Self-Crimping, Biodegradable, Electrospun Polymer Microfibers

Nov 3, 2010 - monomers L-lactide (Purac Biomaterials, Lincolnshire, IL) and ε-ca- prolactone (Lancaster Synthesis, Wyndham, NH) along with the cataly...
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Self-Crimping, Biodegradable, Electrospun Polymer Microfibers Denver C. Surrao,†,§ James W. S. Hayami,†,§ Stephen D. Waldman,†,‡,§ and Brian G. Amsden*,†,§ Departments of Chemical Engineering and Mechanical and Materials Engineering, Queen’s University, Kingston, ON, Canada, and Human Mobility Research Centre, Kingston General Hospital, Kingston, ON, Canada Received September 10, 2010; Revised Manuscript Received October 15, 2010

Semicrystalline poly(L-lactide-co-ε-caprolactone) (P(LLA-CL)) was used to produce electrospun fibers with diameters on the subcellular scale. P(LLA-CL) was chosen because it is biocompatible and its chemical and physical properties are easily tunable. The use of a rotating wire mandrel as a collection device in the electrospinning process, along with high collection speeds, was used to align electrospun fibers. Upon removal of the fibers from the mandrel, the fibers shrunk in length, producing a crimp pattern characteristic of collagen fibrils in soft connective tissues. The crimping effect was determined to be a result of the residual stresses resident in the fibers due to the fiber alignment process and the difference between the operating temperature (Top) and the glass-transition temperature (Tg) of the polymer. The electrospun fibers could be induced to crimp by adjusting the operating temperature to be greater than that of the polymer glass-transition temperature. Moreover, the crimped fibers exhibited a toe region in their stress-strain profile that is characteristic of collagen present in tendons and ligaments. The crimp pattern was retained during in vitro degradation over 4 weeks. Primary bovine fibroblasts seeded onto these crimped fibers attached, proliferated, and deposited extracellular matrix (ECM) molecules on the surface of the fiber mats. These self-crimping fibers hold great promise for use in tissue engineering scaffolds for connective tissues that require fibers similar in structure to that of crimped collagen fibrils.

1. Introduction The soft connective tissues of the musculoskeletal system (e.g., tendons, ligaments, and the intervertebral disk) are loadbearing tissues that primarily consist of highly organized, 3D networks of extracellular matrix (ECM) protein fibers (collagen, elastin) and structural carbohydrates (proteoglycans).1 Each of these tissues has a low propensity for self-repair due to a combination of factors, one of which includes high ECM density and organization along with the presence of few or no blood vessels.2,3 The inability of soft connective tissues to self-repair has led to investigations into creating functional constructs to replace damaged or diseased tissue using tissue engineering principles.2,3 Whereas this approach holds much promise, scaffolds developed to-date do not possess structural similarity to native tissue. The resultant mechanical properties are then insufficient for the demanding physiological conditions of soft connective tissues2,4 and may led to premature failure of the developed constructs. Electrospinning is a material-processing technique5 that has been used to produce fibers in the micrometer-to-submicrometer range for a variety of tissue engineering applications.5,6 This technique is both cost efficient and allows for the creation of fibers from a wide variety of polymers with and without the use of solvents.5 The electrospinning process involves the generation of an electrical field between a charged needle and a grounded collecting mat.6 A polymer solution is pumped * To whom correspondence should be addressed. Address: Brian G. Amsden, Ph.D., P.Eng., Professor Department of Chemical Engineering; Dupuis Hall, 19 Division Street; Queen’s University; Kingston, K7L 3N6, Ontario, Canada. Tel: 613 533 3093. Fax: 613 533 6637. E-mail: [email protected]. † Department of Chemical Engineering, Queen’s University. ‡ Department of Mechanical and Materials Engineering, Queen’s University. § Kingston General Hospital.

through the needle until a droplet is formed. The electrical field pulls the droplet toward the collecting mat, forming a fiber jet in the air gap between the needle and collection mat.5,6 As the fiber jet is stretched, it undergoes an unstable whipping motion to produce very fine fibers with diameters in the micrometerto-nanometer range.6 The electrospun fibers can also be aligned through the use of a rotating drum or mandrel in place of a static collection plate.7-9 We have used a conventional electrospinning device in conjunction with a rotating wire mandrel to align biocompatible P(LLA-CL) fibers. The aligned fibers, with nano- to micrometersized diameters, were discovered to undergo crimping after removal from the mandrel. The resulting aligned, crimped fibers had similar structural characteristics (amplitude and wavelength) to that of native collagen fibrils, making them ideal for soft connective tissue engineering scaffold applications. Herein, we report our findings, propose a mechanism for the self-crimping behavior of these electrospun fibers, and demonstrate that the induced crimp in the fibers is stable, recoverable, and controllable. Additionally, we demonstrate that the crimped fibers can be used to guide the growth of fibroblasts in vitro.

2. Material and Methods 2.1. Polymer Synthesis. High-molecular-weight poly(L-lactide-coε-caprolactone) (P(LLA-CL)) was synthesized via ring-opening polymerization, as described previously,10,11 using stannous 2-ethylhexanoate (Sigma Aldrich, Oakville, Canada) as the catalyst. In brief, the monomers L-lactide (Purac Biomaterials, Lincolnshire, IL) and ε-caprolactone (Lancaster Synthesis, Wyndham, NH) along with the catalyst were added to a flamed-dried 10 mL ampule, purged with argon, vacuum sealed, and placed in an oven at 140 °C for 2 days under vacuum. The synthesized polymer was purified by precipitating in ice cold methanol with constant stirring. A feed copolymer molar ratio of

10.1021/bm101078c  2010 American Chemical Society Published on Web 11/03/2010

Electrospun Polymer Microfibers 80:20 LLA/CL was used to obtain a glass-transition temperature (Tg) of ∼37 °C.12 Initial work with poly(D,L-lactide-co-ε-caprolactone) indicated that fibers prepared with higher ε-caprolactone content were too soft, whereas those with lower ε-caprolactone content were too brittle to be handled effectively.13 2.2. Polymer Characterization. The monomer composition of the P(LLA-CL) copolymer was measured from 1H NMR spectra obtained from a Bruker Avance-400 MHz spectrometer, as previously described.13 In brief, the synthesized P(LLA-CL) polymer samples were dissolved in either dimethyl sulfoxide-d6 (Cambridge Isotope Laboratories) or chloroform-d (Fluka) at 10 mg/mL. Chemical shifts were measured relative to the methyl proton resonance of an internal tetramethylsilane reference. The peaks corresponding to δ 5.2 (methine hydrogen of LLA groups) and δ 4 (methylene hydrogen of CL groups) were used to determine the monomer compositions. A Mettler Toledo DSC1 system was used to determine the thermal properties (glasstransition temperature (Tg), melting point (Tm), and heat of fusion (∆Hm)) of P(LLA-CL). The samples were first cooled to -80 °C from ambient conditions, after which the sample was heated to 180 °C; this marked the first cycle. From 180 °C, the samples underwent cooling to -80 °C; this marked the second cycle. The third cycle was a heating cycle from -80 to 180 °C. A 10 °C/min heating rate with a 3 min hold time at each set point was utilized. The glass-transition temperature (Tg) was determined from the third cycle, whereas Tm and ∆Hm were determined from the first heating cycle. We calculated the crystallinity of P(LLA-CL) by dividing the measured ∆Hm value by the ∆Hm of purely crystalline poly(L-lactide), which is 93.1 J/g.14 The numberaverage molecular weight (Mn) and polydispersity index (PDI) were measured using a Waters 2698 separation module GPC equipped with a Wyatt Technology light scattering (LS) detector and four Styragel 300 × 5 mm columns (HR4.0, HR3.0, HR1.0, HR0.5). Tetrahydrofuran (THF) at 40 °C was used as the mobile phase at a flow of 1 mL/min, with the polymer sample dissolved in THF to a concentration of 8 to 10 mg/mL. Results were analyzed and fitted in Astra v4.90.07 (Wyatt Technology) using a measured P(LLA-CL)/THF dn/dc value of 0.064 mL/g (Wyatt Optilab refractometer). 2.3. Electrospinning. The P(LLA-CL) was dissolved in a 3:1 (volume ratio) solution of dichloromethane/dimethylformamide (DCM/ DMF) (Sigma Aldrich) at a concentration of 5% (w/v). DMF was used as a cosolvent to prevent the rapid evaporation of DCM, thus improving the polymer electrospinning properties. The electrospinning apparatus consisted of a syringe pump (KD Scientific), high voltage generator (Gamma Voltage Research), and a rotating wire mandrel.13 The wire mandrel was constructed using two circular end pieces (diameter 5 cm) that were connected by four equally spaced, 10 cm long connecting rods. The wire mandrel was attached to a horizontally mounted in-line mixer (Barnant, Series 20), which rotated at 1000 rpm (measured with a contact tachometer, Extech). The polymer solution was driven using a syringe pump at a flow rate of 0.03 mL/min through a 21 ga. blunt tip needle (Becton Dickinson & Company). The air gap between the needle tip and the ground plate was set at 15 cm, and a 1 kV/cm positive electric field was applied between the air gap using a voltage generator. The fibers that collected on the wire mandrel were stored at room temperature in a vacuum desiccator until required. 2.4. Crimp Conditions. The electrospun fibers were transferred onto cardboard windows (2 cm × 2 cm) using double-sided adhesive tape (Scotch, 3M, London, Canada). The fibers were crimped at room temperature in an aqueous environment by immersion in 1× phosphatebuffered saline (PBS) for 1 h, with the sides of the window cut. To determine the influence of operating temperature on the crimp parameters of the electrospun fibers, as-spun fiber mats were incubated for 48 h in 1× PBS at the following temperatures: 4 °C, room temperature, and 37 °C. Additionally, as-spun fibers while still on the wire mandrel were vacuum-dried in a desiccator at room temperature for 48 h with sections of the vacuum-dried fibers that were further incubated in 1× PBS at 50 °C for 48 h.

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2.5. Characterization of Electrospun Fibers. Fiber alignment, fiber diameter, and crimp parameters (amplitude and wavelength) were determined from SEM images. The incubated fiber mats were air-dried, mounted on aluminum stubs, pulse sputter-coated (Anatech Hummer VI-A sputter coater), and imaged with a JEOL JSM840 SEM (Peabody) with HKL Flamenco EBSD data acquisition software (version 5.0.6.0). Fiber diameter (n ) 100) and crimp parameters (n ) 20) were determined from SEM images processed with Sigma Scan Pro (calibrated for each magnification with the in image scale). The crimp amplitude was measured as the distance between the peak and the central horizontal line of the crimped fiber, whereas the wavelength was determined as the distance between two consecutive peaks, or troughs. The residual stress in the uncrimped electrospun fibers was measured at 37 °C using a Mach-1 micromechanical tester (Biosyntech, Canada). The fibers were transferred onto cardboard windows using double-sided adhesive tape, mounted between the grips of the mechanical tester, and the sides of the window were then cut. With no actuator movement, the residual force in the fibers was measured for a duration of 10 min at a frequency of 10 Hz. Residual stress in the fibers was then determined by normalizing the measured forced to the total cross sectional area of the fibers mounted in the sample. The mechanical properties of the crimped electrospun fibers were measured in uniaxial tension at 37 °C using the Mach-1. Crimped fibers mounted between the grips of the Mach-1, as previously described, were tested at a rate of 1% strain/s until failure with load and displacement measured at frequency of 10 Hz. Tensile stress in the fibers was calculated as a function of measured force normalized to the total cross sectional area of the fibers mounted in the sample, whereas the applied strain was measured as a function of deformation normalized to the gauge length of the fiber mat sample. The toe region (Rtoe) was taken as the distance between zero strain and the onset of the linear region (elastic region), on the horizontal axis (strain) of the stress-strain curve. The modulus of the crimped fiber mats was determined from a linear regression of the linear region of the stress-strain curve. The yield stress was defined as a point on the vertical axis (stress) of the stress-strain curve that corresponded to the onset of the plastic region (permanent deformation). 2.6. Crimp Stability and Degradation Studies. Electrospun fiber mats of known weight were placed in 1× PBS at 37 °C for 4 weeks. At weekly time points, the modulus, fiber diameter, crimp parameters, and thermal properties of the fiber mats were determined, as explained in Sections 2.2 and 2.5. Crimp recovery in the electrospun fibers was measured in uniaxial tension at 37 °C using a Mach-1. At weekly time points, the fiber mats were tested at a rate of 1% strain/s until the crimp pattern unfolded completely, the fiber mats were then unmounted from the Mach-1 and allowed to recover in 1× PBS for 45 min at 37 °C; after 45 min, the fiber mats were tested to failure at a rate of 1% strain/ s, as explained in Section 2.5. For the degradation studies, fiber mats of known mass were placed in 1× PBS at 37 °C for a duration of 4 weeks, and the weight of the fibers was measured at the end of 4 weeks to determine if the fibers underwent degradation. 2.7. Cell Culture. Bovine fibroblasts were isolated from the central ligament in the lower metacarpal joint of 12-18 month old calves. The dissections were carried out under sterile conditions in a laminar flow hood. The central ligament was trimmed of excess adipose tissue and digested in DMEM culture media (Sigma Aldrich) supplemented with 0.25% (w/v) collagenase (Roche Diagnostics) for 36 h in a tissue culture incubator at 37 °C with 5% CO2 and 95% relative humidity. The cells were isolated, and the Trypan Blue dye exclusion assay (Invitrogen) was used to identify and count viable cells. Fibroblasts were passaged between P1 and P5 before seeding on the fiber mats. The fiber mats were first sterilized by soaking in 70% ethanol for 30 min, after which the fibers were sterilized under UV light from the laminar flow hood for 30 min per side. The sterilized fiber mats (1.5 to 2 cm2) were seeded with a 20 µL cell suspension containing 10 000 cells (P1-P5). The volume of the culture media (DMEM containing

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Surrao et al. Table 1. Properties of P(LLA-CL) monomer ratio Tg (°C) Tm (°C) ∆Hm (J/g) Xc (%) Mn (kDa) PDI 85:15

Figure 1. (A) Aligned fibers on the mandrel and (B) crimped fibers after removal from mandrel (scale bar 100 µm).

5% FBS and 25 mM HEPES) was topped up to 5 mL after 4 h of seeding. The culture media was changed every 2 to 3 days with a total culture period of 30 days. The constructs were harvested periodically at days 1, 3, 5, 14, and 30 and analyzed using an SEM (JEOL JSM 840) at 10 kV. Prior to SEM imaging, the constructs were washed with 1× PBS, fixed with 8% glutaraldehyde (Electron Microscopy Sciences, Canada) for at least 4 h at 4 °C, dehydrated through a series of graded ethanol solutions up to 100% ethanol, and then air-dried overnight in the fume hood. Additionally, the mechanical properties of the constructs were measured at weeks 2 and 4 (n ) 6, for each time point) using the Mach-1, as explained in Section 2.5. 2.8. Physical and Biochemical Evaluation. At the end of 4 weeks, the constructs (fibers + tissue) were harvested, lightly patted dry, and weighed (wet weight) (AT-250 Balance, Mettler Instrument). The tissues were lyophilized overnight, and the dry weight of the tissue was determined. The constructs were then digested in papain (40 µg/ mL in 20 mM ammonium acetate, 1 mM ETDA, and 2 mM DTT) for 48 h at 65 °C and then stored at -20 °C until analyzed. Aliquots of the digest were assayed separately for the proteoglycan, collagen, and DNA contents. We estimated the proteoglycan content by quantifying the amount of sulphated glycosaminoglycans using the dimethylmethylene blue dye binding assay (Polysciences) and spectrophotometry (wavelength: 525 nm).15,16 We estimated collagen content by measuring the hydroxyproline content and assuming that hydroxyproline accounts for ∼10% of the weight of collagen.17 Aliquots of the papain digest were hydrolyzed in 6 N HCl at 110 °C for 18 h, and the hydroxyproline content of the hydrolyzate was determined using chloramine-T/Ehrlich’s reagent assay18 and spectrophometry (wavelength: 561 nm). The DNA content was determined from aliquots of the papain digest using the Hoechst 33258 dye (Polysciences, Washington, PA) binding assay and fluorometry (emission wavelength: 365 nm; excitation wavelength: 458 nm).19 2.9. Statistical Analyses. Experiments generating quantitative numerical data were carried out with a minimum of n ) 6 per group. The numerical data generated is presented as mean ( standard error.

39

150

-14.6

16

125

1.5

crimped, wave-like pattern along the fiber backbone (Figure 1B) resembling the native structure of collagen fibers. Measurements of the crimp structure confirmed this observation, with crimp amplitude and wavelength found to be 9 ( 0.4 and 52 ( 1.5 µm, respectively. These values were similar to our own measurements for bovine ligament collagen crimp (wavelength of 51 ( 1 µm) as well as reported values for crimp in human ligament (amplitude of 5-10 µm and wavelength of 45-60 µm, respectively).21 On the basis of the finding of Grijpma et al.,12 the 85:15 copolymer was expected to be semicrystalline and have a glasstransition temperature (Tg) of ∼40 °C (Table 1). Thermal analysis of the as-spun 15:85 copolymer revealed that the polymer was semicrystalline but with a glass-transition temperature of 10 °C. Given the high boiling point of DMF (153 °C22,23), it was reasoned that the depression in the glasstransition temperature was due to residual DMF in the fibers. This explanation was supported by a 1H NMR analysis of the as-spun fibers in DMSO-d6, which showed characteristic peaks at 7.95 ppm (s, methine) and at 2.73 and 2.89 ppm (s, methyl) corresponding to DMF, and by a thermal analysis that showed a broad endotherm centered at ∼150 °C. After the residual DMF was removed via vacuum drying, the glass-transition temperature of the fibers increased to 39 °C. Moreover, under ambient conditions, these dried fibers did not crimp upon removal from the mandrel. Given these findings and considering the nature of the electrospinning process adopted in this study, it was hypothesized that the crimp in the fibers was created as a result of: (i) the presence of residual stress within the fibers and (ii) an operating temperature (Top) above that of the glass-transition temperature of the polymer (Tg). The electrospinning technique, through the elongational flow of the fiber jet length and order of magnitude decrease in the fiber jet diameter, orients polymer chains in the direction of the fiber.6 Residual stresses within the fibers were also imparted by fiber stretching during aligned collection on the mandrel at high rotational speeds. Stretching the fibers further oriented the polymer chains, thereby decreasing the entropy of the fibers. Rapid solvent (DCM) evaporation from the fibers during the electrospinning process led to fixed polymer chain alignments within the electrospun fibers.24,25 This

3. Results and Discussion The monomer composition of the P(LLA-CL) copolymer as determined from the 1H NMR spectra was 85:15 (LLA/CL). A slightly larger L-lactide content than initially desired was obtained because of the higher reactivity of L-lactide compared with ε-caprolactone.11,20 Various polymer concentrations were studied to produce synthetic ECM to support cell growth via electrospinning. It was determined that a 5 wt % polymer solution in a 3:1 volume ratio of DCM/DMF produced ideal, defect-free fibers with an average diameter of 0.88 ( 0.002 µm (n ) 100). We achieved fiber alignment by collecting the fibers on a rotating wire mandrel (Figure 1A). The wire mandrel augmented fiber drying and minimized fiber-fiber and fiber-collection surface contact. After the fibers were removed from the mandrel and left under ambient conditions in 1× PBS (aqueous crimped fibers) for a short period of time, they shrunk in length and displayed a

Figure 2. Average residual stress released from uncrimped P(LLACL) fibers plasticized with DMF at 37 °C (n ) 10).

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Figure 4. Nonplasticized fibers crimped at temperatures greater than Tg, showing the influence of operating temperature (Top) on crimp parameters (amplitude and wavelength). (A) Nonplasticized fibers incubated at room temperature (22 °C). (B) Nonplasticized fibers incubated at 50 °C (scale bar 100 µm).

Figure 3. Fibers plasticized with DMF crimped at different temperatures. (A) Original aligned fibers on the mandrel. (B) Fibers removed from mandrel and incubated at 4 °C. (C) Fibers removed from mandrel and incubated at room temperature (22 °C). (D) Fibers removed from mandrel and incubated at 37 °C (scale bar 100 µm). Table 2. Influence of Operating Temperature (Top) on the Crimp Parameters of Electrospun P(LLA-CL) Fibers (n ) 10 for Each Operating Temperature Point) P(LLA-CL) Fibers Plasticized with DMF (As-Spun Fibers) crimp parameters operating temperature

amplitude (µm)

wavelength (µm)

∆T ) (Top - Tg) (°C)

uncrimped fibers 4 °C 22 °C 37 °C

no crimp no crimp 9 ( 0.4 4 ( 0.3

no crimp no crimp 52 ( 1.5 17 ( 0.8

e0 e0 12 27

Non-Plasticized P(LLA-CL) Fibers (Vacuum-Dried Fibers)

Figure 5. Stress-strain curve of aqueous crimped fibers showing similar behavior to collagen fibrils with a characteristic toe region (n ) 10). Table 3. Mechanical Properties of Crimped P(LLA-CL) Fibers (n ) 10) toe region (cm/cm) 0.55 ( 0.03

crimp parameters operating temperature

amplitude (µm)

wavelength (µm)

∆T ) (Top - Tg) (°C)

22 °C 50 °C

no crimp 9 ( 0.6

no crimp 51 ( 1.8

e0 11

polymer chain orientation was locked in place as the fibers were kept under tension and resulted in the storage of potential energy. When the electrospun fibers were removed from the mandrel at a temperature well above the glass-transition temperature of the polymer, the polymer chains relaxed to a more random coil orientation to relieve the residual stress by releasing the potential energy and in the process increased their entropy. The mechanism of crimp proposed in this study is similar to the thermal shrinkage mechanism proposed by Urudzhev et al.26 and GingHo et al.27 To test this hypothesis, as-spun P(LLA-CL) fibers plasticized with DMF were transferred onto a cardboard window while still on the mandrel, then mounted between the grips of a mechanical tester. The sides of the window were cut, and the residual stresses in the fibers were then measured at 37 °C. In agreement with the proposed mechanism, the residual forces within the fibers generated stress as they dissipated because of polymer chain relaxation (Figure 2). The average residual stress in the P(LLA-CL) fibers was measured to be 15 ( 3 kPa (n ) 10). Furthermore, the crimp parameters (amplitude and wavelength) of the plasticized P(LLA-CL) fibers were tunable by

yield stress (kPa)

modulus (kPa)

193 ( 44

349 ( 69

adjusting the difference between Top and Tg (Figure 3A-D). The crimp amplitude and wavelength decreased as the difference between Top and Tg increased (Table 2). Similar experiments were conducted on vacuum-dried fibers. Unlike the DMF

Figure 6. Electrospun P(LLA-CL) fibers in 1× PBS at 37 °C showing the crimp induced in the fibers is retained over the period studied. (A) Week 1, (B) week 2, (C) week 3, and (D) week 4 (scale bar 100 µm).

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Figure 7. Crimp recovery in P(LLA-CL) fibers showing that the crimp induced in the fibers is recoverable with time (n ) 10 for each time point).

Figure 8. Change in modulus and Tg of P(LLA-CL) fibers with time (n ) 10, for each time point) during incubation in PBS.

plasticized fibers, the vacuum-dried fibers did not crimp at room temperature (22 °C) (Figure 4A) (Table 2). However, when the operating temperature (Top) was raised to 50 °C, ∼10 °C above the Tg of the vacuum-dried fibers with no DMF, a similar degree of crimp as that seen with the DMF plasticized fibers at room temperature was produced (Figure 4B). The ∆T (Top - Tg) between the two conditions is almost identical, thus further supporting the proposed crimp mechanism (Table 2). This finding establishes the requirement of having an operating temperature (Top) greater than the glass-transition temperature (Tg) of the polymer to produce a crimp pattern in the electrospun polymer fibers.

Surrao et al.

The utility of these fibers for tissue engineering applications was exemplified by the similarity of their stress-strain behavior to that of native collagen. When the aqueous crimped fibers were strained uniaxially, a “J-shaped” stress-strain curve with a “toe region” was observed (Figure 5), which is characteristic of the behavior of collagen.28 The average toe region, yield stress, and modulus were determined from the stress-strain curves of the crimped fibers (Table 3). It should be noted that the modulus of the crimped fibers is not comparable to that of collagen, and work is currently underway to improve the mechanical properties of the crimped fibers. The electrospun fibers maintained their physical morphology (crimp structure) and alignment throughout the crimp stability studies, thus demonstrating that the crimp induced in the fibers via the electrospinning process adopted in this study was stable over a 4 week study at 37 °C in 1× PBS (Figure 6A-D). The crimp amplitude and wavelength, at week 1, was 4.5 ( 0.2 and 17.2 ( 0.8 µm, respectively, after which it remained constant until the end of the study. The crimp induced in the fibers recovered in 45 min in 1× PBS after it was unfolded; the recovery of crimp was characteristic throughout the crimp stability study (Figure 7). The crimp pattern induced in the electrospun fibers recovered after unfolding because there was insufficient strain applied to remove the polymer chain configurational memory. The modulus of the fibers dropped drastically between weeks 0 and 1, after which it remained constant (Figure 8). This drop in modulus is attributed to the plasticizing effect of absorbed water in the electrospun fibers; a study by Blasi et al.29 showed a similar effect of plasticization in poly(L-lactide-co-glycolide) caused by absorbed water. The P(LLA-CL) fibers incubated at 37 °C in 1× PBS did not degrade significantly over the time frame examined because there was no change in fiber diameter and no mass loss (n ) 6) at the end of week 4. Additionally, the Tg of the degrading P(LLA-CL) fibers measured from the second heating cycle was 39 °C, which was identical to the Tg of the freshly synthesized P(LLA-CL) copolymer (Figure 8). Primary bovine fibroblasts attached effectively to the surface of the fiber mats (Figure 9). At day 1, the fibroblasts had a spherical morphology indicating cell attachment and the fibroblasts aligned along the length of the fibers, similar to the behavior of fibroblasts in the native ligament that are aligned along individual collagen fibrils.21 At day 5, cell proliferation and ECM deposition is evident. At the end of the day 14, the fibroblasts proliferated and deposited ECM on 90% of the fiber mat surfaces. The modulus of the construct (fibers + cells) more than doubled in value from 44 ( 2 to 123 ( 9 kPa, from weeks 2 to 4 (n ) 6 for each time point), reflecting the contribution to

Figure 9. Fibroblast attachment and growth on crimped fibers. Day 1: (A) 5 µm, (B) 10 µm; Day 3: (C,D) 10 µm; Day 5: (E) 50 µm, (F) 20 µm. Day 14: (G) 100 µm, (H) (20 µm). The numbers within parentheses represent the length of the scale bar in each image.

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the stiffness of the fiber mats by newly deposited ECM. At the end of week 4, the cells synthesized 23 ( 1.3 and 48 ( 5.9 µg/fibermat (n ) 10) of proteoglycans and collagen, respectively, on the surface of the crimped fibers.

4. Conclusions Using a rotating mandrel as the collection device in the electrospinning process yielded an oriented polymer chain structure in the P(LLA-CL) fibers. This is similar to results reported for melt-drawn and as-spun fibers in the textile industry. The electrospun fibers, with an average diameter of 0.88 µm, crimped when the operating temperature was greater than the glass-transition temperature of the polymer (Top > Tg). This temperature difference could be induced through plasticization to allow crimping at lower temperatures or by increasing the operating temperature. The degree of crimp (amplitude and wavelength) was also controllable by adjusting the difference between the operating temperature and the glass-transition temperature of the polymer. The crimp pattern induced in the fibers remained stable during in vitro incubation in PBS and was recoverable upon complete unfolding. Fibroblasts were able to attach, proliferate, and synthesize ECM on the surface of the crimped fiber mats. Additionally, after the 30 day culture period, the modulus of the constructs (fibers + cells) increased, thus further demonstrating the potential of crimped electrospun fibers as tissue engineering scaffolds. This crimping process should be translatable to any polymer. Acknowledgment. Funding from the Ontario Premier’s Research Excellence Award, the Advanced Foods and Materials Network, and from the Natural Sciences and Engineering Research Council of Canada is gratefully acknowledged.

References and Notes (1) Culav, E. M.; Clark, C. H.; Merrilees, M. J. Connective tissues: matrix composition and its relevance to physical therapy. Phys. Ther. 1999, 79, 308–319. (2) Murphy, W. L.; Grorud, K.; Vanderby, R. Healing of Bone and Connective Tissues. In Encyclopedia of Biomaterials and Biomedical Engineering; Wnek, G. E., Bowlin, G. L., Eds.; Informa Healthcare: New York, 2007; pp 1-14. (3) Vunjak-Novakovic, G.; Altman, G.; Horan, R.; Kaplan, D. L. Tissue Engineering of Ligaments. Annu. ReV. Biomed. Eng. 2004, 6, 131– 156. (4) Hubbell, J. A. Biomaterials in tissue engineering. Nat. Biotechnol. 1995, 13, 565–576. (5) Reneker, D.; Chun, I. Nanometre diameter fibers of polymer, produced by electrospinning. Nanotechnology 1996, 7, 216–223. (6) Doshi, J.; Reneker, D. H. Electrospinning process and applications of electrospun fibers. J. Electrost. 1995, 35, 151–160. (7) Huang, Z. M.; Zhang, Y. Z.; Kotaki, M.; Ramakrishna, S. A review on polymer nanofibers by electrospinning and their applications in nanocomposites. Compos. Sci. Technol. 2003, 63, 2223–2253. (8) Sundaray, B.; Subramanian, V.; Natarajan, T. S.; Xiang, R.-Z.; Chang, C.-C.; Fann, W.-S. Electrospinning of continuous aligned polymer fibers. Appl. Phys. Lett. 2004, 84, 1222–1224.

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