Micron 72 (2015) 1–7
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Micron journal homepage: www.elsevier.com/locate/micron
Quantitative imaging of electrospun fibers by PeakForce Quantitative NanoMechanics atomic force microscopy using etched scanning probes a ´ Adrian Chlanda a,∗ , Janusz Rebis a , Ewa Kijenska , Michal J. Wozniak a,b , a Krzysztof Rozniatowski , Wojciech Swieszkowski a , Krzysztof J. Kurzydlowski a a b
Warsaw University of Technology, Faculty of Material Science and Engineering, 141 Woloska str., 02-507, Warsaw, Poland Warsaw University of Technology, University Research Centre – Functional Materials, 141 Woloska str., 02-507, Warsaw, Poland
a r t i c l e
i n f o
Article history: Received 25 November 2014 Received in revised form 31 January 2015 Accepted 31 January 2015 Available online 9 February 2015 Keywords: Atomic force microscopy PeakForce Quantitative NanoMechanics Electrospun fibers Focused ion beam Mechanical properties
a b s t r a c t Electrospun polymeric submicron and nanofibers can be used as tissue engineering scaffolds in regenerative medicine. In physiological conditions fibers are subjected to stresses and strains from the surrounding biological environment. Such stresses can cause permanent deformation or even failure to their structure. Therefore, there is a growing necessity to characterize their mechanical properties, especially at the nanoscale. Atomic force microscopy is a powerful tool for the visualization and probing of selected mechanical properties of materials in biomedical sciences. Image resolution of atomic force microscopy techniques depends on the equipment quality and shape of the scanning probe. The probe radius and aspect ratio has huge impact on the quality of measurement. In the presented work the nanomechanical properties of four different polymer based electrospun fibers were tested using PeakForce Quantitative NanoMechanics atomic force microscopy, with standard and modified scanning probes. Standard, commercially available probes have been modified by etching using focused ion beam (FIB). Results have shown that modified probes can be used for mechanical properties mapping of biomaterial in the nanoscale, and generate nanomechanical information where conventional tips fail. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction In the last twenty years, a growing interest in new biomaterials for medical applications can be observed. Fibrous polymer scaffolds which can replace traditional metal implants become increasingly important in this field. In order to fulfill their task, fibrous biomaterials should be biocompatible and characterized by suitable mechanical properties. Electrospinning technique offers the ability to control material properties, as well as structure, and mechanical functions of the scaffolds for tissue engineering applications (Baker et al., 2012). Due to their biodegradability, interfibrous pore size, high surface area to volume ratio, immunogenicity, structural similarity to the tissue extracellular matrix (ECM) (Elsabee et al., 2012; Bosworth et al., 2013; Khadka and Haynie, 2012; Raghavan et al., 2012; Sun et al.,
∗ Corresponding author. Tel.: +48 22 662 31 58; fax: +48 22 234 87 50. E-mail address: [email protected] (A. Chlanda). http://dx.doi.org/10.1016/j.micron.2015.01.005 0968-4328/© 2015 Elsevier Ltd. All rights reserved.
´ 2013; Kijenska et al., 2012), electrospun fibers are good candidates for scaffolds. The mechanical properties of the material might have a strong influence on the biological function of the scaffold (Reilly and Engler, 2010). It is also worth to be noted that overall mechanical properties of any fibrous structure is based on three quantities (Carlisle et al., 2009): the architecture of the electrospun mesh, the properties of the single fibers and the junction between the fibers. To study the mechanical properties of the electrospun fibers at the nanoscale atomic force microscopy (AFM) can be used. It is a powerful tool for the visualization, and probing of selected mechanical properties of materials in broadly defined life sciences (Wozniak et al., 2009, 2010). This is mainly due to the ability of the technique for measuring force and distance at a high resolution and exploring surface in air and liquid with minimal sample preparation. It allows to examine topography, and mechanical properties of cells and tissues, as well as engineered biomaterials for tissue engineering. Testing mechanical properties of the electrospun fibers is crucial, and must be taken into account, because fibrous structures should be able to withstand the external forces acting on them after implantation (Chew
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Fig. 1. Force curves (A), and corresponding information that can be obtained from them for PLCL fibers (2 m × 2 m scan area): peak force error data channel (B), DMT modulus data channel (C), adhesion data channel (D), deformation data channel (E), and dissipation data channel (F).
et al., 2006). In addition, different types of cells may need different mechanical properties of the scaffolds (Yang et al., 2008). One can find literature positions describing testing of mechanical properties of electrospun fibers using AFM, with nanoindentation (Zhang et al., 2011), stretching (Baker et al., 2012) and bending method (Croisier et al., 2012), but to the authors’ knowledge it is the first time when new AFM technique – PeakForce Quantitative NanoMechanics (PFQNM) is used to characterize electrospun polymeric fibers. PeakForce Quantitative NanoMechanics is a new atomic force microscopy imaging mode developed recently, which allows to measure some mechanical properties of the material, such as: reduced Young’s modulus, adhesion, deformation and dissipation with high spatial resolution by probing at the nanoscale. Very few publications describing use of PFQNM have been printed, including stiffness mapping of amyloid fibrils (Adamcik et al., 2011), bitumen (Fischer et al., 2013), or polymers deformation testing (Liu et al., 2012). Each series of nanomechanical measurements on a sample is equivalent to 256 × 256 force-separation curves, providing a map of mechanical properties with the same resolution as the topographic image (Fig. 1). This technique can be used with any standard AFM probes, but for quality as well as quantity results, proper calibration must be done. Both – appropriate tip, and suitable reference sample choice are crucial to obtain adequate modulus values. The Derjaguin-Muller-Toporov (DMT) model (Butt et al., 2005) implemented into the dedicated Bruker NanoScope Analysis software is used. If not, the modulus map will only represent qualitative instead of quantitative information (Pittenger et al., 2010). To calculate the reduced Young’s modulus, the retract curve (Fig. 1A) was fitted into the DMT model: F − Fadh =
4 ∗ E 3
R(d − d0 )
3
where F is the force acting on the cantilever, Fadh the adhesion force between probe and the surface, R is the tip radius, d − d0 the sample deformation, and E* is the reduced Young’s modulus. If the sample Poisson ratio is known, the Young’s modulus can also be calculated. Next factor that was taken into account for quantitative analysis was deformation of the sample material. Maximum deformation during the PFQNM examination can be described as the penetration
of the probe into the sample. The deformation channel may include information from both plastic and elastic deformation components (Pittenger et al., 2010). Image resolution for AFM techniques depends mostly on the scanning probe parameters. As probe radius decrease, image resolution increase, thus probe geometry is crucial for high resolution imaging. There are several ways of probes modification described in the literature: carbon nanotube attachment (Lee et al., 2008), PVD (Physical Vapour Deposition) (Chen et al., 2000), and T – CVD (Thermal Chemical Vapour Deposition) methods (Chattopadhyay et al., 2006) or ion sputtering (Bale and Palmer, 2002). According to our knowledge, there is no information regarding work that propose a use of modified AFM probes for PFQNM examination. In this paper the AFM tip probe FIB modification procedure for PeakForce QNM application is described. It has been conducted in order to compare images obtained by standard, and modified probes. 2. Materials and methods 2.1. Materials Poly(l-lactide-co-caprolactone) (PLCL) with molecular weight of 150 000 was purchased from Evonik (Germany). Polycaprolactone (PCL) with a molecular weight of 80 000 g/mol, hydroxyapatite powder of grain size below 200 nm, and 1,1,1,3,3, 3-hexafluoro-2-propanol (HFP) used as a solvent in electrospinning, were purchased from Sigma–Aldrich [USA]. Collagen type 1 has been purchased from Koken (Japan). 2.2. Electrospun fibers fabrication Solutions of 9 w/v P(LCL)/collagen/HAp, and 9 w/v PCL/HAp (both with 90:10 ratio) were prepared by dissolving the polymer and proteins/ceramics together in HFP and stirred overnight at room temperature. P(LLA-CL) solutions with concentrations of 9 w/v, and PCL solutions with concentrations of 9 w/v were prepared as a reference, using the same procedure. Each of prepared solutions were placed in a 3 ml plastic syringe attached with flatended steel needles with 27G inner diameter, and then the syringe were placed in the pump (KD Scientific) and dispensed at a rate of 0.8 ml/h. Fibers were fabricated using electrospinning at 12 kV of
A. Chlanda et al. / Micron 72 (2015) 1–7
3
Fig. 2. Standard scanning probes before (A and B), and after (C and D) FIB etching.
applied voltage from a high voltage power supply system (Gamma High Voltage Research). Fibers were electrospun from a single needle setup at room temperature, and 30% relative humidity. For collecting fibers, a flat grounded steel plate covered with aluminum foil was used, which was placed 10 cm from the tip of the needle. Fibers were collected on 15 mm glass cover slips for further examination. After electrospinning all the fibrous meshes were dried under vacuum for 72 h. 2.3. SEM observation of fibers The morphology and diameter of electrospun fibers was determined from images obtained by scanning electron microscopy (SEM) measurements (Phenom Pro X, FEI). Before imaging samples were coated with gold using high vacuum sputter coater (Leica EM SCD 500). 2.4. FIB nanofabrication Method described by Rozniatowski and Rebis (2013) for the AFM probes modification was utilized to modify the shape of standard rectangular silicon AFM probes. Focused ion beam (Hitachi FB2100) milling was used. Probes for modification were taken from new, unopened container. Special, custom holder was engineered to mount AFM probe in the FIB, that allows to change the position of the probe in relation to the beam of gallium ions. This allows by etching probe to change the tip cone angle (Fig. 2). All milling steps were performed with ion beam energy predefined in the FIB software for silica cutting. The beam spot size was decreased from tens (for coarse cutting) to few nanometers for high accuracy. 2.5. AFM nanomechanical mapping Atomic force microscopy experiments were performed using a Bruker MultiMode AFM NanoScope 8 microscope, equipped with standard probes (APP NANO – ACST type), as well as FIB modified probes. The probes were selected based on the recommendation of AFM microscope producer (Bruker, 2011) based on expected
range of reduced modulus values (5–500 MPa) of tested polymeric fibers. The nominal probe spring constant was 7 N/m and the actual spring constant of the probe cantilever was measured by thermal tune method (approx. 5 N/m for standard, and 4 N/m for modified probe). Nominal standard tip radius suggested by the producer was smaller than 10 nm, however the actual size estimated by the dedicated NanoScope Analysis (ver. 1.40) software was bigger, and was estimated at 35 nm. The tip radius after FIB-sharpening, estimated using the same software was approximately 15 nm. Relative calibration method was used to obtain quantitative results from experiment, and for that reason dedicated low-density polyethylene reference sample was chosen. Examination was conducted using the PeakForce Quantitative NanoMechanics AFM mode. All measurements were carried out in air, under ambient conditions and each set of nanomechanical measurements on a sample corresponds to 256 × 256 force-separation curves taken over different areas (20 m × 20 m, and 500 nm × 500 nm). Data analysis was made using NanoScope Analysis ver. 1.40, professional, dedicated software provided by the microscope producer. Individual force curves were also analyzed using SPIP 6.3.3 software.
3. Results and discussion 3.1. Topography of the fibers The topography of four kinds of electrospun fiber meshes made of polycaprolactone (PCL), polycaprolactone with hydroxyapatite (PCL/HAp), poly(l-lactide-co-caprolactone) (PLCL), and poly(l-lactide-co-caprolactone) with collagen and hydroxyapatite (PLCL/COL/HAp) was characterized using scanning electron microscope (Fig. 3), and atomic force microscope (Fig. 4) equipped with standard, commercially available, and FIB-modified probes. To illustrate the structural differences between examined meshes, the diameters, and surface roughness was calculated. Morphology of the electrospun meshes can be influenced by various parameters (Zandén et al., 2012; Lee et al., 2012; Sencadas et al., 2012). As shown in Figs. 5 and 7–9 fibers have a smooth surface and after addition of hydroxyapatite, surface morphology changes radically
Fig. 3. SEM images of electrospun fibers: (A) PCL, (B) PCL/HAp, (C) PLCL, and (D) PLCL/COL/HAp; magnification for all images is 5000×.
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Fig. 4. AFM topography images of electrospun fibers: (A) PCL, (B) PCL/HAP, (C) PLCL, and (D) PLCL/COL/HAp.
Fig. 5. Representative force curve recorded during examination fitted to DMT model.
(Fig. 6), which is reflected in both the mat surface roughness, as well as the average diameter of the individual fibers (Table 1). In case of PCL meshes, addition of 10 wt% HAp increased both fiber diameter and average mesh roughness (from approx. 840 to 940 nm for standard tip), whereas for PLCL composite opposite tendency can be observed – fiber diameter, and surface roughness decreased (from approx. 540 to 440 nm for standard tip). This phenomenon can be explained by the lower content of HAp in PLCL based composite 5 wt%, in comparison to 10 wt% in PCL based fibers. Smaller amount of HAp allowed particles to incorporate in the fiber material, and despite the smaller diameter of the PLCL fibers HAp is not visible on the surface. Moreover it is believed, that collagen smoothed the fibers surface, and therefore roughness is reduced. Analyzing quantitative roughness data obtained from measurements with modified AFM tips, it can be seen that the dependency described in the study using standard probes applies also to them. Furthermore comparing the data for the same materials (even with the caution that after the tip change, it was impossible to image exactly the same area) can be seen that every time, modified tips provides higher surface roughness value, which may indicate a better surface projection (Rozniatowski and Rebis, 2013). 3.2. Mechanical properties of the fibers Figs. 6–10 show the data from the different channels obtained during PFQNM examination. The data were used for qualitative as well as quantitative analysis of the meshes. The changes in
nanomechanical properties are indicated by the color changes on images from different data channels – the brighter the color, the higher the value. It can be seen, that in general images obtained with modified tips are less grainy and have better quality in comparison to the images acquired with standard tips probes. These differences can be observed especially well for PLCL-based meshes, where during measurements with commercial tips the dissipation data channel is empty (Fig. 8D). Representative force curve for PLCL mesh fitted to DMT model is presented in Fig. 5. No suitable information was recorded, whereas after tip modification it was possible to obtain valuable image (Fig. 9D). This may be due to the fact, that sharper shape of the modified scanning probe, leads to an easier distortion of the material. The sudden change in mechanical properties of the meshes on the joints can be observed. In the merging place, the boundaries with the width of about 30–40 nm are formed (Figs. 6 and 10). It can therefore be concluded, that on the joints of the fibers the value of reduced Young’s modulus, and dissipation increase, opposite to the decrease of the deformation value. However, further studies are required to explain this phenomenon. Similar dependence is visible for images taken on PCL/HAp sample (Fig. 7), where HAp particles in comparison to PCL surface have higher (brighter) modulus value, and lower (darker) other measured mechanical properties. This is a consequence of different physical and mechanical characteristic of ceramics and polymers – ceramics are stiffer, thus more difficult to deform (Wang et al., 2010).
Table 1 Fiber diameter and average mat roughness (Ra ) examined using standard and modified (*) AFM tips. Measured quantity
Material PCL
PCL/HAp
PLCL
PLCL/COL/HAp
Fiber diameter [nm] Average mat roughness [nm] Average mat roughness* [nm]
747 ± 142 841 ± 80 870 ± 72
817 ± 94 936 ± 121 1056 ± 55
319 ± 79 537 ± 53 576 ± 60
361 ± 40 440 ± 30 465 ± 33
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Fig. 6. PFQNM images of PCL electrospun fibers made with standard tips [(A) DMT modulus and (B) deformation] and with modified tips [(C) DMT modulus and (D) deformation]; 500 nm × 500 nm scan size.
Fig. 7. PFQNM images of PCL/HAp electrospun fibers made with standard tips [(A) DMT modulus and (B) deformation] and with modified tips [(C) DMT modulus and (D) deformation]; 500 nm × 500 nm scan size.
Fig. 8. PFQNM images of PLCL electrospun fibers made with standard tips, 500 nm × 500 nm scan size: (A) DMT modulus, (B) adhesion, (C) deformation and (D) dissipation.
Fig. 9. PFQNM images of PLCL electrospun fibers made with modified tips, 500 nm × 500 nm scan size: (A) DMT modulus, (B) adhesion, (C) deformation and (D) dissipation. Table 2 Quantitative analysis of selected nanomechanical properties of electrospun fibers measured using standard and modified AFM tips. Tested parameter
Material PLCL
DMT modulus [MPa] Deformation [nm]
PLCL/COL/HAp
PCL
PCL/HAp
Standard tip
Modified tip
Standard tip
Modified tip
Standard tip
Modified tip
Standard tip
Modified tip
12 ± 4 31 ± 5
19 ± 4 40 ± 3
46 ± 32 18 ± 4
117 ± 35 35 ± 6
3±1 43 ± 6
6±4 46 ± 7
17 ± 5 27 ± 3
48 ± 15 38 ± 1
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Fig. 10. PFQNM images of PLCL/COL/HAp electrospun fibers made with standard tips [(a) DMT modulus and (b) deformation] and with modified tips [(c) DMT modulus and (d) deformation]; 500 nm × 500 nm scan size.
Data obtained from the quantitative analysis are shown in Table 2. Reduced Young’s modulus of electrospun PCL and PLCL meshes has value of 3 MPa, and 12 MPa, respectively (measured with standard tips). Literature reports describe similar Young’s modulus results of the electrospun PCL, and PLCL meshes: 3.8 MPa for PCL (Croisier et al., 2012), and 1.22 MPa for PLCL (Kim et al., 2006). These values are lower than those obtained for electrospun composites. Similar correlation exists for examination run by modified AFM tips. The analysis of data for this same material indicate that the DMT modulus value obtained via etched tips is always higher than for conventional tip. This can be explained by referring to the shape of the probe – commercial probes are more spherical, while the probe after sharpening are more conical, and the DMT theory is applied with assumption of the spherical shape of the end of the scanning probe (Butt et al., 2005). As the reduced Young’s modulus value of the composites increases and the value of the deformation decreases – the more rigid is the material and the more difficult to deform. Furthermore, for the same material, value of deformation is always higher when measured with modified tips. This can be attributed to a sharper shape of the probe, that facilitates deformation of the material, despite lower spring constant for etched tip. 4. Conclusion To our knowledge, this is the first study applying PeakForce Quantitative NanoMechanics AFM technique to investigate electrospun fibers. Based on obtained results, it can be stated that PFQNM method shows huge potential for applications as high resolution technique of identification of morphology, and related nanomechanical properties of electrospun fibrous meshes. Due to its versatility it can be employed to other types of fibers and features at the nanoscale. Despite its many advantages, it should be noted that the described experimental method is more qualitative than quantitative, caused by complex, multi-step calibration procedure. The calibration even if done properly may not produce fully correct (quantitative) results, especially if the DMT model may be not appropriate. Available literature shows it is also the first examination using FIB-modified AFM probes for this kind of measurements and the experimental results allow us to conclude that the sharpened tips can be successfully used for the PFQNM measurements. Furthermore, images obtained with etched tips are less grainy, thus have better quality in comparison to the images acquired with standard tips and even can provide nanomechanical information on where conventional probes failed. Acknowledgements This study is financially supported by: (1) National Science Center of Poland (NCN) under grant: “Three-dimensional composite
scaffold based on biodegradable polymers and bioceramic with incorporated growth factors for bone tissue engineering. Research on the manufacturing process and the material influence on living cells function”, number: UMO-2011/01/B/ST8/07559. (2) Ministry of Science and Higher Education, Republic of Poland (MNiSW) under grant: “Polymer-ceramic composite, extracellular matrix mimicking biomaterials for tissue engineering needs: processes of degradation of the structure and mechanical properties”, number: 0616/IP2/2011/71. (3) National Science Center of Poland (NCN) under grant: “Hybrid growth factors delivery system supporting bone tissue regeneration”, number DEC-2011/01/M/ST8/07742.
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A. Chlanda et al. / Micron 72 (2015) 1–7 Liu, H., Chen, N., Fujinami, S., Louzguine-Luzgin, D., Nakajima, K., Nishi, T., 2012. Quantitative nanomechanical investigation on deformation of poly(lactic acid). Macromolecules, 8770–8779. Pittenger, B., Erina, N., Su, C., 2010. Quantitative mechanical property mapping at the nanoscale with PeakForce QNM. Veeco. Raghavan, P., Lim, D.H., Ahn, J.H., Nah, C., Sherrington, D.C., Ryu, H.S., Ahn, H.J., 2012. Electrospun polymer nanofibers: the booming cutting edge technology. React. Funct. Polym. 72 (12), 915–930. Reilly, G.C., Engler, A.J., 2010. Intrinsic extracellular matrix properties regulate stem cell differentiation. J. Biomech. 43, 55–62. Rozniatowski, K., Rebis, J., 2013. Atomic force microscopy – new quality of measurements. Towaroznawcze Problemy Jakosci 34. Sencadas, V., Correia, D.M., Areias, A., Botelho, G., Fonseca, A.M., Neves, I.C., Gomez Ribelles, J.L., Lanceros Mendez, S., 2012. Determination of the parameters affecting electrospun chitosan fiber size distribution and morphology. Carbohydr. Polym. 87, 1295–1301. Sun, B., Long, Y.Z., Zhang, H.D., Li, M.M., Duvail, J.L., Jiang, X.Y., Yin, H.L., 2013. Advances in three-dimensional nanofibrous macrostructures via electrospinning. Prog. Polym. Sci.
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Contents lists available at ScienceDirect
Micron journal homepage: www.elsevier.com/locate/micron
Quantitative imaging of electrospun fibers by PeakForce Quantitative NanoMechanics atomic force microscopy using etched scanning probes a ´ Adrian Chlanda a,∗ , Janusz Rebis a , Ewa Kijenska , Michal J. Wozniak a,b , a Krzysztof Rozniatowski , Wojciech Swieszkowski a , Krzysztof J. Kurzydlowski a a b
Warsaw University of Technology, Faculty of Material Science and Engineering, 141 Woloska str., 02-507, Warsaw, Poland Warsaw University of Technology, University Research Centre – Functional Materials, 141 Woloska str., 02-507, Warsaw, Poland
a r t i c l e
i n f o
Article history: Received 25 November 2014 Received in revised form 31 January 2015 Accepted 31 January 2015 Available online 9 February 2015 Keywords: Atomic force microscopy PeakForce Quantitative NanoMechanics Electrospun fibers Focused ion beam Mechanical properties
a b s t r a c t Electrospun polymeric submicron and nanofibers can be used as tissue engineering scaffolds in regenerative medicine. In physiological conditions fibers are subjected to stresses and strains from the surrounding biological environment. Such stresses can cause permanent deformation or even failure to their structure. Therefore, there is a growing necessity to characterize their mechanical properties, especially at the nanoscale. Atomic force microscopy is a powerful tool for the visualization and probing of selected mechanical properties of materials in biomedical sciences. Image resolution of atomic force microscopy techniques depends on the equipment quality and shape of the scanning probe. The probe radius and aspect ratio has huge impact on the quality of measurement. In the presented work the nanomechanical properties of four different polymer based electrospun fibers were tested using PeakForce Quantitative NanoMechanics atomic force microscopy, with standard and modified scanning probes. Standard, commercially available probes have been modified by etching using focused ion beam (FIB). Results have shown that modified probes can be used for mechanical properties mapping of biomaterial in the nanoscale, and generate nanomechanical information where conventional tips fail. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction In the last twenty years, a growing interest in new biomaterials for medical applications can be observed. Fibrous polymer scaffolds which can replace traditional metal implants become increasingly important in this field. In order to fulfill their task, fibrous biomaterials should be biocompatible and characterized by suitable mechanical properties. Electrospinning technique offers the ability to control material properties, as well as structure, and mechanical functions of the scaffolds for tissue engineering applications (Baker et al., 2012). Due to their biodegradability, interfibrous pore size, high surface area to volume ratio, immunogenicity, structural similarity to the tissue extracellular matrix (ECM) (Elsabee et al., 2012; Bosworth et al., 2013; Khadka and Haynie, 2012; Raghavan et al., 2012; Sun et al.,
∗ Corresponding author. Tel.: +48 22 662 31 58; fax: +48 22 234 87 50. E-mail address: [email protected] (A. Chlanda). http://dx.doi.org/10.1016/j.micron.2015.01.005 0968-4328/© 2015 Elsevier Ltd. All rights reserved.
´ 2013; Kijenska et al., 2012), electrospun fibers are good candidates for scaffolds. The mechanical properties of the material might have a strong influence on the biological function of the scaffold (Reilly and Engler, 2010). It is also worth to be noted that overall mechanical properties of any fibrous structure is based on three quantities (Carlisle et al., 2009): the architecture of the electrospun mesh, the properties of the single fibers and the junction between the fibers. To study the mechanical properties of the electrospun fibers at the nanoscale atomic force microscopy (AFM) can be used. It is a powerful tool for the visualization, and probing of selected mechanical properties of materials in broadly defined life sciences (Wozniak et al., 2009, 2010). This is mainly due to the ability of the technique for measuring force and distance at a high resolution and exploring surface in air and liquid with minimal sample preparation. It allows to examine topography, and mechanical properties of cells and tissues, as well as engineered biomaterials for tissue engineering. Testing mechanical properties of the electrospun fibers is crucial, and must be taken into account, because fibrous structures should be able to withstand the external forces acting on them after implantation (Chew
2
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Fig. 1. Force curves (A), and corresponding information that can be obtained from them for PLCL fibers (2 m × 2 m scan area): peak force error data channel (B), DMT modulus data channel (C), adhesion data channel (D), deformation data channel (E), and dissipation data channel (F).
et al., 2006). In addition, different types of cells may need different mechanical properties of the scaffolds (Yang et al., 2008). One can find literature positions describing testing of mechanical properties of electrospun fibers using AFM, with nanoindentation (Zhang et al., 2011), stretching (Baker et al., 2012) and bending method (Croisier et al., 2012), but to the authors’ knowledge it is the first time when new AFM technique – PeakForce Quantitative NanoMechanics (PFQNM) is used to characterize electrospun polymeric fibers. PeakForce Quantitative NanoMechanics is a new atomic force microscopy imaging mode developed recently, which allows to measure some mechanical properties of the material, such as: reduced Young’s modulus, adhesion, deformation and dissipation with high spatial resolution by probing at the nanoscale. Very few publications describing use of PFQNM have been printed, including stiffness mapping of amyloid fibrils (Adamcik et al., 2011), bitumen (Fischer et al., 2013), or polymers deformation testing (Liu et al., 2012). Each series of nanomechanical measurements on a sample is equivalent to 256 × 256 force-separation curves, providing a map of mechanical properties with the same resolution as the topographic image (Fig. 1). This technique can be used with any standard AFM probes, but for quality as well as quantity results, proper calibration must be done. Both – appropriate tip, and suitable reference sample choice are crucial to obtain adequate modulus values. The Derjaguin-Muller-Toporov (DMT) model (Butt et al., 2005) implemented into the dedicated Bruker NanoScope Analysis software is used. If not, the modulus map will only represent qualitative instead of quantitative information (Pittenger et al., 2010). To calculate the reduced Young’s modulus, the retract curve (Fig. 1A) was fitted into the DMT model: F − Fadh =
4 ∗ E 3
R(d − d0 )
3
where F is the force acting on the cantilever, Fadh the adhesion force between probe and the surface, R is the tip radius, d − d0 the sample deformation, and E* is the reduced Young’s modulus. If the sample Poisson ratio is known, the Young’s modulus can also be calculated. Next factor that was taken into account for quantitative analysis was deformation of the sample material. Maximum deformation during the PFQNM examination can be described as the penetration
of the probe into the sample. The deformation channel may include information from both plastic and elastic deformation components (Pittenger et al., 2010). Image resolution for AFM techniques depends mostly on the scanning probe parameters. As probe radius decrease, image resolution increase, thus probe geometry is crucial for high resolution imaging. There are several ways of probes modification described in the literature: carbon nanotube attachment (Lee et al., 2008), PVD (Physical Vapour Deposition) (Chen et al., 2000), and T – CVD (Thermal Chemical Vapour Deposition) methods (Chattopadhyay et al., 2006) or ion sputtering (Bale and Palmer, 2002). According to our knowledge, there is no information regarding work that propose a use of modified AFM probes for PFQNM examination. In this paper the AFM tip probe FIB modification procedure for PeakForce QNM application is described. It has been conducted in order to compare images obtained by standard, and modified probes. 2. Materials and methods 2.1. Materials Poly(l-lactide-co-caprolactone) (PLCL) with molecular weight of 150 000 was purchased from Evonik (Germany). Polycaprolactone (PCL) with a molecular weight of 80 000 g/mol, hydroxyapatite powder of grain size below 200 nm, and 1,1,1,3,3, 3-hexafluoro-2-propanol (HFP) used as a solvent in electrospinning, were purchased from Sigma–Aldrich [USA]. Collagen type 1 has been purchased from Koken (Japan). 2.2. Electrospun fibers fabrication Solutions of 9 w/v P(LCL)/collagen/HAp, and 9 w/v PCL/HAp (both with 90:10 ratio) were prepared by dissolving the polymer and proteins/ceramics together in HFP and stirred overnight at room temperature. P(LLA-CL) solutions with concentrations of 9 w/v, and PCL solutions with concentrations of 9 w/v were prepared as a reference, using the same procedure. Each of prepared solutions were placed in a 3 ml plastic syringe attached with flatended steel needles with 27G inner diameter, and then the syringe were placed in the pump (KD Scientific) and dispensed at a rate of 0.8 ml/h. Fibers were fabricated using electrospinning at 12 kV of
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Fig. 2. Standard scanning probes before (A and B), and after (C and D) FIB etching.
applied voltage from a high voltage power supply system (Gamma High Voltage Research). Fibers were electrospun from a single needle setup at room temperature, and 30% relative humidity. For collecting fibers, a flat grounded steel plate covered with aluminum foil was used, which was placed 10 cm from the tip of the needle. Fibers were collected on 15 mm glass cover slips for further examination. After electrospinning all the fibrous meshes were dried under vacuum for 72 h. 2.3. SEM observation of fibers The morphology and diameter of electrospun fibers was determined from images obtained by scanning electron microscopy (SEM) measurements (Phenom Pro X, FEI). Before imaging samples were coated with gold using high vacuum sputter coater (Leica EM SCD 500). 2.4. FIB nanofabrication Method described by Rozniatowski and Rebis (2013) for the AFM probes modification was utilized to modify the shape of standard rectangular silicon AFM probes. Focused ion beam (Hitachi FB2100) milling was used. Probes for modification were taken from new, unopened container. Special, custom holder was engineered to mount AFM probe in the FIB, that allows to change the position of the probe in relation to the beam of gallium ions. This allows by etching probe to change the tip cone angle (Fig. 2). All milling steps were performed with ion beam energy predefined in the FIB software for silica cutting. The beam spot size was decreased from tens (for coarse cutting) to few nanometers for high accuracy. 2.5. AFM nanomechanical mapping Atomic force microscopy experiments were performed using a Bruker MultiMode AFM NanoScope 8 microscope, equipped with standard probes (APP NANO – ACST type), as well as FIB modified probes. The probes were selected based on the recommendation of AFM microscope producer (Bruker, 2011) based on expected
range of reduced modulus values (5–500 MPa) of tested polymeric fibers. The nominal probe spring constant was 7 N/m and the actual spring constant of the probe cantilever was measured by thermal tune method (approx. 5 N/m for standard, and 4 N/m for modified probe). Nominal standard tip radius suggested by the producer was smaller than 10 nm, however the actual size estimated by the dedicated NanoScope Analysis (ver. 1.40) software was bigger, and was estimated at 35 nm. The tip radius after FIB-sharpening, estimated using the same software was approximately 15 nm. Relative calibration method was used to obtain quantitative results from experiment, and for that reason dedicated low-density polyethylene reference sample was chosen. Examination was conducted using the PeakForce Quantitative NanoMechanics AFM mode. All measurements were carried out in air, under ambient conditions and each set of nanomechanical measurements on a sample corresponds to 256 × 256 force-separation curves taken over different areas (20 m × 20 m, and 500 nm × 500 nm). Data analysis was made using NanoScope Analysis ver. 1.40, professional, dedicated software provided by the microscope producer. Individual force curves were also analyzed using SPIP 6.3.3 software.
3. Results and discussion 3.1. Topography of the fibers The topography of four kinds of electrospun fiber meshes made of polycaprolactone (PCL), polycaprolactone with hydroxyapatite (PCL/HAp), poly(l-lactide-co-caprolactone) (PLCL), and poly(l-lactide-co-caprolactone) with collagen and hydroxyapatite (PLCL/COL/HAp) was characterized using scanning electron microscope (Fig. 3), and atomic force microscope (Fig. 4) equipped with standard, commercially available, and FIB-modified probes. To illustrate the structural differences between examined meshes, the diameters, and surface roughness was calculated. Morphology of the electrospun meshes can be influenced by various parameters (Zandén et al., 2012; Lee et al., 2012; Sencadas et al., 2012). As shown in Figs. 5 and 7–9 fibers have a smooth surface and after addition of hydroxyapatite, surface morphology changes radically
Fig. 3. SEM images of electrospun fibers: (A) PCL, (B) PCL/HAp, (C) PLCL, and (D) PLCL/COL/HAp; magnification for all images is 5000×.
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Fig. 4. AFM topography images of electrospun fibers: (A) PCL, (B) PCL/HAP, (C) PLCL, and (D) PLCL/COL/HAp.
Fig. 5. Representative force curve recorded during examination fitted to DMT model.
(Fig. 6), which is reflected in both the mat surface roughness, as well as the average diameter of the individual fibers (Table 1). In case of PCL meshes, addition of 10 wt% HAp increased both fiber diameter and average mesh roughness (from approx. 840 to 940 nm for standard tip), whereas for PLCL composite opposite tendency can be observed – fiber diameter, and surface roughness decreased (from approx. 540 to 440 nm for standard tip). This phenomenon can be explained by the lower content of HAp in PLCL based composite 5 wt%, in comparison to 10 wt% in PCL based fibers. Smaller amount of HAp allowed particles to incorporate in the fiber material, and despite the smaller diameter of the PLCL fibers HAp is not visible on the surface. Moreover it is believed, that collagen smoothed the fibers surface, and therefore roughness is reduced. Analyzing quantitative roughness data obtained from measurements with modified AFM tips, it can be seen that the dependency described in the study using standard probes applies also to them. Furthermore comparing the data for the same materials (even with the caution that after the tip change, it was impossible to image exactly the same area) can be seen that every time, modified tips provides higher surface roughness value, which may indicate a better surface projection (Rozniatowski and Rebis, 2013). 3.2. Mechanical properties of the fibers Figs. 6–10 show the data from the different channels obtained during PFQNM examination. The data were used for qualitative as well as quantitative analysis of the meshes. The changes in
nanomechanical properties are indicated by the color changes on images from different data channels – the brighter the color, the higher the value. It can be seen, that in general images obtained with modified tips are less grainy and have better quality in comparison to the images acquired with standard tips probes. These differences can be observed especially well for PLCL-based meshes, where during measurements with commercial tips the dissipation data channel is empty (Fig. 8D). Representative force curve for PLCL mesh fitted to DMT model is presented in Fig. 5. No suitable information was recorded, whereas after tip modification it was possible to obtain valuable image (Fig. 9D). This may be due to the fact, that sharper shape of the modified scanning probe, leads to an easier distortion of the material. The sudden change in mechanical properties of the meshes on the joints can be observed. In the merging place, the boundaries with the width of about 30–40 nm are formed (Figs. 6 and 10). It can therefore be concluded, that on the joints of the fibers the value of reduced Young’s modulus, and dissipation increase, opposite to the decrease of the deformation value. However, further studies are required to explain this phenomenon. Similar dependence is visible for images taken on PCL/HAp sample (Fig. 7), where HAp particles in comparison to PCL surface have higher (brighter) modulus value, and lower (darker) other measured mechanical properties. This is a consequence of different physical and mechanical characteristic of ceramics and polymers – ceramics are stiffer, thus more difficult to deform (Wang et al., 2010).
Table 1 Fiber diameter and average mat roughness (Ra ) examined using standard and modified (*) AFM tips. Measured quantity
Material PCL
PCL/HAp
PLCL
PLCL/COL/HAp
Fiber diameter [nm] Average mat roughness [nm] Average mat roughness* [nm]
747 ± 142 841 ± 80 870 ± 72
817 ± 94 936 ± 121 1056 ± 55
319 ± 79 537 ± 53 576 ± 60
361 ± 40 440 ± 30 465 ± 33
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Fig. 6. PFQNM images of PCL electrospun fibers made with standard tips [(A) DMT modulus and (B) deformation] and with modified tips [(C) DMT modulus and (D) deformation]; 500 nm × 500 nm scan size.
Fig. 7. PFQNM images of PCL/HAp electrospun fibers made with standard tips [(A) DMT modulus and (B) deformation] and with modified tips [(C) DMT modulus and (D) deformation]; 500 nm × 500 nm scan size.
Fig. 8. PFQNM images of PLCL electrospun fibers made with standard tips, 500 nm × 500 nm scan size: (A) DMT modulus, (B) adhesion, (C) deformation and (D) dissipation.
Fig. 9. PFQNM images of PLCL electrospun fibers made with modified tips, 500 nm × 500 nm scan size: (A) DMT modulus, (B) adhesion, (C) deformation and (D) dissipation. Table 2 Quantitative analysis of selected nanomechanical properties of electrospun fibers measured using standard and modified AFM tips. Tested parameter
Material PLCL
DMT modulus [MPa] Deformation [nm]
PLCL/COL/HAp
PCL
PCL/HAp
Standard tip
Modified tip
Standard tip
Modified tip
Standard tip
Modified tip
Standard tip
Modified tip
12 ± 4 31 ± 5
19 ± 4 40 ± 3
46 ± 32 18 ± 4
117 ± 35 35 ± 6
3±1 43 ± 6
6±4 46 ± 7
17 ± 5 27 ± 3
48 ± 15 38 ± 1
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Fig. 10. PFQNM images of PLCL/COL/HAp electrospun fibers made with standard tips [(a) DMT modulus and (b) deformation] and with modified tips [(c) DMT modulus and (d) deformation]; 500 nm × 500 nm scan size.
Data obtained from the quantitative analysis are shown in Table 2. Reduced Young’s modulus of electrospun PCL and PLCL meshes has value of 3 MPa, and 12 MPa, respectively (measured with standard tips). Literature reports describe similar Young’s modulus results of the electrospun PCL, and PLCL meshes: 3.8 MPa for PCL (Croisier et al., 2012), and 1.22 MPa for PLCL (Kim et al., 2006). These values are lower than those obtained for electrospun composites. Similar correlation exists for examination run by modified AFM tips. The analysis of data for this same material indicate that the DMT modulus value obtained via etched tips is always higher than for conventional tip. This can be explained by referring to the shape of the probe – commercial probes are more spherical, while the probe after sharpening are more conical, and the DMT theory is applied with assumption of the spherical shape of the end of the scanning probe (Butt et al., 2005). As the reduced Young’s modulus value of the composites increases and the value of the deformation decreases – the more rigid is the material and the more difficult to deform. Furthermore, for the same material, value of deformation is always higher when measured with modified tips. This can be attributed to a sharper shape of the probe, that facilitates deformation of the material, despite lower spring constant for etched tip. 4. Conclusion To our knowledge, this is the first study applying PeakForce Quantitative NanoMechanics AFM technique to investigate electrospun fibers. Based on obtained results, it can be stated that PFQNM method shows huge potential for applications as high resolution technique of identification of morphology, and related nanomechanical properties of electrospun fibrous meshes. Due to its versatility it can be employed to other types of fibers and features at the nanoscale. Despite its many advantages, it should be noted that the described experimental method is more qualitative than quantitative, caused by complex, multi-step calibration procedure. The calibration even if done properly may not produce fully correct (quantitative) results, especially if the DMT model may be not appropriate. Available literature shows it is also the first examination using FIB-modified AFM probes for this kind of measurements and the experimental results allow us to conclude that the sharpened tips can be successfully used for the PFQNM measurements. Furthermore, images obtained with etched tips are less grainy, thus have better quality in comparison to the images acquired with standard tips and even can provide nanomechanical information on where conventional probes failed. Acknowledgements This study is financially supported by: (1) National Science Center of Poland (NCN) under grant: “Three-dimensional composite
scaffold based on biodegradable polymers and bioceramic with incorporated growth factors for bone tissue engineering. Research on the manufacturing process and the material influence on living cells function”, number: UMO-2011/01/B/ST8/07559. (2) Ministry of Science and Higher Education, Republic of Poland (MNiSW) under grant: “Polymer-ceramic composite, extracellular matrix mimicking biomaterials for tissue engineering needs: processes of degradation of the structure and mechanical properties”, number: 0616/IP2/2011/71. (3) National Science Center of Poland (NCN) under grant: “Hybrid growth factors delivery system supporting bone tissue regeneration”, number DEC-2011/01/M/ST8/07742.
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