ARTICLE pubs.acs.org/JPCC
Hydroxyapatite Needle-Shaped Particles/Poly(L-lactic acid) Electrospun Scaffolds with Perfect Particle-along-Nanofiber Orientation and Significantly Enhanced Mechanical Properties Fei Peng,† Montgomery T. Shaw,‡ James R. Olson,§ and Mei Wei*,† †
Department of Chemical, Materials, and Biomolecular Engineering and ‡Polymer Program, Institute of Materials Science, University of Connecticut, Storrs, Connecticut 06269, United States § Teleflex Medical, 1295 Main Street, P.O. Box 219, Coventry, Connecticut 06238, United States ABSTRACT: Electrospun hydroxyapatite (HA)/biopolymer fibrous composites have attracted much interest in the field of hard-tissue engineering. Unfortunately, poor mechanical property is a continuing problem and limits their usefulness in many applications. In this study, we attempted to improve the mechanical strength, modulus, and toughness of the HA/poly(L-lactic acid) (PLLA) electrospun scaffold by aligning the needle-shaped HA particles within the PLLA nanofibers. Three types of HA particles with different aspect ratios and sizes were incorporated into PLLA nanofibrous scaffolds with a random or aligned fibrous assembly. It was the first time that the needle-shaped HA particles with high aspect ratios were perfectly oriented along the long axes of the nanofibers. All HA particles significantly enhanced the tensile modulus, strength, and toughness of the corresponding scaffold, but to different extents. The dramatic reinforcement effects for different morphologies were rationalized using the HalpinTsai and shear-lag models for composites.
1. INTRODUCTION Human bone is a ceramic/biopolymer hybrid with a threedimensional woven structure made up of 6570 wt % hydroxyapatite (HA, Ca10(PO4)6(OH)2) crystals and 3035 wt % type I collagen fibers.13 As for its microstructure, the matured bone is constructed with lamellae of HA crystals (2528 nm wide and 4 nm thick) oriented along the long axes of collagen nanofibers (diameter around 80100 nm).4,5 This HA-oriented-in-collagenfiber structure contributes the most to the superior mechanical properties of bone.4,5 In bone, the HA crystals mainly contribute to its compressive strength and stiffness, while collagen fibers provide the corresponding tensile properties.4 The development of scaffolds mimicking the composition, structure, and mechanical properties of human bone matrix has become one of the major goals in the bone regeneration and tissue engineering fields. Recently, electrospinning has been used widely to fabricate bioceramic/biopolymer nanofibrous composites for biomedical applications.610 Many synthetic biopolymers have been employed to fabricate tissue engineering scaffolds due to their relatively low cost, high flexibility, and stable chemical and physical properties compared to some natural biopolymers. Among them, poly(L-lactic acid) (PLLA) is one of the most extensively used biopolymers, as it has good biocompatibility and is one of the few bioresorbable polymers that has been approved by the U.S. Food and Drug Administration for in vivo applications.1113 In addition, PLLA has a much higher elastic compliance than HA, and its mechanical properties can be tailored by adjusting its crystallinity and molecular weight through synthesis and variations in the manufacturing process.1215 r 2011 American Chemical Society
In recent years, inorganic particles, such as HA, carbon nanotubes, FeO, and TiO2, have been widely co-electrospun into polymer nanofibers for enhanced mechanical properties.10,1619 However, current reported works on orienting needle-shaped bioceramic particles along nanosized organic fibers are still far from perfect.6,7,20,21 The bioceramic needle-oriented-in-polymer fiber structure is challenging but very important for enhancing the mechanical properties of the scaffold. In the study described herein, highly crystalline pure HA particles with a spherical or a needle shape have been incorporated into PLLA nanofibers by electrospinning. The morphologies of the resulting nanofibrous scaffolds and the orientation of the HA particles within the electrospun nanofibers have been studied in depth. The HAPLLA adhesion and the influence of different HA particles on the thermal, crystallization, and mechanical properties of the HA/PLLA electrospun scaffolds have also been investigated systematically. As is well-known, it is very difficult to evaluate precisely the mechanical properties of the electrospun scaffolds due to their highly porous and interconnected fibrous structures. It is more complicated to directly and accurately interpret the reinforcement effect of the oriented HA particles with different morphologies. In order to overcome these challenges, the mechanical properties of the electrospun scaffolds have been evaluated using the void-excluded tensile data. The reinforcement effects of different HA particles have been Received: February 11, 2011 Revised: April 26, 2011 Published: July 27, 2011 15743
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The Journal of Physical Chemistry C compared with predictions from the HalpinTsai and shear-lag models.
2. EXPERIMENTAL METHODS 2.1. Synthesis of HA Particles. The nanosized, sphericalshaped HA nanoparticles with an aspect ratio (a) of around 3 (Nano3) and the nanosized, needle-shaped HA nanoparticles with a ≈ 7 (Nano7) were synthesized using a metathesis method. Typically, calcium nitrate (99%, Sigma) and diammonium phosphate (99%, Sigma) solutions were prepared at a Ca/P molar ratio of 1.67. After the pH of each solution was brought up to 11 with ammonia hydroxide solution (2830%, Alfa Aesar), the phosphate solution was added to the calcium nitrate solution, leading to the gradual precipitation of HA particles. By controlling the reagent concentrations, heating temperature, and the heating and aging times, HA nanoparticles with different sizes and aspect ratios were obtained. To synthesize microsized, needle-shaped HA microparticles with a ≈ 17 (Micro17), the pH of calcium nitrate and diammonia phosphate solutions were adjusted to 4 using dilute nitric acid solution. The two solutions were then mixed with urea (certified ACS reagent, Fisher Scientific) as an additive.22 The mixed solution was heated at 8595 °C with mild stirring, resulting in precipitation of rodshaped octacalcium phosphate (OCP).23 Through a crystal transformation from OCP rods, the Micro17 HA particles were obtained.23 All HA particles were washed using ethanol, dried, and ground into fine powders for later use. 2.2. Characterization of HA Particles. The morphologies of HA particles were observed using a field emission scanning electron microscope (FESEM, JEOL JSM-6335F) at an acceleration voltage of 5 kV. The length and diameter of randomly chosen single HA particles, which were lying flat on the sample holder, were measured using a software analysis (Image Tool, version 2.00, University of Texas Health Science Center in San Antonio, TX, USA) from the FESEM images. The aspect ratio of each HA particle type was calculated by dividing the mean particle length by the mean particle diameter. The composition of the HA particles was examined using powder wide-angle X-ray diffraction (WAXD) (XRD, Bruker AXS D5005) over a 2θ range from 10 to 60°. 2.3. Electrospinning PLLA and HA/PLLA Scaffolds. To prepare PLLA electrospun scaffolds, PLLA (Mn = 60 145 Da, Mw/Mn = 1.64) pellets were dissolved in dichloromethane (DCM)/N,N-dimethylformamide (DMF) (5/5, v/v) at 12 wt %. To prepare HA/PLLA (20/80, w/w) electrospun scaffolds, HA particles were weighed, dispersed in DMF, sonicated for 45 min, and then mixed with the PLLA solution. The PLLA or HA/ PLLA spinning dope was loaded into a 20-mL plastic syringe connected to a stainless steel capillary, whose feeding rate was controlled at 2.0 mL/h by a micro infusion pump (WZ-50C2, Zhejiang University Medical Instrument Co., China). A positive DC high-voltage power supply (DW-P303-1AC, Tianjin Dongwen High-Voltage Power Supply Factory, China) was connected to the capillary to generate a high electric field of 2.0 kV/cm within the 10 cm distance between the capillary and the grounded collector. A flat plate wrapped with aluminum foil was used as the collector to spin scaffolds with a random fibrous assembly (i.e., random scaffold). A drum (diameter = 25.0 cm) wrapped with aluminum foil, rotating at 800 m/min, was used to prepare scaffolds with an aligned fibrous assembly. The spinning time for each loaded spinning dope was kept within 2.5 h, and multiple freshly prepared spinning dopes were used to prepare scaffolds.
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The electrospun scaffolds were dried under vacuum at room temperature for 72 h. 2.4. Characterization of PLLA and HA/PLLA Electrospun Scaffolds. 2.4.1. Morphologies of Electrospun Scaffolds and the Layout of HA Particles within Them. The morphologies of electrospun scaffolds were observed using FESEM. The diameters of the electrospun nanofibers within the scaffolds were measured from their FESEM images. The layout of HA particles within HA/PLLA scaffolds was directly observed using transmission electron microscopy (TEM, JEM-1200EX, Japan). The specimens for TEM were prepared by direct deposition of the electrospun fibers onto a copper grid coated with a carbon film, and investigated using low electron dose imaging at an acceleration voltage of 90 kV. To examine the HA orientation within the electrospun scaffolds at a macroscale, HA/PLLA random scaffolds were examined using powder WAXD. 2.4.2. Thermal and Crystallization Behaviors of PLLA within Electrospun Scaffolds. The thermal properties and crystallization behavior of PLLA within the as-spun PLLA and HA/PLLA scaffolds were studied using a differential scanning calorimeter (DSC Q20, TA Instruments, USA). The scaffold specimens with a mass around 5 mg were first cooled from room temperature to 50 °C and then heated to 200 °C at 5 °C/min (first heat-up cycle). The temperature of cold crystallization (Tc), the enthalpy change at Tc (ΔHc), and the heat of fusion at the melting temperature (ΔHm) were determined from the DSC trace for the first heat-up cycle. For HA/PLLA specimens, the ΔHc and ΔHm data were normalized to the mass of PLLA within the composite scaffolds. The fraction crystallinity of PLLA formed during electrospinning (Xc) was calculated by dividing ΔHm ΔHc by the reported heat of fusion of a perfect PLLA crystal (ΔHmp = 93.6 J/g), i.e., Xc = (ΔHm ΔHc)/ΔHmp.24 The crystallinity and crystal phase behavior of PLLA within PLLA and Nano7/PLLA electrospun scaffolds were studied further by annealing the as-spun scaffolds at their respective Tc values. At the end of a 3-h annealing, each scaffold was quenched to room temperature and examined using powder WAXD. The net area underneath each peak of interest was integrated using the evaluation software EVA (Eva Application, version 6.0.0.1, Socabim, Austria). 2.4.3. Mechanical Properties of Electrospun Scaffolds. The mechanical properties of the electrospun scaffolds were evaluated using a uniaxial tensile test carried out on a dynamic mechanical analyzer (DMA 2980, TA Instruments, USA) under a controlled-force tension mode. Each bend-shaped scaffold specimen (with a width around 7 mm, a length around 15 mm, and an apparent thickness around 1 mm) was tested with a preload of 0.010 N and at a force ramping rate of 0.050 N/min at 25.0 ( 0.5 °C. The scaffold with aligned fibrous assembly was tested in the fiber direction. In order to study the reinforcement effect of different HA particles to the scaffolds, the void-excluded cross-sectional area (S) of each tensile specimen was determined using eq 1: S¼
M dL
ð1Þ
where M is the mass of each specimen; d is the density of the scaffold (for PLLA scaffolds, d = dPLLA = 1.269 g/cm3; for HA/ PLLA scaffolds, d = dHA/PLLA = 1.647 g/cm3 with the density of HA as 3.17 g/cm3); L is the length of the scaffold.25,26 Using S, the void-excluded tensile data were calculated from the experimental results. Each reported value is the average of 815 15744
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Figure 1. Morphologies of (a) Nano3, (b) Nano7, and (c) Micro17 particles.
independent observations, and is presented as the mean value and the sample standard deviation. 2.5. Modeling of Elastic Moduli of HA/PLLA Electrospun Scaffolds. The void-excluded tensile moduli for the electrospun scaffolds (EE) were compared to the moduli calculated using the shear-lag model (ESL) and the HalpinTsai model (EHT).27,28 Generally, ESL was calculated according to the shear-lag model for the longitudinal tensile modulus of aligned short fiber (i.e., HA particles) reinforced composite using eq 2:27 ESL ¼ ηEHA VHA þ EPLLA ð1 VHA Þ
ð2Þ
where EPLLA is the elastic modulus of the PLLA matrix. In order to exclude the influences from the complicated interactions of the electrospun fibers as well as the large void volume among them, EPLLA was taken from the observed EE of neat-PLLA electrospun scaffold for modeling. EHA is the elastic modulus of HA particles. As the studied HA particles were too stiff and short for direct modulus determination using an atomic force microscope, different EHA values ranging from 3 to 180 GPa were used for the modeling.2,29,30 VHA = 0.0921 is the volume fraction of HA particles within the HA/PLLA scaffold. η is a factor which corrects the modulus for the shortness of HA particles: η ¼ 1
tanhðnaÞ na
ð3Þ
where a is the aspect ratio of HA particle and n represents a dimensionless group of constants: sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 2GPLLA n¼ ð4Þ EHA lnð2R=dÞ R is the averaged half-distance between two adjacent HA particles, and 2R/d was calculated using eq 5: rffiffiffiffiffiffiffiffiffiffi π 2R=d ¼ ð5Þ 4VHA GPLLA is the shear modulus of PLLA, which was calculated using Poisson’s ratio of PLLA (υPLLA) as 0.35 (ESL was not sensitive to υPLLA values ranging from 0.3 to 0.50, which are typical values for semicrystalline polymers) and eq 6: GPLLA ¼
EPLLA 2ð1 þ υPLLA Þ
ð6Þ
EHT was calculated according to the HalpinTsai model for the elastic modulus of a two-dimensional random short fiber
reinforced HA/PLLA composite using eq 7:28 EHT ¼
3 5 EL þ ET 8 8
ð7Þ
where EL is the longitudinal modulus of the composite: EL ¼ EPLLA
1 þ 2aηL VHA 1 ηL VHA
ð8Þ
and ηL ¼
EHA =EPLLA 1 EHA =EPLLA þ 2a
ð9Þ
ET is the transverse modulus of an aligned short fiber composite: ET ¼ EPLLA
1 þ 2ηT VHA 1 ηT VHA
ð10Þ
and ηT ¼
EHA =EPLLA 1 EHA =EPLLA þ 2
ð11Þ
3. RESULTS 3.1. Morphologies of HA Particles. As shown in Figure 1, Nano3 particles demonstrate a nanosize (length l = 88 ( 33 nm) and a low aspect ratio around 3 (a = 2.7). Nano7 particles are also nanosized (l = 287 ( 42 nm) but with a needle shape and a higher aspect ratio around 7 (a = 6.8). Micro17 particles are microparticles with lengths ranging from less than 1 μm to several micrometers (l = 2117 ( 729 nm) and an average aspect ratio around 17 (a = 16.6). 3.2. Morphologies of PLLA and HA/PLLA Electrospun Scaffolds. All the scaffolds, including PLLA, HA/PLLA random, and aligned electrospun scaffolds, comprise smooth nanofibers with a diameter between 300 and 500 nm (Figure 2 and Table 2). No knoblike nodes with agglomerated HA particles can be found in these scaffolds. With the tangential force of the rotating collector, the electrospun fibers have been stretched and aligned with each other in the aligned scaffolds (Figure 2eh).31 Nano3 and Nano7 nanoparticles are buried within the electrospun fibers, but their orientations are hard to discern using the FESEM images (Figure 2b,c,f,g). In contrast, Micro17 microparticles can be clearly seen to be well oriented in the axial direction of the nanofibers (indicated by arrows in Figure 2d,h). 15745
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Figure 3. Layout of (a) Nano3, (b) Nano7, and (c, d) Micro17 particles within electrospun nanofibers.
Figure 2. Morphologies of electrospun (a) PLLA, (b) Nano3/PLLA, (c) Nano7/PLLA, and (d) Micro17/PLLA random scaffolds and (e) PLLA, (f) Nano3/PLLA, (g) Nano7/PLLA, and (h) Micro17/PLLA aligned scaffolds. Arrows in (d) and (h) indicate visible Micro17 microparticles well oriented in the axial direction of the nanofibers.
3.3. Layout and Orientation of HA Particles within the HA/ PLLA Electrospun Scaffolds. To view more clearly the orienta-
tion of HA particles within PLLA fibers, TEM was employed. Some of the Nano3 particles are well dispersed and oriented axially, but the others are not oriented or partially oriented agglomerates (Figure 3a). In comparison, most of the Nano7 particles exhibit almost perfect axial orientation (Figure 3b). Although the length of some Micro17 particles goes up to several micrometers (Figure 3d), Micro17 particles are all encased within the PLLA fiber matrix and are perfectly oriented in the fiber’s long axial direction. Figure 4a shows the powder WAXD pattern of different HA particles. As illustrated in Figure 4c, these particles are randomly laid down with all possible orientations. Therefore, their patterns in Figure 4a clearly show that the locations and relative intensities of almost all the allowed diffraction peaks match very well with those of the standard powder pattern of pure HA particles (Figure 4a). The pattern for Micro17 particles exhibits the sharpest peaks due to their large crystal size and high crystallinity. The patterns for Nano3 and Nano7 nanoparticles are a bit more diffuse but still sharp, indicating good crystallinity. Figure 4b shows the powder WAXD patterns of PLLA and HA/PLLA
electrospun scaffolds. The scaffold specimens were mounted as illustrated in Figure 4d. Apparently, the intensity of some HA peaks changed dramatically in the scaffolds (indicated by black solid or gray open triangles in Figure 4b). Because Micro17 particles and the Micro17/PLLA scaffold have the sharpest HA peaks, their patterns were chosen for a quantitative study. The quantitative diffraction intensities for some Micro17 peaks in Figure 4a,b were obtained by integrating the net area underneath each peak. To eliminate the influence from the scanning time, sample amount, and mounting of each sample, the intensity data were normalized by fixing the intensity of the (300) peak to 60 (Table 1). Obviously, the intensities for the diffractions of the planes related to the l index decrease dramatically or even disappear, especially those from (111), (002), (211), and (004). Meanwhile, the intensities for diffractions from high h or k index involved planes are still high. In particular, the diffraction intensity for the (100) peak increases from 9.0 to 14.6. Unfortunately, no apparent increase of the (200) peak was observed, which may be because it is too weak. 3.4. Thermal and the Crystallization Behavior of PLLA in Electrospun Scaffolds. Summarized in Table 2 are DSC results of electrospun scaffolds from their first heat-up DSC cycles. This first cycle can reflect the influences of the electrospinning process and the original orientation of HA particles on the crystalline morphology of PLLA. No consistent correlation has been found between the fiber diameter ranging from 300 to 500 nm and the thermal properties of the PLLA. The glass transition temperatures (Tg) for PLLA within all the studied scaffolds are clustered randomly around 63.3 °C. Regardless of their fibrous assembly, the PLLA and the HA/PLLA scaffolds crystallize around Tc = 98.499.7 °C and Tc = 80.283.4 °C, respectively (Figure 5a). The Xc data demonstrate that HA particles have introduced more cold crystallization of PLLA during heating. The melting of PLLA provides two adjacent endothermal peaks (indicated by gray dashed lines in Figure 5a). The first peak at Tm1 is assigned to the melting of PLLA in the β crystalline form,25,26,32 and the second peak at Tm2 is assigned to the melting of PLLA in the R phase crystalline form.26,32 For the PLLA random scaffold, the Tm1 peak is around 148 °C, which is distinct from Tm2 around 156 °C. The Tm1 peaks of HA/PLLA random 15746
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Figure 4. Powder WAXD patterns of (a) HA particles and (b) HA/PLLA electrospun random scaffolds. In (b), the solid triangles indicate HA peaks that kept good intensities and the gray open triangles indicate peaks showing decreased intensities. The HA particles and electrospun scaffold specimens were mounted as illustrated in (c) and (d), respectively.
Table 1. Comparison of the Diffraction Intensities of Some Significantly Changed Peaks in the Powder WAXD Patterns of Micro17 Particles and Micro17/PLLA Electrospun Random Scaffold, Respectively Miller indices
integrated net area reference
2θ, deg
h
k
l
intensity
Micro17
Micro17/PLLA
10.82
1
0
0
12
9.0
14.6
21.72
2
0
0
10
3.3
2.5
22.90
1
1
1
10
4.8
0.0
25.88
0
0
2
40
17.2
0.0
28.97
2
1
0
18
14.9
9.0
31.77
2
1
1
100
63.7
5.8
32.90
3
0
0
60
60.0
60.0
39.85
3
1
0
20
19.9
15.4
51.29
4
1
0
12
9.8
6.1
53.15
0
0
4
20
8.8
0.0
Figure 5. (a) DSC traces of the first heat-up cycle for PLLA and HA/ PLLA electrospun random scaffolds. The black traces are for as-spun scaffolds. The gray traces are for the PLLA (i.e., no HA) and Nano7/ PLLA scaffolds annealed for 3 h at different temperatures (indicated with gray arrows). (b) Powder WAXD patterns of PLLA and Nano7/PLLA random scaffolds.
Table 2. DSC Results of PLLA and HA/PLLA Electrospun Scaffolds with Random or Aligned Fibrous Assembly scaffold
fibrous assambly
fiber diameter, nm
Tg, °C
Tc, °C
ΔHc, J/g
ΔHm ΔHc, J/g
Xc, %
PLLA
random
299 ( 28
63.6
99.7
15.9
16.2
17.3
Nano3/PLLA
random
498 ( 61
63.4
82.6
16.2
10.9
11.6
Nano7/PLLA
random
375 ( 55
63.2
83.4
17.0
11.4
12.2
Micro17/PLLA
random
314 ( 54
63.6
80.8
18.5
16.1
17.2
PLLA
aligned
312 ( 78
63.2
98.4
16.7
18.7
20.0
Nano3/PLLA Nano7/PLLA
aligned aligned
411 ( 67 418 ( 64
63.2 63.3
81.9 82.1
18.1 18.7
12.2 13.9
13.0 14.8
Micro17/PLLA
aligned
385 ( 79
63.7
80.2
19.2
16.3
17.4
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Table 3. Void-Excluded Tensile Results of PLLA and HA/PLLA Electrospun Scaffolds with Random or Aligned Fibrous Assemblya HA
a
fibrous assembly
fiber diameter, nm
σ, MPa
EE, MPa 60 ( 19
ε, %
EHT, MPa
ESL, MPa
random
299 ( 28
5.4 ( 0.6
21.8 ( 7.5
Nano3
random
498 ( 61
103 ( 4
5.2 ( 0.6
19.5 ( 3.0
86
64
Nano7
random
375 ( 55
359 ( 57
11.1 ( 0.7
17.6 ( 2.7
104
112
147
388
Micro17
random
314 ( 54
538 ( 42
17.3 ( 1.0
20.4 ( 1.8
aligned
312 ( 78
299 ( 40
16.5 ( 3.1
20.2 ( 6.0
Nano3
aligned
411 ( 67
522 ( 76
18.1 ( 4.3
10.6 ( 2.2
426
317
Nano7
aligned
418 ( 64
795 ( 255
22.9 ( 3.5
5.9 ( 0.7
513
552
Micro17
aligned
385 ( 79
1185 ( 257
31.0 ( 4.2
4.8 ( 0.3
707
1722
Presented EHT and ESL data are predicted using EHA = 120 GPa.29
scaffolds move up to around 154 °C and have sizes comparable to their Tm2 peaks around 157 °C. These results collectively indicate that addition of HA particles increases the melting point and the content of β phase PLLA. However, these particles do not show a pronounced influence on the R phase PLLA. The data for ΔHm ΔHc and ΔHc demonstrate that the crystallization of PLLA during both the spinning and the first heat-up cycle follows the order PLLA g Micro17/PLLA > Nano7/PLLA > Nano3/ PLLA. Also, the PLLA in aligned scaffolds consistently exhibits higher crystallinity than that in random scaffolds. To study the crystallization behavior of PLLA within HA/ PLLA scaffolds and avoid strong diffractions from large HA particles, the Nano7/PLLA scaffold was chosen for the annealing study. The DSC traces of the first heat-up cycles for the annealed scaffolds are illustrated in gray in Figure 5a. These traces do not show any PLLA cold crystallization. Higher crystallinity has been evidenced in PLLA (29.1%) and Nano7/PLLA (32.5%) scaffolds after a 3-h annealing at their respective Tc values. When annealed at the same temperature (99.7 °C), the Nano7/PLLA scaffold developed higher crystallinity (35.6%) in PLLA than the neat PLLA scaffold (29.1%). These results demonstrate that HA particles promote the crystallization of PLLA during annealing. Annealed at its Tc = 99.7 °C, the PLLA scaffold shows four peaks in its WAXD pattern (Figure 5b), indicating the presence of a mixture of R and β phases. In detail, the diffractions at 2θ = 15.4, 19.3 and 22.8° are from the (010), (203), and (015) planes of R phase, respectively.26,33 The diffraction at 2θ = 16.8° is assigned to the (110) plane of R phase and the (110) and (200) planes of the β phase.26,33 Although R and β phase PLLA could have different diffraction abilities at the same 2θ angle, the relative crystallinity of R phase (labeled as RR/(Rþβ)) can be estimated by the ratio of the integrated net area under the peaks at 2θ = 19.3° to that at 2θ = 16.8°. PLLA scaffold annealed at 99.7 °C has an RR/(Rþβ) of 17.5%. Annealed at its Tc = 83.4 °C, the Nano7/PLLA scaffold only shows two clear peaks at 2θ = 16.8 and 19.3°, respectively, and has an RR/(Rþβ) of 12.9%. Annealed at 99.7 °C, it also exhibits two weak peaks at 2θ = 15.4 and 22.8°, and its RR/(Rþβ) becomes 13.8%, indicating more R phase PLLA was formed. In conclusion, these data show that HA particles moved the cold crystallization of PLLA to a lower temperature, promoted the PLLA crystallization, and introduced more β phase. 3.5. Mechanical Properties of PLLA and HA/PLLA Electrospun Scaffolds. In this study, the mechanical properties of the electrospun scaffolds were evaluated using their void-excluded tensile data, which were obtained using the uniaxial tensile test results of the scaffolds and the void-excluded cross-sectional area of these. The calculated void-excluded tensile moduli (EE) and strength (σ), along with the tensile strain at break (ε) for the
Figure 6. Void-excluded elastic moduli of HA/PLLA electrospun scaffolds with (a) random or (b) aligned fibrous assembly predicted with HalpinTsai (EHT, black solid symbols and lines) and shear-lag (ESL, gray open symbols and lines) models using EHA values ranging from 3 to 180 GPa.
electrospun scaffolds, are summarized in Table 3. For random scaffolds, the EE of HA/PLLA scaffolds exhibits much higher values than that of the PLLA scaffolds, demonstrating pronounced reinforcement effects of various HA particles on PLLA scaffolds. Especially, the EE of the needle-shaped HA reinforced scaffolds, Micro17/PLLA and Nano7/PLLA, are 6 and 9 times as high as the modulus of the PLLA scaffold, respectively. The addition of HA particles also improves the tensile strengths of the random scaffolds to different extents (Micro17/PLLA . Nano7/PLLA > Nano3/PLLA ≈ PLLA), but does not show a significant impact on the tensile strain. Apparently, HA/PLLA random scaffolds have a higher toughness. To predict the void-excluded EHT and ESL of HA/PLLA random scaffolds, the EPLLA was taken from the EE of random PLLA scaffold (i.e., EPLLA = 60 MPa). As demonstrated in Figure 6a, all the EHT and ESL predictions level off when EHA > 60 GPa. Although the EE value of Nano3/PLLA scaffold is close to its EHT and ESL values predicted using EHA = 120 GPa, the EE data of Nano7- or Micro17-containing scaffolds are much higher than their predictions.29 The EE value of the PLLA aligned scaffold is 5 times as high as that of the PLLA random scaffold (Table 3). The tensile moduli and strengths of all the aligned scaffolds also follow the order Micro17/PLLA > Nano7/PLLA > Nano3/PLLA > PLLA. 15748
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The Journal of Physical Chemistry C Meanwhile, the aligned scaffolds are brittle due to the incorporation of HA particles. Their ultimate tensile strains decrease in the following order: Micro17/PLLA ≈ Nano7/PLLA < Nano3/PLLA , PLLA. The EHT and ESL for the aligned scaffolds with HA were calculated using EPLLA = 299 MPa, i.e., the EE of the PLLA aligned scaffold. Most EHT and ESL predictions level off when EHA > 60 GPa, but the ESL for the aligned Micro17/PLLA continues to increase with EHA within the studied range (Figure 6b). In Table 3, the observed EE values of both the scaffolds with Nano3 and Nano7 are much higher than their EHT and ESL values predicted using EHA = 120 GPa. The EE of Micro17/PLLA scaffold (1185 ( 257 MPa) is higher than its EHT prediction (707 MPa), but still lower than its ESL prediction at 1722 MPa.
4. DISCUSSION 4.1. Morphologies of Different HA Particles and Their Orientations within the HA/PLLA Electrospun Scaffolds.
Despite their dramatic differences in size and aspect ratio, Nano3, Nano7, and Micro17 particles were all highly crystalline. In both random and aligned HA/PLLA electrospun scaffolds, 9.2 vol % (i.e., 20 wt %) HA particles were successfully incorporated into the PLLA nanofibers using the electrospinning method. It was observed by TEM that only a portion of the Nano3 particles showed some orientation along the long axes of the electrospun nanofiber, while almost all the needle-shaped Nano7 and Micro17 particles were homogenously dispersed and perfectly oriented along the electrospun nanofibers (Figure 3). When observed by TEM at 200000, electrospun fibers were thinned by the focused high-energy electron beam and their diameter decreased to less than 200 nm (Figure 3ac vs Figure 2bd). This indicated good adhesion between the HA particles and the PLLA matrix, because no void was observed around the edge of HA particles during this process. Meanwhile, it is hard to tell whether the observed HA orientation was obtained during the electrospinning or as a result of fiber thinning.16 In comparison, it is apparent that the perfect orientation of Micro17 particles within Micro17/PLLA scaffold was attained unstretched, as shown in Figures 2h and 3d. To investigate the orientation of all HA particles, their layout within the whole scaffold was examined by scanning the as-spun HA/PLLA electrospun random scaffolds using powder WAXD. It was found that the HA diffractions from the planes related to the l index (especially the (00l) planes) were hardly collected by the detector. It is well-known that HA has a hexagonal crystal cell, in which the a and b axes are identical and the c axis lies in the long axis of the HA particle. Also considering the mounting of the specimens (Figure 4d), the WAXD results suggest that most of the HA particles had lain parallel to the plane of the scaffold. Moreover, most of the HA particles must have been well oriented along the fibers. Otherwise, considerable diffractions from the l index involved planes should have been collected from those particles oriented to form big angles with the fiber long axis (as the white HA particles illustrated in Figure 4d). Therefore, it can be concluded that most of the three types of HA particles, especially the needle-shaped Nano7 and Micro17 particles, were well oriented along the fibers during the electrospinning process. 4.2. HAPLLA Adhesion in Electrospun Scaffolds. There were both physical trapping and chemical nonbonding interactions between the PLLA nanofibers and all studied HA particles, creating stable adhesion between them. The physical trapping
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came from the continuous stretching of the electrospun fiber during the spinning process, when high compressive stress had been continuously working on PLLA and HA from the sides and made them tightly adhered together. The chemical nonbonding interactions should be mainly attributed to two possibilities: (1) the interactions between the Ca2þ ions on HA surface and the carboxyl/hydroxyl end groups on PLLA chains; (2) nonbonding interactions between the Ca2þ ions on HA surface and abundant carbonyl moieties on PLLA chains. The stable adhesion between PLLA and HA particles can be well demonstrated by TEM images in Figure 3. Figure 3 shows that no voids could be found at the ends of HA particles when the PLLA/HA nanofibers with about 300-nm diameters were further thinned to less than 100 nm underneath the high-energy electron beam in the TEM. In other words, no de-adhesion happened at the PLLAHA interface around the particle ends when high tensile stress had been present to separate them. 4.3. Thermal and Crystallization Properties of PLLA in Electrospun Scaffolds. The DSC results did not show any consistent trend regarding the effect of the fiber diameter on the thermal properties of the electrospun scaffolds within the studied range. Regardless of the fibrous assembly, the addition of HA particles decreased the crystallization of PLLA during the electrospinning process in the order Nano3 < Nano7 < Micro17. This result is similar to the phenomenon reported by Yu et al., who found that the addition of talc decreased the cold crystallization temperature of PLA in talc/PLA composite films.34 It may be because the well-dispersed HA particles (especially the nanosized ones) acted as physical cross-linking points and increased the viscosity of the fiber during spinning. In turn, this would lead to decreased chain mobility, impeding crystallization. However acting as nucleation sites, all types of HA particles led to more cold crystallization of PLLA during the first heat-up process of DSC tests. The melting of PLLA provides two adjacent endothermal peaks corresponding to PLLA in β and R crystalline forms, respectively. PLLA in β phase has an orthorhombic crystal cell and a fibrillar structure, and PLLA in R phase has a pseudoorthorhombic crystal cell and a lamellar crystalline structure.26,32 Although the HA particles showed no apparent influence on the melting of the R phase PLLA, they increased the melting temperature and the content of β phase PLLA. In other words, HA particles preferentially nucleated the formation of β phase PLLA crystals.34,35 The WAXD results also suggest that HA particles promoted the crystallization of PLLA during annealing, leading to a higher fraction of β phase PLLA crystals (Figure 5b). According to the results and discussion above, it is proposed that the incorporation of HA particles may increase the formation of β phase PLLA crystals during the electrospinning process and tensile deformation. 4.4. Mechanical Properties of PLLA and HA/PLLA Electrospun Scaffolds. Pronounced reinforcement effects from the HA particles were evident in both the random and the aligned HA/ PLLA electrospun scaffolds following the order of Micro17 > Nano7 > Nano3. Assuming similar elastic moduli of three types of HA particles and good HAPLLA adhesion, their different reinforcement effects cannot be simply explained by considering their specific surface areas (SA), because their SA values follow the order NRHA > NNHA . MNHA, which is the opposite of the reinforcement effect. To investigate these effects in depth, both the HalpinTsai and shear-lag models were used to predict the void-excluded moduli of the HA/PLLA scaffolds. 15749
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The Journal of Physical Chemistry C EHA was not found to have an obvious influence on EHT or ESL predictions of HA/PLLA scaffolds at EHA > 60 GPa, except for ESL of Micro17/PLLA aligned scaffold. The low dependence was due to the relatively low HA volume fraction and the relatively low aspect ratios of Nano3 and Nano7 particles. Compared to the HalpinTsai model, the shear-lag model underestimated the reinforcement effect of Nano3 particles. Particle end effect is not considered in the shear-lag model, which may not be applicable to particles with a low aspect ratio, such as Nano3. The shear-lag model yielded higher modulus predictions for Nano7/PLLA and Micro17/PLLA scaffolds than the HalpinTsai model, because the shear-lag model not only accounts for the stress transfer through the needle-shaped particles, it also predicts the longitudinal moduli of scaffolds with good particle orientation along the test direction.28 ESL predictions were closer to the EE data of Nano7/PLLA and Micro17/PLLA random scaffolds, indicating that lots of fibers in random scaffolds had aligned along the test direction either from the beginning or during the test. As a result, considerable Nano7 or Micro17 particles ended up aligned in the test direction. The modulus predictions for all the HA/PLLA random scaffolds were lower than their experimental data. It is wellknown that more crystalline PLLA, especially in β phase, can enhance the tensile modulus and strength of PLLA.32 The use of EE of PLLA random scaffold as EPLLA for HA/PLLA random scaffolds might underestimate the modulus of the PLLA matrix. As discussed in section 4.3, HA particles, especially the needleshaped Nano7 and Micro17 particles, might have introduced higher content of β phase PLLA crystals during the spinning and more PLLA crystallization during the tensile test. The EE (299 MPa) of the aligned PLLA scaffold is 5 times as high as that (60 MPa) of the random PLLA scaffold. This difference can be explained by the following two reasons. First, the aligned scaffolds had higher PLLA crystallinity than the random ones (Table 2). Second, in the aligned scaffold, many more fibers contribute their longitudinal moduli to the scaffold instead of their transverse ones. The longitudinal modulus of the electrospun PLLA fiber is higher than its transverse modulus. Kakade et al. also reported a higher longitudinal modulus of their aligned electrospun scaffold compared to the transverse modulus. And they suggested that electrospinning improves the alignment of polymer chains along the fiber axis, indicating anisotropic mechanical properties of the electrospun nanofibers.36 The aligned HA/PLLA scaffolds were also stiffer and stronger than the random ones. It should be assigned to the above two factors. In addition, there were more HA particles aligned in the test direction in the aligned scaffolds. These HA particles shared more stress transferred from the PLLA matrix. Thus, the HA particles contributed their longitudinal moduli more to the scaffold, and the longitudinal moduli of HA particles are slightly higher than their transverse moduli.30 EE (299 MPa) of the aligned PLLA scaffold was used for EPLLA to calculate the ESL and EHT predictions for the aligned HA/PLLA scaffolds. These predictions were much higher than those for the random scaffolds, showing that the modulus of the PLLA matrix has a strong impact on the moduli of the composite scaffolds. The relationships found among the EE, ESL, and EHT data of Nano3/ PLLA (EE > EHT > ESL) and Nano7/PLLA (EE > ESL > EHT) aligned scaffolds were similar to those of the random scaffolds. However, a slight difference presented for Micro17/PLLA, which followed a trend of ESL > EE > EHT. This can be explained as follows: in electrospun fibers, the PLLA matrix layer around the side surface of Micro17 particles was thin, especially that around
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large Micro17 particles (Figure 2d and Figure 3c,d). As a result, the stress transfer between the PLLA matrix and the Micro17 particles was not as sufficient as estimated by the shear-lag model. As to tensile strength of the composite scaffolds, the Nano3 particles with a length around 100 nm brought a modest reinforcement effect due to their small size. Nano7 with a length around 300 nm and Micro17 particles with an average length more than 2000 nm promoted the tensile strength of the composite scaffolds markedly. In the composites reinforced with needle-shaped particles (i.e., short fiber), the stress supported by the particle increases with its length before its length reaches the critical value, at which the maximum stress supported by the reinforcement can be achieved.28 According to the tensile strengths of the HA/PLLA scaffolds, which followed the order Nano3 < Nano7 < Micro17, it can be concluded that the critical particle length for the studied composites should be larger than 300 nm.28
5. CONCLUSION Three types of highly crystalline and pure HA particles, including the nanosized and more sphere-shaped Nano3, nanosized and needle-shaped Nano7, and microsized and needleshaped Micro17 particles, have been well dispersed and homogenously incorporated into PLLA nanofibers using the electrospinning method. On the basis of the TEM and WAXD results, the needle-shaped HA particles were perfectly oriented within the PLLA nanofibers in the axial direction. To the best of our knowledge, this perfect particle-along-nanofiber orientation has not been previously achieved. It has been found that HA particles acted as physical crosslinking points and decreased the crystallization of PLLA during the spinning process. On the other hand, HA particles introduced a strong nucleation effect and promoted PLLA crystallization and β phase content during the heat-up and annealing processes. Pronounced mechanical reinforcement effects have been evident with the addition of HA particles. The tensile moduli of both the random and the aligned electrospun scaffolds increased dramatically following the order Nano3 < Nano7 < Micro17. The aligned PLLA scaffolds had much higher elastic moduli than the random ones due to their fiber alignment and higher crystallinity. Moreover, the aligned HA/PLLA scaffolds were much stiffer and stronger than the random ones, as more fibers and therefore more HA particles were oriented in the extension direction. Compared to the short round-shaped Nano3 particles, the long needle-shaped Nano7 and Micro17 particles resulted in a much better reinforcement effect to the electrospun scaffolds. ’ AUTHOR INFORMATION Corresponding Author
*Tel.: (860) 486-9253. Fax: (860) 486-4745. E-mail: m.wei@ ims.uconn.edu.
’ ACKNOWLEDGMENT This paper is based upon work supported by NSF Grant BES0503315 and Connecticut Innovations under the Yankee Ingenuity Technology Competition. We appreciate the valuable discussion and help from Prof. Zhi-Kang Xu’s group at Zhejiang University, China. We thank Bill Eichner of Teleflex Medical for setting up the electrospinning facility for aligned scaffolds. We also appreciate Erica Kramer’s assistance in proofreading the manuscript. 15750
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