Electrospinning and mechanical characterization of gelatin nanofibers

Electrospinning and mechanical characterization of gelatin nanofibers

Polymer 45 (2004) 5361–5368 www.elsevier.com/locate/polymer Electrospinning and mechanical characterization of gelatin nanofibers Zheng-Ming Huanga,*...

457KB Sizes 0 Downloads 8 Views

Polymer 45 (2004) 5361–5368 www.elsevier.com/locate/polymer

Electrospinning and mechanical characterization of gelatin nanofibers Zheng-Ming Huanga,*, Y.Z. Zhangb,c, S. Ramakrishnab,c,d, C.T. Limb,c,d a

School of Aeronautics, Astronautics and Mechanics, Tongji University, 1239 Siping Road, Shanghai 200092, People’s Republic of China b Department of Mechanical Engineering, National University of Singapore, Singapore 117576, Singapore c Division of Bioengineering, National University of Singapore, Singapore 117576, Singapore d Nanoscience and Nanotechnology Initiative, National University of Singapore, Singapore 117576, Singapore Received 23 January 2004; received in revised form 3 April 2004; accepted 3 April 2004 Available online 17 June 2004

Abstract This paper investigates electrospinning of a natural biopolymer, gelatin, and the mass concentration-mechanical property relationship of the resulting nanofiber membranes. It has been recognized that although gelatin can be easily dissolved in water the gelatin/water solution was unable to electrospin into ultra fine fibers. A different organic solvent, 2,2,2-trifluoroethanol, is proven suitable for gelatin, and the resulting solution with a mass concentration in between 5 and 12.5% can be successfully electrospun into nanofibers of a diameter in a range from 100 to 340 nm. Further lower or higher mass concentration was inapplicable in electrospinning at ambient conditions. We have found in this study that the highest mechanical behavior did not occur to the nanofibrous membrane electrospun from the lowest or the highest mass concentration solution. Instead, the nanofiber mat that had the finest fiber structure and no beads on surface obtained from the 7.5% mass concentration exhibited the largest tensile modulus and ultimate tensile strength, which are respectively 40 and 60% greater than those produced from the remaining mass concentration, i.e. 5, 10, and 12.5%, solutions. q 2004 Elsevier Ltd. All rights reserved. Keywords: Electrospinning; Gelatin; Nanofiber

1. Introduction Electrospinning technique has been recognized as an efficient processing method to manufacture nanoscale fibrous structures for a number of applications [1]. In biomedical field, for example, this technique can be used to make wound dressings [2,3], drug delivery platforms [4,5], tissue engineering scaffolds [6 – 8], and so forth. For tissue scaffold application, the fiber mats produced from biodegradable polymers have a diameter ranging from several microns down to a few nanometers. Such small size fibers could physically mimic the structural dimension of the extracellular matrix of various native tissues and organs, which are deposited and proliferated on essentially fibrous structures realigning from nanometers to millimeters. As human cells can attach and organize well around fibers with diameters smaller than those of the cells [9], the fibrous * Corresponding author. Tel.: þ 86-21-65985373; fax: þ 86-2165982914. E-mail address: [email protected] (Z.M. Huang). 0032-3861/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.polymer.2004.04.005

scaffolds prepared from electrospinning can be considered as ideal candidates. So far, the majority of work related with electrospinning biodegradable polymers has been focused on synthetic materials, mostly on PLA, PGA, PLGA, and PCL. For many biomedical applications, the most important characteristics that should be targeted for include biocompatibility and mechanical performance. In comparison with synthetic counterparts, natural biopolymers generally have better biocompatibility and hence are more suitable for human body. However, to convert a natural biopolymer into submicron or nanometer fibers through electrospinning is usually more difficult than to do a synthetic polymer. Due to this reason, only in the recent literature have we found a few reports addressing electrospinning of some natural biopolymers [3,6,10]. In this paper, we investigate electrospinning of a different biopolymer, gelatin material, not done yet in the literature. Gelatin is a natural biopolymer derived from collagens and has almost identical compositions and biological properties as those of collagens. It is an aqueous polymer, i.e. dissolvable in water. Unfortunately, gelatin/water system


Z.-M. Huang et al. / Polymer 45 (2004) 5361–5368

cannot be processed with electrospinning. Moreover, when dissolved in water at a temperature around or above 37 8C, gelatin becomes a kind of a colloidal sol and hence without a special treatment (e.g., cross-linking) is not suitable for tissue scaffold application. However, gelatin does have an important merit that it is a cheap biopolymer. By some posttreatment or mixed with another (synthetic) biodegradable polymer [11], gelatin can be used alone or as a blend component to prepare nanofibrous membranes for tissue scaffolds, wound healing and health caring devices, and other biomedical applications. In this paper, the electrospinning of gelatin has been achieved by dissolving it an uncommonly used solvent, 2,2,2-trifluoroethanol. With this solvent, the gelatin solutions of mass concentrations in a range of 5– 12.5 wt% have been successfully electrospun into ultra fine fibers at room temperature. However, further lower or higher concentrations were difficult to process. Another purpose of this paper is to study the mass concentration-mechanical property relationship of the resulting ultra fine fiber mats. As aforementioned, mechanical behavior is another important property that should be considered in developing new biomaterials. Most current researches on electrospinning have dealt with processing phenomena involved and physical and geometrical characterizations. Limited work has been done on the relationship between the processing parameters-mechanical property of an electrospun polymer. It has been found in this paper that the mechanical parameters (tensile modulus and ultimate tensile strength) of the nanofibrous membranes electrospun from different gelatin solutions were not varied in a fixed manner with the gelatin mass concentrations. An optimal mass ratio existed in this regard. This finding would be useful to a material design with electrospinning.

2. Processing 2.1. Solvent selection In electrospinning, polymer liquids in solution or molten form are forced under a high DC voltage to become ultra fine fibers with diameters from a few microns down to several nanometers [1]. The solvent used to prepare the polymer solution has a predominant influence on its spinnability. Most current research has been on electrospinning of synthetic polymers, and limited on natural biopolymers, which are usually polyelectrolyte polymers. Essentially no such report has been found in the open literature on some more useful biopolymers such as chitosan, gelatin, and alginate. Fibers with diameters of hundreds of microns of those biopolymers have been manufactured through wet or dry spinning in industry. Gelatin can be easily dissolved in water at temperature of above 40 8C as an aqueous solution. Such an aqueous solution can be used to produce large diameter gelatin fibers for arresting bleeding purpose by means of wet spinning

[12,13]. Due to the poor fiber-formation ability of gelatin, usually some other synthetic polymers are added to improve this ability. Nevertheless, the gelatin solution dissolved in water is unable to electrospin into ultra fine fibers even under heated and non-gelation condition. This may be probably attributed to its polyelectrolyte characteristic. Unlike synthetic polymers that are generally nonionic and can be dissolved in organic solvents through nonionic interactions between solute and solvents, gelatin is a kind of polyelectrolyte polymers, which possess many ionizable groups. Its amine and carboxylic functional groups can be ionized by acidic agents or hydrolyzed to carry positive or negative charges. In aqueous solution, such ionization (pH dependent) gives rise to a polyion bearing many charges, accompanied by many small counterions as shown below:

In addition, the strong hydrogen bonding constituting the gelatin behavior results in a 3-D macromolecule network which causes the mobility of the polymer molecule chains reduced tremendously. Therefore, although water is commonly used to make gelatin solution, searching for an alternative organic solvent plays a key role in successfully electrospinning this biopolymer. Gelatin is a biopolymer with strong polarity. There are very few high polarity organic solvents available for dissolving this biopolymer. It has been known that fluorinated alcohols such as trifluoroethanol and hexafluoro isopropanol (HFIP) are good solvents for polypeptide biopolymers such as collagen. Recently, Matthews et al. used HFIP as a solvent to have successfully electrospun collagen into nanofibers [14]. As gelatin can be considered as denatured collagen, similar fluorinated solvents would be applicable. In this study, we found that 2,2,2-trifluorothanol (TFE) is efficient for this purpose, which affords the ability to directly electrospin gelatin into ultra fine fibers without adding any other fiber formation material. 2.2. Solution preparation Polymer gelatin of type A from porcine skin in powder form was purchased from Aldrich (Milwaukee, USA). Dissolving solvent of TFE is a product from Fluke. To prepare spinning solutions, the gelatin was dissolved in TFE stirring at room temperature for 6 h to make transparent solutions. Different gelatin concentrations of 2.5, 5.0, 7.5, 10, 12.5, and 15% w/v were prepared in order to investigate spinnability, fiber morphology, and mechanical performance of the resulting ultra fine fiber mats. Here, for instance, 10% w/v means that 1 g of gelatin powder was mixed with 10 ml of TFE solvent. The polymer and the solvent were used as provided without further purification.

Z.-M. Huang et al. / Polymer 45 (2004) 5361–5368


Fig. 1. Schematic electrospinning set-up in this study.

2.3. Electrospinning The experimental setup used for electrospinning process of this study is schematically shown in Fig. 1, which consisted of an adjustable DC power supply (Gamma High Voltage Research, USA) capable of generating DC voltage in a range of 0– 50 kV, a syringe pump on which a 5 ml syringe was connected to a stainless steel needle using a teflon tube having an inner diameter of around 1.0 mm, and a horizontally to and fro moving collector wrapped with aluminum foil. When the needle, whose inner diameter was 0.85 mm, was charged to a high DC voltage of positive polarity from the power supply, the gelatin solutions were ejected from the tip of the needle to generate ultra fine fibers, and the resulting non-woven fiber mat was collected on the collector that was connected to the ground (a negative polarity). The syringe pump was used to push the syringe in such a way that a constant and stabilized mass flow of the polymer solution can be delivered during the electrospinning. In this study, a constant mass flow rate of 0.8 ml/h was applied to all of the concentration solutions. The horizontally to and fro moving collector was a specially designed set-up [15] in order that a relative large piece of the ultra fine fiber mat with even thickness can be obtained. Such kind of mat is essential for the preparation of mechanical characterization samples. For the solutions of different gelatin concentrations, the processing parameters used such as humidity, temperature, voltage, and the distance between the needle tip and the collector were slightly different. Detailed information of those parameters for the different concentration solutions is

Fig. 2. SEM photograph of an electrospun gelatin/TFE fiber mat with 2.5% w/v concentration.

given in Table 1. In the present study, the electrospinning was essentially processed at ambient conditions. However, the DC voltages were adjusted from 0 value to the levels so that the corresponding solutions could be electrospun without visible liquid drops by naked eyes. It has been recognized by previous researchers that as long as a polymer solution can be electrospun into ultra fine fibers a lower electric potential will favor finer fiber formation [16]. After the electrospinning, all the fiber mats from the different concentration solutions collected with the aluminum foils on the collector were kept at room temperature for 24 h before being placed in a vacuum drying oven for a couple of days’ drying treatment.

3. Characterization The fiber morphology was observed under scanning electronic microscopy (SEM) using a JEOL JSM-5800LV machine with an acceleration voltage of 15 kV. Before SEM observation, all of the samples cut from the electrospun fiber mats of 2.5 – 15% w/v mass concentrations were sputter coated with gold under a JEOL JFC-1200 fine coater for

Table 1 Electrospinning conditions for different gelatin/TFE solutions Concentration% (w/v)

Humidity (%)

Temperature (8C)

Voltage (kV)

ha (cm)

2.5 5.0 7.5 10.0 12.5 15.0

82 84 80 83 84 83

20.8 20.3 19.9 19.5 20.3 19.5

16.0 12.5 10.5 10.0 10.0 10–15

12 12 12 12 12 12

(mass flow rate ¼ 0.8 ml/h, the inner diameter of the needle ¼ 0.85 mm). a h ¼ Distance between the needle tip and the collector.

Fig. 3. SEM photograph of an electrospun gelatin/TFE fiber mat with 5.0% w/v concentration.


Z.-M. Huang et al. / Polymer 45 (2004) 5361–5368

Fig. 4. SEM photograph of an electrospun gelatin/TFE fiber mat with 7.5% w/v concentration.

Fig. 6. SEM photograph of an electrospun gelatin/TFE fiber mat with 12.5% w/v concentration.

60 s. Typically representative SEM photographs of all the fiber mats with different gelatin concentrations are shown in Figs. 2 – 7. Based on these SEM photos, the fiber diameters of the different concentration nanofibrous mats were analyzed using an image visualization software ImageJ developed by Upper Austria University of Applied Sciences [17]. Mechanical characterization was achieved by applying tensile test loads to specimens prepared from the electrospun ultra fine non-woven fiber mats. As a single polymer nanofiber is very weak, the resulting non-woven mat would be so delicate that any direct touch on the mat surface during manipulation can damage the fibers. As such, sufficient care must be taken in preparing and then gripping the tensile specimens in order to avoid severe damage. In this study, we prepared the specimens in the following way. First, a white paper template was cut as shown in Fig. 8, and double-side tapes were glued onto the top and bottom areas of one side. The template was then glued onto topside of the fiber mat, and was cut into rectangular pieces along the vertical lines. After the aluminum foil was carefully peeled off, single side tapes were applied onto the gripping areas as end-tabs. The

resulting specimens had a planar dimension of width £ gauge length ¼ 10 mm £ 30 mm. Whereas the planar dimensions of all the specimens with different gelatin concentrations were the same, their thicknesses were different; see Table 2 for detail. As can be seen from the table, the lower the concentration the thinner the thickness. This was because nearly the same amount (around 3 ml) of polymer solutions was used to electrospin the non-woven fiber mats. The specimen thicknesses were measured using a digital micrometer, having a precision of 1 mm. The tensile testing was performed using a tabletop Instron tester (Model 3345) with a load cell of 10 N. A

Fig. 7. SEM photograph of an electrospun gelatin/TFE fiber mat with 15.0% w/v concentration.

Fig. 5. SEM photograph of an electrospun gelatin/TFE fiber mat with 10.0% w/v concentration.

Fig. 8. A paper template used to prepare tensile specimens of the electrospun non-woven fiber mat.

Z.-M. Huang et al. / Polymer 45 (2004) 5361–5368 Table 2 Tensile properties of the nanofiber membranes electrospun from gelatin/TFE solutions of different gelatin concentrations 5% w/v 7.5% w/v 10% w/v Specimen thickness (mm) 20 Modulus (MPa) 117 Ultimate strength (MPa) 2.93

41 174 4.79

12.5% w/v

50 97 116 (134)a 123 2.60 3.40

(Tensile specimen width £ gauge length ¼ 10 mm £ 30 mm). 116 ¼ The averaged modulus using all of the four testing stress–strain curves, 134 ¼ the averaged modulus using the three testing stress–strain curves which were close to each others. a

cross-head speed of 10 mm/min was used for all of the specimens tested. The machine-recorded data were used to process the tensile stress – strain curves of the specimens. Namely, the tensile strains were obtained by dividing the crosshead displacements with the original gauge length (30 mm). It is noted that the tensile tests were performed only for those specimens from the gelatin/TFE solutions having gelatin concentrations of 5% w/v to 12.5% w/v. The amount of the fibers collected from the other two solutions, i.e. the 2.5 and 15% w/v concentration solutions, was too small to prepare membrane-like tensile specimens.

4. Results and discussion 4.1. Spinnability In the present case when the electrospinning was performed at room temperature, the spinnability depended mainly on the gelatin mass concentration. Continuous fibers were successfully electrospun from the gelatin/TFE solutions of 5 –12.5% w/v. The SEM photographs of Figs. 3 –6 clearly show that whereas beads-on-string were involved with the 5% w/v concentration essentially no beads occurred on the fibers electrospun from the gelatin/TFE solutions with the mass concentrations from 7.5 to 12.5% w/v. It has been found in this study that the remaining two solutions, i.e., those with the lowest and the highest gelatin concentrations, were difficult to be processed into nanofibers through the electrospinning technique. For the 2.5% w/ v concentration, essentially no continuous fibers except for beads and bead assemblages could be obtained, although some fiber segments seemed to still exist. Fig. 2 shows the fiber morphology electrospun from the 2.5% w/v concentration. In contrast to the 2.5% w/v concentration, no beads were found with the fibers electrospun from the solution of


the 15% w/v concentration as shown in Fig. 7. However, the difficulty in electrospinning using the highest concentration solution was that highly viscous fluid balls would be gradually gathered outside the tip of the needle after the electrospinning had started for a while no matter how high an electric voltage had been applied and hence a few fibers were resulted. Moreover, only a portion of the electrically charged solution jets seemed to have been solidified into ultra fine fibers. The remaining portion must have been still in the fluid form when reaching the aluminum foil. Thus, although the continuous jets were obviously ejected towards the collector even when there were the fluid balls formed outside the needle tip, much less fibers than those from the solutions of the 5– 12.5% w/v concentrations were obtained. It is likely that varying some processing conditions such as increasing the solidification temperature could result in better spinnability for the 15% w/v concentration solution. 4.2. Fiber diameter Measured fiber diameters for the solutions with gelatin concentrations from 5 to 15% w/v are listed in Table 3. As long as the gelatin solutions were spinnable, the resulting ultra fine fibers had a diameter generally below 350 nm. As expected, the 5% w/v concentration solution resulted in the smallest fiber diameter, being around 100 nm. However, significant beads occurred on the fiber surface with this concentration. On the other hand, the 7.5% w/v concentration gave no beads, and the nanofibers electrospun from this solution had an averaged diameter of 140 nm. As seen subsequently, the nanofibrous membrane from this latter concentration solution exhibited the best mechanical performance. 4.3. Mechanical performance The tensile stress – strain curves of the electrospun gelatin/TFE nanofiber membranes with gelatin mass concentrations from 5 to 12.5% w/v are shown in Figs. 9 –12. From these figures, the averaged tensile modulus and the ultimate tensile strength of the respective concentrations are obtained and summarized in Table 2. Typical curves each from a different gelatin concentration membrane are plotted in Fig. 13 for an obvious comparison. From Table 2 and Fig. 13, one can clearly see an interesting phenomenon that the highest mechanical performance of the fiber membrane did not correspond to the lowest or the highest mass concentration. Instead, the nanofiber membrane

Table 3 Fiber diameters of electrospun gelatin/TFE solutions with different gelatin concentrations

Diameter range (nm) Averaged diameter (nm)

5% w/v

7.5% w/v

10% w/v

12.5% w/v

15% w/v

50–175 100

80 –300 140

130–540 240

200 –750 340

630–4800 1900


Z.-M. Huang et al. / Polymer 45 (2004) 5361–5368

Fig. 9. Tensile stress–strain curve of electrospun gelatin/TFE solution with a gelatin concentration of 5% w/v.

Fig. 10. Tensile stress–strain curve of electrospun gelatin/TFE solution with a gelatin concentration of 7.5% w/v.

Fig. 11. Tensile stress –strain curve of electrospun gelatin/TFE solution with a gelatin concentration of 10.0% w/v.

Z.-M. Huang et al. / Polymer 45 (2004) 5361–5368


Fig. 12. Tensile stress–strain curve of electrospun gelatin/TFE solution with a gelatin concentration of 12.5% w/v.

resulted from the 7.5% w/v concentration exhibited the largest tensile modulus and the ultimate tensile strength, whereas the nanofiber mats from the other three mass concentrations had comparable stiffness and strength. Quantitatively, the modulus and the strength from the 7.5% w/v concentration were, respectively, 40 and 61% higher than the modulus and tensile strength averaged from those of the other three mass concentrations. This may be attributed to the fact that the 7.5% w/v concentration provided the finest fiber mat compared with the 10 and 12.5% w/v concentrations (see Table 3), and resulted in the tightest cohesion thereby. Although the 5% w/v concentration resulted in an even smaller fiber diameter, it at the same time gave rise to too many beads than the 7.5% w/v concentration did (see Figs. 3 and 4). The beads on fiber surface might have considerably reduced the cohesive force between the fibers of the non-woven fiber mat and hence a poorer mechanical performance of the nanofiber mat from the 5% w/v concentration than from the 7.5% w/v concentration was obtained. Furthermore, the 5% w/v nanofiber mat even displayed smaller modulus and ultimate tensile strength than the 12.5% w/v nanofiber mat.

5. Conclusion Electrospinning of a natural biopolymer, gelatin, was investigated in this paper. Although this biopolymer can be well dissolved in water, the resulting solution is recognized unspinnable through an electrospinning technique. A different organic solvent, 2,2,2-trifluoroethanol (TFE), has been found to be suitable. At the ambient condition, the gelatin/TFE solution with a mass concentration less than 5% or greater than 12.5% was difficult to electrospin into nanofibers. Mechanical characterization indicates that both the fiber diameter and the beads on the fiber surface can influence the mechanical performance of the electrospun nanofiber membranes. The finest fiber mat generally exhibits higher tensile modulus and ultimate tensile strength. However, if there are beads on the fiber surface, the situation may be different. The nanofiber mat with the smallest fiber diameter but having beads on the fiber surface even exhibited poorer mechanical performance than that having the largest fiber diameter but without any beads.

Fig. 13. Comparison between the measured tensile results of the electrospun gelatin/TFE solutions with different gelatin concentrations.


Z.-M. Huang et al. / Polymer 45 (2004) 5361–5368

Acknowledgements The authors would like to thank Mdm X. L. Zhong and Mr. K. Fujihara in technical help in performing the SEM and mechanical tests. ZMH acknowledges the financial support of the NanoSciTech Promote Center, the Shanghai Science and Tech. Committee of China under Grant 0352nm091.

References [1] Huang ZM, Zhang YZ, Kotaki M, Ramakrishna S. A review on polymer nanofibers by electrospinning and their applications in nanocomposites. Compos Sci Technol 2003;63:2223–53. [2] Khil MS, Cha DI, Kim HY, Kim IS, Bhattarai N. Electrospun nanofibrous polyurethane membrane as wound dressing. J Biomed Mater Res 2003;67B(2):675–9. [3] Kim SH, Nam YS, Lee TS, Park WH. Silk fibroin nanofiber. Electrospinning, properties, and structures. Polym J 2003;35(2): 185–90. [4] Zeng J, Xu X, Chen X, Liang Q, Bian X, Yang L, Jing X. Biodegradable electrospun fibers for drug delivery. J Controlled Release 2003;92:227–31. [5] Verreck G, Chun I, Rosenblatt J, Peeters J, Dijck AV, Mensch J, Noppe M, Brewster ME. Incorporation of drugs in an amorphous state into electrospun nanofibers composed of a water-insoluble, nonbiodegradable polymer. J Controlled Release 2003;92:349–60. [6] Jin HJ, Fridrikh SV, Rutledge GC, Kaplan DI. Electrospinning Bombyx mori silk with poly(ethylene oxide). Biomacromolecules 2002;3(6):1233–9.

[7] Zong X, Ran S, Fang D, Hsiao SB, Chu B. Control of structure, morphology and property in electrospun poly(glycolide-co-lactide) non-woven membranes via post-draw treatments. Polymer 2003;44: 4959–67. [8] Fertala A, Han WB, Ko FK. Mapping critical sites in collagen II for rational design of gene-engineered proteins for cell-supporting materials. J Biomed Mater Res 2001;57:48–58. [9] Laurencin CT, Ambrosio AMA, Borden MD, Cooper Jr JA. Tissue engineering: orthopedic applications. Annu Rev Biomed Engng 1999; (1):19– 46. [10] Wnek GE, Carr ME, Simpson DG, Bowlin GL. Electrospinning of nanofiber fibrinogen structures. Nano Lett 2003;3(2):213–326. [11] Giusti P, Barbani N, Lazzeri L, Polacco G, Cristallini C, Cascone MG. Gelatin-poly(vinyl alcohol) blends as bioartificial polymeric materials. Proceedings of the Fourth International Conference on Frontiers of Polymers and Advanced Materials, 4–9 January, Cairo, Egypt; 1997. [12] Liu S, Niu J, Ge H, Liu H. Clinical hemostatic effects of gelatin fleece. Chin J Trauma 2000;16(3):181–3. [13] Nagura M, Yokota H, Ikeura M, Gotoh Y, Ohkoshi Y. Structures and physical properties of cross-linked gelatin fibers. Polymer J 2002; 34(10):761–6. [14] Matthews JA, Wnek GE, Simpson DG, Bowlin GL. Electrospinning of collagen nanofibers. Biomacromolecules 2002;3:232–8. [15] Chia SS, Lim CT, Zhang YZ, Ramakrishna S. Highly productive electrospinning apparatus with multiple spinneret system, US Patent provisional application no. 60/475921; 2003. [16] Deitzel JM, Kleinmeyer J, Harris D, Tan NCB. The effect of processing variables on the morphology of electrospun nanofibers and textiles. Polymer 2001;42:261–72. [17] http://rsb.info.nih.gov/ij/.