen, Shaobing Zhou, and Jie Weng Key Laboratory of AdVanced Technologies of Materials, Ministry of Education, School of Materials Science and Engineering, Southwest Jiaotong UniVersity, Chengdu 610031, P. R. China

CRYSTAL GROWTH & DESIGN 2008 VOL. 8, NO. 12 4576–4582

ReceiVed June 17, 2008; ReVised Manuscript ReceiVed August 6, 2008

ABSTRACT: Development of fibrous nanocomposites of hydroxyapatite (HA) and poly(DL-lactide) (PDLLA) offers great potential as tissue engineering scaffolds. Attempts have been made to solve problematic issues of the agglomeration of HA crystallites in fibrous mats to enhance the mechanical property and bioactivity. A novel method was introduced in the present study to use electrospun PDLLA nanofibers with surface modifications as the induction sites for composite fabrication. Free amino groups were created on electrospun fibers through an optimized aminolysis process, and gelatin was further grafted on the aminolyzed fibers through glutaraldehyde coupling. In the aminolysis and gelatin grafting process, the amount of amino groups and gelatin could be modeled with quantitative equations as a function of the aminolysis time. The formation of nonstoichiometric nanostructured HA and good dispersion on electrospun fibers were determined. Gelatin was demonstrated to control the nucleation and growth of crystals on fibrous templates, and kinetic equations of HA growth were drafted as a function of the incubation time for fibrous mats with different gelatin contents. The amount of HA formed on the fibrous composites and the crystal size were regulated through the content of grafted gelatin, which could be further determined by the aminolysis process. Introduction Much attention has been paid to investigations into the preparation, mechanical property, and biocompatibility of the composites of hydroxyapatite (HA) and poly(lactic acid) (PLA).1 The presence of mineralized coatings or inclusions within polymer scaffolds can augment the mechanical strength2 and neutralize the acidic degradation products because of the alkalinity of the calcium phosphate.3 According to practical requirements, several methods have been developed to fabricate the composites, such as hot press molding, plasma spraying, in situ polymerization, and solution compounding.4 But the mechanical integrity of manmade composite scaffolds is still at least 1 order of magnitude lower than that of cancellous or cortical bone,5 and the homogeneous distribution of bioceramic particles in the composites is thought to be important for achieving high mechanical performance. Electrospinning is a unique technology that can produce nonwoven fibrous articles with fiber diameters ranging from tens of nanometers to microns. Electrospun nanofibrous scaffolds possess an extremely high surface-to-volume ratio, tunable porosity, and malleability to conform over a wide variety of sizes and shapes,6 which resulted in suitable substrates for tissue engineering, drug carriers, wound dressing, immobilized enzymes and catalyst.7,8 Fibrous nanocomposites of HA and PLA are potential tissue growth scaffolds, which combine the ostesconductivity and bone bonding ability of HA and the highly porous microstructure with interconnected pores, ensuring accommodation of a large number of cells and uniform distribution of cells within scaffolds. Several strategies have been developed in the fabrication of nanofibrous composites of HA and PLA. Fan et al. prepared composite fibers by blending electrospinning of β-tertiary calcium phosphate and PLA matrix,9 and the incorporation of calcium phosphate increased the hydrophilicity of the scaffold and improved cell adhesion and proliferation. However, the poor dispersion or easy agglomeration of the ceramic component was * Corresponding author. Tel.: +86 28 87634023; fax: +86 28 87634649; e-mail: [email protected].

indicated within the polymeric matrix. A surfactant, hydroxysteric acid, was added in the system to effectively disperse hydrophilic HA powders into PLA solution in chloroform.10 Although this surfactant-mediation approach was effective in dispersing HA nanoparticles in PLA matrix and creating continuous fibers during the electrospinning stage, cellular assay experiments indicated that this scaffold had lower cell attachment and proliferation properties compared with fibrous HA/ PLA mats. Xu et al. modified HA nanoparticles through grafting with low molecular weight PLA, and composite fibers were fabricated from the blending electrospinning of the modified HA nanoparticles and PLA matrix.11 The nanoparticles were dispersed uniformly in the fibers at lower HA content of about 4 wt%, and the composite fibrous mats exhibited higher strength properties compared with the pristine PLA fibrous mats and HA/PLA blend mats. But HA nanoparticles began to aggregate with the increase in the HA content, which resulted in the deterioration of the mechanical properties. Therefore, attempts have been made to solve problematic issues of the agglomeration of HA crystallites in electrospun fibrous mats. We previously applied electrospun nanofibers as the reaction confinement for composite fabrication. Poly(DL-lactide) (PDLLA) ultrafine fibers with calcium nitrate entrapment were prepared by electrospinning, and then incubated in phosphate solution to form in situ calcium phosphate on the polymer matrix. The formation of nanostructured HA and good dispersion of HA particles on the electrospun fibers were observed.12 But the mechanical properties were not significantly improved because of the polymer degradation in the process of HA growth under weak alkaline conditions and lack of interaction between formed HA and polymer matrix. The biomineralization process is defined as a method whereby a bone-like apatite layer is formed on a substrate after immersion in a simulated body fluid (SBF) with composition similar to that of human blood plasma. Many factors such as the surface chemistry, ionic concentrations, and components of the SBF solution can influence the growth rate of the apatite layer.13 Collagen and gelatin were used in the biomineralization process showing the same self-assembly profiles as natural bone, and it

10.1021/cg800641s CCC: $40.75  2008 American Chemical Society Published on Web 11/12/2008

Hydroxyapatite on Electrospun Poly(DL-lactide) Fibers

was suggested that controlled nucleation and growth of crystals on organic templates should play an important part in the biomineralization process.14 Therefore, a novel method was exploited in the current investigation to use electrospun PDLLA nanofibers with surface modifications as the induction sites for fabricating scaffold composites. Free amino groups were created on electrospun PDLLA fibers through an aminolysis process, which were transferred into aldehyde groups by a treatment of glutaraldehyde. Gelatin was covalently coupled via Schiff base formation between the aldehyde groups and the amino groups on gelatin molecules. The nucleation and growth of HA were supposed to be controlled through gelatin molecules, and the average size of HA can be modulated by changing the concentration of gelatin on the surface of electrospun fibers. The influences of the process parameters of aminolysis, gelatin grafting and SBF incubation on the mineral growth rate and crystal size were also quantitatively evaluated with kinetic models. Experimental Section Materials. PDLLA (Mw )147 kDa, Mw/Mn )1.35) was synthesized in our laboratory. The molecular weight was determined by gel permeation chromatography (GPC, waters 2695 and 2414, Milford, MA) using polystyrene as a standard. The column used was a Styragel HT 4 (7.8 × 300 mm). The mobile phase consisted of tetrahydrofuran (THF) using a regularity elution at a flow rate of 1.0 mL/min. Gelatin (porcine skin, type B powder) was purchased from Sigma (St. Louis, MO). Ultrapure water from a Milli-Q biocel purification system (UPIIV-20, Shanghai UP Scientific Instrument Co., Shanghai, China) was used. All other chemicals and solvents were of reagent grade or better, and purchased from Changzheng Regents Company (Chengdu, China) unless otherwise indicated. Preparation of Electrospun Fibrous Mats. The electrospinning process was performed as described elsewhere.15 Briefly, the electrospinning apparatus was equipped with a high-voltage statitron (Tianjing High Voltage Power Supply Co., Tianjing, China) of maximal voltage of 50 kV. The polymer solution was added in a 2-mL syringe attached to a circular-shaped metal syringe needle as the nozzle. An oblong counter electrode was located about 15 cm from the capillary tip. The flow rate of the polymer solution was controlled by a precision pump (Zhejiang University Medical Instrument Co., Hangzhou, China) to maintain a steady flow from the capillary outlet. Electrospun PDLLA fibrous mats were vaccuum-dried at room temperature for 2 days to completely remove any solvent residue. Aminolysis and Gelatin Grafting on Electrospun PDLLA Fibers. Electrospun PDLLA fibrous mats was immersed in 1,6-hexanediamine solution in isopropanol. The 1,6-hexanediamine concentration, incubation temperature and time period were optimized, and the amount of amino groups on the fibers surface and the residual molecular weight of PDLLA matrix were set as the end points. Aminolyzed fibrous mats were rinsed with pure water to remove free 1,6-hexanediamine, and dried as before. The aminolyzed mats were immersed in 0.5 wt% glutaraldehyde solution for 1 h at room temperature to transform amino groups into aldehyde groups, followed by rinsing with large amounts of pure water to remove free glutaraldehyde. The fibrous mats were then incubated in 2 mg/mL gelatin solution in phosphate buffered saline (PBS, pH ) 7.4) for 24 h at room temperature. Gelatin grafted fibrous mats were washed with PBS until no free gelatin could be detected in the washing solution, then, rinsed with pure water to remove inorganic salts before vaccuum-dried at room temperature. Characterization of Aminolyzed and Gelatin Grafted Fibers. The diameters of electrospun fibers and morphologies of fibrous mats were determined by a scanning electron microscope (SEM, FEI Quanta 200, The Netherlands) equipped with a field-emission gun (20 kV) and Robinson detector after 2 min of gold coating to minimize charging effect. The fibers diameter was measured from SEM images with the magnification of 10 000, and five images were used for each fibrous sample. From each image, at least 20 different fibers and 200 different segments were randomly selected and their diameter was measured to generate an average fiber diameter by using the tool of Photoshop 10.0 edition.15 The polymer matrices were subjected to degrade during the

Crystal Growth & Design, Vol. 8, No. 12, 2008 4577 aminolysis process, and triple samples of each aminolyzed fibers were dissolved into THF and centrifuged to remove inorganic salts. The molecular weight of the matrix polymer was determined by GPC as described above. To clarify the effect of surface modification on the hydrophilicity of fibrous mats, a drop of purified water was deposited onto the fibrous mat surface using a microsyringe. The water contact angles (WCA) of water drops on the electrospun fibrous mat were measured on Kruss GmbH DSA 100 Mk 2 goniometer (Hamburg, Germany) followed by image processing of sessile drop with DSA 1.8 software. The final results were obtained by averaging at least five separate runs. Attenuated total reflectance-Fourier transform infrared (ATR-FTIR, Thermo Nicolet 5700, Madison, WI) was performed to analyze the chemical group transformation, and the spectra were collected over the range of 4000-400 cm-1. Chemical compositions of the mats surface were determined by X-ray photoelectron spectroscopy (XPS, XSAM800, Kratos Ltd., Britain) using Mg KR1,2 radiation, and data was processed by using Kratos VISION 2000. The total acquisition time was 15 min for each sample. The overlapping peaks were resolved by the peak synthesis method, applying Gaussian peak components after Shirley type background subtraction. Determination of the Amount of Amino Groups and Grafted Gelatin. The indicator, ninhydrin, was used to quantitatively detect the amount of amino groups on the aminolyzed fibers.16 Briefly, the fibrous mats were immersed in 1.0 mol/L ninhydrin solution in ethanol for 1 min, and then transferred into a glass tube, followed by heating at 80 °C for 10 min to accelerate the reaction between ninhydrin and amino groups. After removal of the adsorbed ethanol, 5 mL 1,4-dioxane was added into the tube to dissolve the fibrous mats when the mats surface displayed purple, followed by the addition of another 5 mL of isopropanol to stabilize the purple compound. The amino groups content was detected at 540 nm with an ultraviolet-visible spectrophotometer (UV-2550, Shimadzu, Japan), in which the calibration curve was obtained with 1,4-dioxane/isopropanol (1/1, v/v) solution containing 1,6-hexanediamine of known concentrations. The hydroproline analysis was employed to quantitatively detect the amount of gelatin on the fibrous mats.17 Briefly, the gelatin grafted fibrous mats were placed in 2 mL of 6 mol/L HCl solution, and was incubated at 120 °C for 24 h to degrade the gelatin and PDLLA matrix completely under reduced pressure. After removal of HCl at 70 °C, the residues were dissolved in 2 mL of water. One milliliter 50 mM chloramine-T solution was added and reacted with this solution at 25 °C for 20 min, followed by the addition of 1 mL of 3.15 mol/L perchloric acid solution. Five minutes later, the mixture was treated with 1 mL of 10% dimethylaminobenzaldehyde solution in ethylene glycol monomethyl ether at 60 °C for 40 min. The absorbance at 562 nm was measured on the UV-vis spectrophotometer. The gelatin content was quantified by referring to a calibration curve obtained with pure gelatin under the same procedures. Mineralization Process. SBF with the concentrations of Ca2+ and PO43- ions 1.5 times larger than those of 1.0 SBF was chosen to trigger homogeneous nucleation and growth.18 The electrospun PDLLA fibers, aminolyzed and gelatin grafted fibrous mats were sectioned into 80 × 80 mm with a thickness of 0.5 mm. Each specimen was immersed in 50 mL of SBF at 37 °C, which was maintained using the thermostatted water bath (Taichang Medical Apparatus Co., Jiangsu, China). The suspensions were changed with fresh SBF solution every other day. Upon removal from SBF at predetermined intervals, the fibrous mats were gently rinsed with pure water and dried as above. Characterization of Mineralized Fibers. The morphology of mineralized fibers and the HA dispersion profiles within the composite were investigated by SEM as described above. The HA formation process and the interactions with fiber matrix were checked by FTIR as described above. To investigate the crystalline phase of calcium phosphate precipitates formed on the PDLLA fibers, samples (20 × 20 mm) were analyzed with X-ray diffraction (XRD, Philips X’Pert PRO, The Netherlands) over the 2 theta range from 20 to 50° with a scanning speed of 0.35°/min, using Cu KR radiation (λ ) 1.54060 Å). The diffraction peak broadening due to small crystallites can be semiquantitatively estimated from the Scherrer equation: β1/2 ) (Kλ)/ (Dcos θ),19 where β1/2 is the full-width at half-maximum in 2 h calculated by the XRD equipment software, K a constant set to 1, λ the X-ray wavelength in Angstroms, D roughly the average crystallite size, and θ the diffraction angle of the corresponding reflex. Thermogravimetric analysis (TGA, Netzsch STA 449C, Bavaria, Germany) was employed to determine the actual yield of HA in the composite

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Figure 1. SEM images of electrospun PDLLA fibers (a), aminolyzed (b), and gelatin grafted fibers (c). material. Approximately 10 mg of sample was heated from 20 to 500 °C with a heating rate of 10 °C/min in perforated and covered aluminum pans under a nitrogen purge. The electrospun fibrous mats were punched into small strips (70.0 × 7.0 × 0.6 mm), and the uniaxial tensile properties were characterized using an all-purpose mechanical testing machine (Instron 5567, MA, USA). The stress-strain curves of the fibrous mats were constructed from the load deformation curves recorded at a stretching speed of 0.5 mm/s. Five separate runs were performed for each sample, and the Young’s modulus, tensile strength, and elongation at break were obtained from the stress-strain curves.

Results and Discussion Aminolysis Process of Electrospun PDLLA Fibers. Figure 1a shows the morphology of electrospun PDLLA fibers, illustrating porous fibrous mats with bead-free and randomly arrayed fibers. The average diameter was 1.55 ( 0.42 µm. To stably immobilize gelatin onto electrospun PDLLA fibers, pretreatment of the surfaces is necessary because of the intrinsic hydrophobicity and absence of active sites in the molecules. Aminolysis was indicated as a convenient and effective method to introduce free amino groups onto polyester materials, accompanied by enhancement of their surface hydrophilicity.16 This method was used for the reaction between the ester groups of polyester and diamine. Hence, aminolysis in principle was an alkaline catalyzed degradation of PDLLA matrix, which resulted in a decrease of molecular weight even at rather short reaction time and destroyed the fibrous structure. The aminolysis process, such as the incubation time period, temperature and diamine concentration, was optimized with regard to the amount of amino groups and the molecular weight residual of PDLLA matrix. After aminolyzed in 0.02 g/mL 1,6-hexanediamine solution in isopropanol at 37 °C for 10 min, the density of surface NH2 groups on the obtained fibers was 2.0 ( 0.2 nmol/ cm2. There was 21.3 ( 2.8% of molecular weight decrease of the matrix polymer, and the morphology of aminolyzed fibrous mats is shown in Figure 1b. The aminolyzed fibers were analyzed by ATR-FTIR for structural changes of the matrix polymer and by XPS for the chemical groups transformation on the fibers surface. As shown in Figure 2A, new absorption peaks appeared in the ATR-FTIR spectrum of aminolyzed fibers at 1680 cm-1 (νCdO in -CONH-) and 1540 cm-1 (νN-H in -CONH-), which were characteristic of the amide bonds, and stronger peaks at 3500-3700 cm-1 (νN-H in -NH2), which was characteristic of amino groups (Figure 2A, a2). Figure 2B,C showed the XPS spectra of electrospun PDLLA and aminolyzed fibrous mats. Electrospun fibers displayed two peaks corresponding to C1s (284.8 eV) and O1s (530.0 eV), as expected. As shown in the amplified images in Figure 2C, c2, the aminolyzed fibers indicated an additional peak at 399.6 eV (N1s), which was attributed to the N element coming from the amino groups (-NH2) and amide bonds (-CONH-). These results suggested that amino groups had covalently bound to the backbone of the matrix polymer.

Figure 2. ATR-FTIR spectra (A), XPS whole spectra (B), magnified N1s peaks (C), and atomic concentrations (D) of electrospun PDLLA fibers (a1, b1, c1, and d1), aminolyzed fibers (a2, b2, c2, and d2), and gelatin grafted fibers (a3, b3, c3, and d3).

Gelatin Immobilization Process on the Aminolyzed Fibers. The free amino groups on fibers surface can not only modify the hydrophilicity, but also provide the necessary active sites, and other bioactive components can be further immobilized. Gelatin was immobilized onto the aminolyzed fibers using glutaraldehyde as a coupling agent, and Figure 1c shows SEM observations of the gelatin grafted fibers. As shown in Figure 2A, a3, stronger absorption peaks appeared in the ATR-FTIR spectrum of gelatin grafted fibers at 1600-1700 cm-1 (νCdO in -CONH-) and 3500-3700 cm-1 (νO-H in -COOH), which were the main peaks characteristic of gelatin. On the basis of the XPS spectra of Figure 2B,C, the N1s of gelatin grafted fibers had a larger peak area, which was attributed to the amine groups and amide bonds of gelatin. Figure 2D shows the quantitative results of the percentage of O1s, N1s and C1s on the surface of electrospun PDLLA fibers, the aminolyzed and gelatin grafted fibers. There was a significant increase in the N1s concentration after gelatin immobilization. The gelatin amount was quantified by a hydroproline colorimetry analysis, indicating that the amount of grafted gelatin was 0.21 ( 0.04 µg/cm2 on the surface of electrospun PDLLA fibers. It indicated that chemical groups with lower binding energy, for example, methyl groups, were enriched on the surface of electrospun PDLLA fibers due to the high voltage of the electrospinning process, which resulted in hydrophobic surface of electrospun fibrous mats.20 The water contact angles measured

Hydroxyapatite on Electrospun Poly(DL-lactide) Fibers

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Figure 3. SEM images of electrospun PDLLA fiber (a), aminolyzed (b) and gelatin grafted fibers (c) after incubation in SBF for 2 (a1, b1, and c1), 4 (a2, b2, and c2) and 6 days (a3, b3, and c3) (insets showing higher magnifications).

with the sessile drop method, and electrospun PDLLA fibers, the aminolyzed and gelatin grafted fibers had WCA values of 137.6 ( 3.1°, 106.7 ( 3.4° and 0°, respectively. There were terminal amino and hydroxyl groups on the fibers surface after aminolysis, and less hydrophobic surface was shown for aminolyzed fibrous mats. At the surface of gelatin grafted mats, the hydrophilic parts of the gelatin would reorganize their molecular structure to generate a hydrophilic parts dominating region, resulting in the superhydrophilic surface. Mineralization Process of Functionalized Fibers. The formation of calcium phosphate on electrospun PDLLA fibers, aminolyzed and gelatin grafted fibers was observed by SEM. Figure 3 shows SEM images of electrospun fibers with surfaces precipitates after immersion in SBF for 2, 4 and 6 days, respectively. There was no calcium phosphate precipitate formed on electrospun PDLLA fibers during the incubation (Figure 3a1, a2, and a3), due to the lack of bioactivity of PDLLA to induce nucleation and growth of calcium phosphate.21 Amino groups on organic templates showed low bioactivity to induce calcium phosphate formation from SBF medium.22 As shown in Figure 3b1, b2, and b3, no precipitate was found during the initial incubation, but finely dispersed crystals were formed on the aminolyzed fibers after 6 days incubation. The growth process of calcium phosphate on the surface of gelatin grafted fibers could be found from nonexistent on day 2 (Figure 3c1), to uniformly dispersed on day 4 (Figure 3c2) and a layer of enwrapping around fibers surface on day 6 after incubation (Figure 3c3). It was difficult to estimate the exact size of calcium phosphate crystals from SEM images, but it did appear that the formation of calcium phosphate was made up of very ultrafine particles with the size of below 100 nm, which were homogeneously distributed throughout the fibers surface. The formation of calcium phosphate crystals on electrospun fibers were characterized by FTIR, XRD and TG analysis, respectively. FTIR spectra of electrospun PDLLA fibers, aminolyzed and gelatin grafted fibers after incubation in SBF for 4

Figure 4. FTIR spectra (A), XRD patterns (B), TGA profiles (C), and stress-strain curves (D) of electrospun PDLLA fibers (a1, b1, c1 and d1), aminolyzed fibers (a2, b2, c2 and d2) and gelatin grafted fibers (a3, b3, c3 and d3) after incubation in SBF for 4 days.

days are shown in Figure 4A. The characteristics of the apatite phase, the ν4 PO4 bending band at about 550 cm-1 and the OH band at about 3500 cm-1, were present in the FTIR spectra of the composites.23 There were four vibrational modes theoretically present for phosphate ions: ν1, ν2, ν3, and ν4. The ν1 and ν3 phosphate modes appeared in the region 1200-900 cm-1 and another two bands of ν2 and ν4 modes appeared in the region 700-450 cm-1.24 Figure 4A, a1 was the typical PDLLA spectrum, which explained there was no calcium phosphate peaks on the fibers. As seen from the spectra of the formed composites in Figure 4A, a2 and a3, the -OH stretching band at 3572 cm-1 belonged to the -OH group along the c-column of HA lattice, and the -OH liberational bands were at 632 cm-1.

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The broad peak at around 3400 cm-1 band was arising from the H-O-H vibration of water molecules absorbed on HA and polymer surface. The ν3 PO4 stretching mode at about 1050 cm-1 was overlapped by the PDLLA spectrum (Figure 4A, a1, a2 and a3). Phosphate ν4 mode was present in the region of 660 and 520 cm-1, and was well-defined and sharp bands observed in the hydroxyapatite. Several sites were detected in this region at 633, 602, and 566 cm-1 as shown in Figure 4A, a2 and a3. This splitting of the ν4 vibrational band indicated that the low site symmetry of molecules, as two and three observed bands confirmed the presence of more than one distinction site for the phosphate group.25 Phosphate ν2 band was observed in the region of 475 and 440 cm-1 and had two sites. These were weak bands, not as strong as the ν3 and ν4 bands. As shown in Figure 4A, a3, stronger absorptions appeared on phosphate modes indicating the larger content and higher crystallinity degree of HA phase for gelatin grafted fibers. The XRD patterns of electrospun PDLLA fibers, aminolyzed and gelatin grafted fibers after incubation in SBF for 4 days are shown in Figure 4B. No distinct peak appeared for electrospun PDLLA fibers (Figure 4B, b1). Figure 4B, b3 shows the XRD profile of gelatin grafted fibrous composite with a big broad peak centered at 2θ ) 31.8°, a broad peak centered at 2θ ) 25.8° and smaller broad peaks centered at 2θ ) 28°, 42° and 47°, which are characteristic patterns of HA.26 The broad peaks were seemingly designated as nonstoichiometric HA and low-crystalline apatitic phase, which may be a mixture of amorphous calcium phosphate (ACP) and crystalline HA. In the composite there were also other forms of crystalline calcium phosphate phases, such as octacalcium phosphate (OCP), as seen from the XRD patterns of the composites (Figures 4B, b2 and b3).27 The strength of peaks indicated that higher content and crystallinity of HA were formed in the composite from gelatin grafted fibers than those from aminolyzed fibers (Figures 4B, b2 and b3). To check the component of the formed composite, TGA was conducted on the composite scaffolds of electrospun PDLLA fibers, aminolyzed and gelatin grafted fibers after incubation in SBF for 4 days.12 As shown in Figure 4C, pure polymeric fibrous mat lost nearly all the weight with the temperature increasing up to about 350 °C (Figure 4C, c1). There were about 5.3% and 15.1% of mineral contents for aminolyzed and gelatin grafted fibers, respectively, after incubation in SBF for 4 days (Figure 4C, c2 and c3). The mechanical properties of electrospun PDLLA fibers, aminolyzed and gelatin grafted fibers after incubation in SBF for 4 days were detected, and the strain-stress curves are shown in Figure 4D. It was noted that the composite formed from gelatin grafted fibers had significantly higher tensile strength (9.5 ( 0.5 MPa) than those from aminolyzed fibers (6.5 ( 0.4 MPa) and electrospun PDLLA fibers (5.2 ( 0.3 MPa). The Young’s moduli were 32.4 ( 3.9, 86.4 ( 12.7 and 113.1 ( 16.8 MPa for composite scaffolds from electrospun PDLLA fibers, aminolyzed and gelatin grafted fibers, respectively. But the strains at the failure, which were 46.1 ( 9.2, 39.5 ( 5.6 and 25.1 ( 4.4%, respectively, decreased gradually with the increase in the amount of mineral contents. The composite formed from gelatin grafted fibers had higher elastic modulus and lower strain at failure, which was explained by the fact that HA minerals rendered the electrospun fiber matrix more stiff and less plastic in its deformation, in a manner which was typical of hard inorganic phases. These above results showed that gelatin grafted fibers was favorable for HA nucleation and growth in SBF solution

Cui et al.

Figure 5. The amount of amino groups of aminolyzed fibers (a) and the gelatin contents (b) as a function of aminolysis time in 0.02 g/mL 1,6-hexanediamine solution in isopropanol at 37 °C.

compared to aminolyzed fibers. The electrostatic interaction between carboxyl groups (-COO-) of gelatin and calcium ions should occur in the initial incubation to induce the further formation of calcium phosphate nanocrystals. The gelatin grafted fibers were examined by ATR-FTIR spectra after incubation in SBF for 4 h. The asymmetric stretching vibration of a dissociated carboxyl group -COO-, observed at 1340 cm-1 for gelatin, shifted to lower wavenumber at 1331 cm-1 in the case of the composite. This red shift meant that two equivalent C-O 1.5fold bonds in -COO- were weakened because of the formation of a new bond between Ca2+ ions. The gelatin grafted on electrospun PDLLA fibers facilitated the nucleation and growth of HA. And the composite formation should be affected by the amount of gelatin grafted on electrospun fibers, which were determined by the aminolysis process as indicated above. Effect of the Amount of Grafted Gelatin on the Composite Formation. In order to quantitatively characterize the effect of the amount of grafted gelatin on HA formation, electrospun fibers were aminolyzed for different time periods. Figure 5 showed the amount of amino groups and grafted gelatin versus the aminolysis time. The amounts of amino groups and grafted gelatin were found to be characterized by “S”-shape of sigmoidal kinetic curves when plotted as a function of aminolysis time. This type of kinetics may be described by eq 1:28

Y ) Cmax(1 - (1 + e(t-tm)⁄k)-1

(1)

where Y is the conversion representing the fraction of grafted content, and t is the variable of the aminolysis time, Cmax is the theoretical maximum value of obtaining results, tm is the time of maximum growth rate, and k is the apparent rate constant. The curves shown in Figure 5 were drafted by statistic software of SPSS 12.0 for the amount of amino groups (eq 2) and gelatin (eq 3) against the aminolysis time. Coefficients of correlation (R2) of above two curves equation were relatively high, which was in agreement with the requirements of statistic analysis. Using the kinetic model of equations set up for the aminolysis system, the amount of amino groups and grafted gelatin can be obtained through designing the aminolysis time.

Hydroxyapatite on Electrospun Poly(DL-lactide) Fibers

Y ) 4.11(1 - (1 + e(t-9.40)⁄3.22)-1) (R2 ) 0.9998) (t-9.44)⁄3.47)-1)

Y ) 0.41(1 - (1 + e

(R ) 0.9998) 2

Crystal Growth & Design, Vol. 8, No. 12, 2008 4581

(2) (3)

Figure 6 summarizes the amount of HA for fibrous composites grafted with different amounts of gelatin after incubation in SBF. For quantitative analysis of the HA growth kinetics, the curves in Figure 6 were drafted for fibers with the gelatin contents of 0.02 (eq 4), 0.09 (eq 5) and 0.21 µg/cm2 (eq 6).

Y ) 76.83(1 - (1 + e(t-15.36)⁄2.89)-1) (R2 ) 0.9986)

(4)

Y ) 76.48(1 - (1 + e(t-8.43)⁄2.80)-1) (R2 ) 0.9988)

(5)

(t-6.54)⁄2.19)-1)

Y ) 76.70(1 - (1 + e

(R ) 0.9991) 2

(6)

where Y is HA content formed in the composite (%) and t is the incubation time in SBF (days). Based on these equations, the times of maximum HA weight growth rate could be calculated,29 and they were 6.5, 8.4, and 15.4 days after incubation for fibers with the gelatin contents of 0.21, 0.09, and 0.02 µg/cm2, respectively. The maximum growth rate reflected the ability of inducing HA and could also control the increasing rate of HA contents in the composite. Higher amounts of gelatin may result in higher degrees of binding of calcium and phosphate ions. Furthermore, the increased wettability of fibers with higher contents of grafted gelatin should increase the driving force and lower the interfacial energy for HA nucleation and growth. Previous study has shown that more calcium could bind to amelogenin proteins with increasing protein concentrations because protein alone can lower the interfacial energy for nucleation relative to homogeneous nucleation and growth.30 Therefore, the amount of HA content grown on electrospun fibrous mats could be quantitatively controlled by the gelatin content and incubation time. The XRD patterns of composites formed from gelatin grafted fibers with different gelatin contents are shown in Figure 7. For quantitative purposes, the line broadening of the (0 0 2) reflection range from 25° to 27° was used to evaluate the mean crystallite size, because this peak was well resolved and showed no interference. The crystal size (0 0 2) values were related to the crystal size in the wide dimension of the HA crystallites.29 The crystal size of HA was calculated by Scherer’s formula, and they were 10.2, 16.1, and 33.6 nm for composites from gelatin grafted fibers with the gelatin contents of 0.21, 0.09, and 0.02 µg/cm2, respectively. It indicated that the sizes decreased with the increase in the gelatin contents grafted on the fibers, and the HA crystals could be controlled in the size range of 10-20 nm, similar to the apatite crystals found in bone. The size of HA crystals was related to the available nucleating sites.31 With the increasing amounts of gelatin, the abundant

Figure 7. XRD patterns of composites formed from gelatin grafted fibers with gelatin contents of 0.02(a), 0.09 (b) and 0.21 µg/cm2 (c) after incubation in SBF for 4 days.

supply of coordination sites COO- available for complexation with Ca2+ ions led to a very large number of nuclei for the growth of HA crystalline so that the crystalline could not grow larger. Therefore, the amount of HA formed on the fibrous composites and the crystal size of formed HA were regulated through the content of gelatin grafted on the fibers, which could be further controlled by the aminolysis process. Conclusions A novel strategy was developed to prepare fibrous composites of HA and PDLLA. Gelatin was grafted on electrospun fibers to control the nucleation and growth of HA crystals. The formation of nonstoichiometric nanostructured HA and good dispersion on electrospun fibers were observed. In the aminolysis and gelatin grafting process, the amount of amino groups and gelatin could be modeled with quantitative equations as a function of the aminolysis time. The amount of HA formed on the fibrous composites and the crystal size of formed HA were regulated through the content of gelatin grafted on the fibers, which could be further determined by the aminolysis process. The fibrous nanocomposites should have potential applications as coating materials on medical devices, as scaffolds for tissue engineering, and as fillers for fiber-enforced composites. Acknowledgment. This work was supported by National Natural Science Foundation of China (20774075), Program for New Century Excellent Talents in University Funded by MOE (NECT-06-0801), and National Key Project of Scientific and Technical Supporting Programs Funded by MSTC (2006BAI16B01).

References

Figure 6. The amount of HA grown on fibrous mats with gelatin contents of 0.02, 0.09, and 0.21 µg/cm2 after incubated in SBF at 37 °C.

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