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Chemical composition and molecular structure of fibers from transgenic flax producing polyhydroxybutyrate, and mechanical properties and platelet aggregation of composite materials containing these fibers
Composites Science and Technology 69 (2009) 2438–2446
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Chemical composition and molecular structure of fibers from transgenic flax producing polyhydroxybutyrate, and mechanical properties and platelet aggregation of composite materials containing these fibers Jan Szopa a,*, Magdalena Wróbel-Kwiatkowska a,b, Anna Kulma a, Magdalena Zuk a, Katarzyna Skórkowska-Telichowska c, Lucyna Dymin´ska d, Mirosław Ma˛czka e, Jerzy Hanuza d,e, Jacek Zebrowski f, Marta Preisner a a
Faculty of Biotechnology, University of Wrocław, Poland Laboratory of Polymers, Institute of Mechanical Engineering and Automation, Wrocław University of Technology, Wrocław, Poland c Department of Endocrinology, 4th Military Hospital, Wrocław, Poland d Department of Bioorganic Chemistry, Institute of Chemistry and Food Technology, Faculty of Economics and Engineering, University of Economics, Wrocław, Poland e Institute of Low Temperatures and Structure Research, Polish Academy of Sciences, Wrocław, Poland f Faculty of Biotechnology, University of Rzeszow, Poland b
a r t i c l e
i n f o
Article history: Received 22 October 2008 Received in revised form 15 June 2009 Accepted 20 June 2009 Available online 26 June 2009 Keywords: A. Flax fiber polypropylene composite B. Fiber/matrix bond B. Mechanical properties D. Infra-red (IR) spectroscopy D. Scanning electron microscopy (SEM)
a b s t r a c t In order to improve the properties of flax fibers so that they interact better with the matrix material in composites, several lines of transgenic flax were produced over-expressing the bacterial polyhydroxybutyrate (PHB) synthesis genes. Infra-red spectrophotometry revealed that the cellulose in fibers from the transgenic plants was more highly structured than in fibers from the control plants and PHB was strongly bound to the cellulose of the fibers by covalent ester and hydrogen bonds. The composite containing fibers from transgenic plants was significantly stronger and stiffer than the composites containing fibers from the control plants. Scanning electron microscopy of the fracture surface of composite sheets indicated that fibers from transgenic plants adhered to the polypropylene matrix significantly better. The composite containing fibers from transgenic plants induced almost no platelet aggregation and so may therefore be useful in the construction of biomedical devices that come in contact with blood. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction There has recently been a surge of interest in the industrial and biomedical uses of composites containing bio-fibers reinforced with polymers. The bio-fibers most often used for industrial purposes are cellulose fibers, which may be used to replace fiberglass in plastics used to produce a variety of products in many industries. For example, composites reinforced with bio-fibers have been used in the manufacture of automobile parts such as door panels, seat backs and glove compartments. The main advantage of these fibers is that they are more biodegradable and less reliant on petrochemical resources than most man-made polymers [1]. A valuable source of high-quality ligno-cellulose fibers is flax. The quality of flax fibers highly depends on the fine structure of the cellulose molecules, the growing conditions, and the retting method. * Corresponding author. E-mail address: [email protected] (J. Szopa). 0266-3538/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.compscitech.2009.06.017
During retting, bast fiber bundles are separated from the core, epidermis and cuticle [2]. This is brought about by enzymatic degradation of pectin and hemicellulose in the flax cell-wall by various microorganisms, including filamentous fungi [3]. Flax fibers contain 70% cellulose, which is a polymer of D-glucopyranosyl units strongly linked by intra-molecular and intermolecular hydrogen bonds. Because of these bonds, cellulose does not melt before it undergoes thermal degradation. One of the main problems in using bio-fibers such as flax fibers in composite materials is that natural fibers are less strong than currently used materials such as fiberglass (the tensile strength of glass fiber is 2400 106 N/m2 and of flax fiber, which is the best among natural fibers, only 800–1500 106 N/m2 [1]). Also components made from unmodified polypropylene or polyethylene and natural fibers are less strong that polypropylene on its own [4,5]. In composite materials, bio-fibers adhere poorly to hydrophobic matrices, often to the point that the composite is mechanically inferior to either the bio-fibers or the matrix material on their own. On the other hand, when the fibers adhere well to the matrix, which may be achieved via fiber or matrix modification,
J. Szopa et al. / Composites Science and Technology 69 (2009) 2438–2446
mechanical stress is transferred from the matrix to the fibers, which improves the mechanical properties of the composite [5,6]. Polypropylene is a thermoplastic polymer used in a wide variety of products including textiles and plastic items for medical or laboratory use [7]. It is often used as a matrix in composites that include natural fibers. However, polypropylene is hydrophobic, and therefore does not bind strongly to hydrophilic natural fibers. Modification of polypropylene such as malonylation can help to overcome this effect [8]. Proper adhesion of the fibers to the matrix is essential for ensuring optimal mechanical properties in the composite. Better contact between the fibers and the matrix also enhances the hydrophobicity of the composite. Additionally several approaches have been used to improve the properties of cellulose fibers so that they interact better with the matrix material in composites. Chemical, physical and enzymatic treatment changes the solubility of the cellulose in the fiber mainly by polymorphically transforming cellulose I into cellulose II [9]. Other methods include surface fibrillation and electric discharge of the fibers, de-waxing, alkali treatment, acetylation and grafting. Coupling agents such as silanes and dihydric phenols can also be used to improve adhesion. These agents chemically react with the fibers and the matrix, promoting the formation of covalent and hydrogen bonds between them [6]. Unfortunately, all of these methods complicate the production of industrial fiber by introducing additional steps to the process. Therefore, genetic engineering is currently being evaluated as a safe and relatively inexpensive alternative method for modifying and improving fiber quality. Polyhydroxybutyrate (PHB) is a biodegradable bacterial polymer which has been extensively studied because of its potential biomedical applications. The polymer itself is fragile, which severely limits its usefulness. Its mechanical properties are significantly improved when it is incorporated into bio-fibers. Furthermore, PHB may improve adhesion between flax fibers and the matrix in composite materials. In previous studies, we produced several lines of transgenic flax that over-express the bacterial genes responsible for PHB synthesis and evaluated the biochemical and mechanical properties of stems and fibers from field grown specimens [10,11]. The transgenic plants had a normal phenotype and produced reasonable amounts of PHB. Fibers from the transgenic plants did not significantly differ from those of the control plants in terms of cellulose, lignin and pectin content. This indicates that the synthesis of these substances was not affected by the synthesis of PHB. Fibers from transgenic plants were stronger and stiffer than fibers from untransformed flax. They also contained more phenolic acids, which are potent anti-oxidants. This may be significant when these fibers are used for biomedical purposes. The improvement in fiber quality was attributed to changes either in the chemical composition of the fibers or in the molecular structure of the cellulose in the fibers. The hydroxyl group of PHB can participate in intra-molecular and inter-molecular hydrogen bonds. The arrangement of these bonds may affect the fine structure of flax fibers. Changes in the chemical composition of flax fibers can be detected by infra-red spectroscopy. This method can also be used to detect changes in molecular structure [12]. Composites containing bio-fibers also have properties that may be useful in several fields of medicine [13]. They may serve as an artificial scaffold in tissue engineering. They may also be useful in constructing drug-release systems, cardiovascular patches and nerve cuffs [14]. Materials that induce platelet aggregation on their surface cannot be used in devices that are to be implanted such as heart valves and stents. On contact with blood, these materials adsorb plasma proteins that induce platelet aggregation. This can lead to serious
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thrombo-embolic complications. On the other hand, these materials also may be useful in some applications because they promote the formation of bone tissue [15]. The aim of the present study was to use infra-red spectroscopy to evaluate the chemical composition and molecular structure of fibers from flax plants that have been genetically modified to produce PHB. Composites containing flax fibers from transgenic and control plants in a polypropylene matrix were also compared in terms of their mechanical properties and their ability to induce blood platelet aggregation.
2. Materials and methods 2.1. Transgenic plants Flax plants (Linum usitatissimum L., fibrous cultivar Nike) were genetically modified to over-express the three bacterial genes that code for all of the enzymes required for the synthesis of PHB [10]. The plants were transformed using the binary vector pBinARHygABC 14-3-3 containing three cDNA sequences from Ralstonia eutropha: phbA (EMBL/GenBank accession number J04987) encoding bketothiolase; phbB (EMBL/GenBank accession number J04987) encoding acetoacetyl-CoA reductase; and phbC (EMBL/GenBank accession number J05003) encoding the last enzyme of the route i.e. PHB synthase. The genes were placed under the control of a stem specific promoter of the 16R isoform of the gene for 14-3-3 protein of Solanum tuberosum [16] and the plastid directing sequence of the Rubisco gene. The vector was introduced into flax explants using Agrobacterium tumefaciens. The inoculated explants were then grown on callus induction medium and shoot regeneration medium. Transgenic plants were preselected using PCR (polymerase chain reaction), and selected using northern blotting [10]. Three lines of transgenic plants were used in this study: M13, M42 and M50. They differed in the concentration of PHB and they accumulated, respectively, 1.33 lg PHB/g of a fresh weight (FW), 4.35 lg/g FW and 4.62 lg/g FW. 2.2. Retting Transgenic and control plants were grown in the same field near Wrocław. The plants were harvested at the same time: 4 months after planting and were retted using the dew method [11]. Briefly, the harvested plants were spread out in an open field and left exposed for at least 40 days. The plants were turned every 2 weeks. During this time, bacteria and fungi grew on the plants and degraded the cell-wall polysaccharide and middle lamella, thereby releasing fibers from the stem matrix. 2.3. Infra-red spectrophotometry of fibers from transgenic and control plants In order to determine the chemical composition and molecular structure of fibers from transgenic and control plants, fibers were analyzed using infra-red spectrometry. Spectra were measured at room temperature using a Biorad 575C FT–IR spectrometer. Data were collected over a spectral range from 50 to 4000 cm 1 with a resolution of 2 cm 1. In the mid infra-red part of this range, samples were prepared in a KBr pellet. In the far infra-red part of this range, samples were suspended in Nujol.
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2.4. Isolation of PHB from transgenic plants PHB was isolated as described in a previous paper [10]. Samples of transgenic plants (25–250 mg) were extracted for 1 h at 55 °C with 1 ml of methanol per gram of plant material, and then extracted twice for 1 h at 55 °C with 1 ml of ethanol per gram of plant material. This ensured the dissolution and removal of lipids and 3hydroxybutyrate monomers. The samples were then suspended in 1 ml of chloroform per gram of plant material. 3-Hydroxy-valeratemethyl ester was added as an internal standard. The samples were then esterified by adding 17 ml ethanol and 0.2 ml 1 N HCl per gram of plant material. The samples were incubated for 4 h at 100 °C. The extracts were filtered and washed with 0.9 N NaCl. The organic phase was removed and analyzed using infra-red spectrophotometry.
of a Merlin software and used to calculate E from a formulae E = (F/x) l3/4ah3, where l – the distance between the supports, a – the specimen width and h – the specimen thickness. Representative load–displacement curves for tensile and bending tests are presented in Fig. 1. Seven to ten samples were evaluated in each test for each composite. Sheets of pure polypropylene were used as a reference. 2.7. Scanning electron microscopy In order to determine the degree of adhesion between the fibers and the matrix, the surface morphology of fractured composite sheets was examined using a JEOL JSM-5800LV scanning electron microscope. Samples were coated with graphite before analysis. 2.8. Aggregation of platelets on the surface of composite sheets
2.5. Preparation of composites Three kinds of composite material were prepared using a matrix of commercially available polypropylene granules (HP 548E). The first was made with fibers derived from M50 transgenic plants, the second with fibers from control plants, and the third with fibers from control plants supplemented with bacterial PHB (Sigma–Aldrich). In all cases, the ratio of fiber to polypropylene was 1:4 (weight ratio). Finally pulverized combed flax fibers were mixed with polypropylene granules at 170 °C. The mixture was mechanically pressed into sheets. These were cut into smaller pieces and heat-pressed between Teflon sheets for 75 s at 175 °C under 20 tons of pressure to produce finished composite sheets that were 1 mm thick. 2.6. Mechanical testing of composite sheets The tensile strength and the effective Young’s modulus in bending of the composite sheets were measured using a computer-driven Instron 5542 system (High Wycombe, UK) as described in a previous paper [11]. In a tensile test, 25 mm long sections of composite sheet cut into a ‘‘dog bone” shape were fixed between clamps at the initial distance of 5 mm. The samples were stretched at a rate of 20 mm/min up to failure and the maximum load, Fmax, was recorded. The ultimate tensile strength, rmax, was calculated as rmax = Fmax/ah, where a – the actual width of the specimen at the cross section location where failure occurred and h – the sheet thickness. To determine the effective Young’s modulus, E, a threepoint flexure test was performed at the loading rate of 20 mm/min. Maximum slope of the load–displacement curve inclination to the displacement axis, (F/x), was automatically determined by means
The aggregation of platelets on composite sheets was examined using scanning electron microscopy as described by Okroj et al. [17]. Sheets of pure polypropylene were used as a reference. Venous blood was collected from healthy volunteers in tubes containing 3.8% sodium citrate. The tubes were centrifuged for 10 min at room temperature at 1000g, after which the plasma was drawn off. Samples of composite were incubated in the plasma for 12 h at 4 °C, after which the samples were rinsed with phosphobuffered saline. The samples were then incubated with citrated whole blood for 1 h with gentle agitation, after which they were washed with phospho-buffered saline. The samples were then fixed for 1 h at 4 °C with 3% glutaraldehyde in phospho-buffered saline. The fixed samples were serially dehydrated in ethanol, air-dried, and coated with a 20 nm thick layer of metallic gold in a Jeol JEE-4X sputtering apparatus. Platelets that adhered to the surface of the composite sheets were visualized using a Hitachi S-3000 N scanning electron microscope. 3. Results 3.1. Infra-red spectrophotometry of fibers from transgenic and control plants The infra-red spectra of fibers from transgenic and control plants are presented in Fig. 2. All of the spectra had a broad band at 3400 cm 1, which corresponds to free OH groups on cellulose molecules and OH groups involved in intra-molecular and intermolecular hydrogen bonds [9]. The bands for fibers from both the transgenic and control plants contained four Lorentzian components at 3466, 3410, 3353, and 3301 cm 1 (Fig. 2, inserts).
Fig. 1. Representative load–displacement curves for bending (a) and tensile (b) tests. The mechanical tests were conducted as specified in Section 2.
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wt
37 00
3 60 0
3 50 0
3 40 0
33 00
3 2 00
3 10 0
3 00 0
3301
3408 3460
3353
Absorbance
3301
3466
3353
3410
Absorbance
M 50
3 700
360 0
-1
3 500
340 0
33 00
320 0
31 00
3 000
-1
M42
w aven um ber [cm ]
wavenum ber [cm ]
Absorbance
M13
M50
wt
3700
3600
3500
3400
3300
3200
3100
3000
-1
wavenumber [cm ] 1
for fibers from M50
3470
3549
3388
3297 3115
3201
2942 2678
Absorbance
A b s o rb a n c e
1724
Fig. 2. Infra-red spectra of fibers from the transgenic and control plants. Inserts show the Lorentzian components in the region from 3000 to 3700 cm transgenic plants and the control plants.
2000
2500
3000
3500
4000
-1
2935 2976 2999
1539
1653
1734
wavenumber [cm ]
M50
bacterial PHB 1500
2000
2500
3000
3500
4000
-1
wavenumber [cm ] Fig. 3. Infra-red spectra of PHB from M50 transgenic plants and bacterial PHB. Inserts show the Lorentzian components in the region from 2500 to 3700 cm
1
.
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The amplitude of the broad band, on the other hand, was significantly higher in fibers from the transgenic plants than in fibers from the control plants. The amplitude was highest in fibers from M42 and M13 transgenic plants.
PHB in fibers from the transgenic plants than in fibers from the control plants (Fig. 3). This indicates that the PHB interacts strongly with the cellulose of the fibers, forming a stable and strong complex. In fibers from the control plants, the broad contour consisted of four Lorentzian components at 3466, 3410, 3353 and 3301 cm 1 (Fig. 2, insert). In fibers from M50 transgenic plants, these four components were also present. The first three components correspond to inter-molecular hydrogen bonds of the type m (OH O) in the cellulose molecule. The fourth component corresponds to inter-molecular hydrogen bonds [9]. In PHB isolated from the transgenic plants, the broad contour contained six Lorentzian components at 3549, 3470, 3388, 3297, 3201 and 3115 cm 1 (Fig. 3, insert). This indicates that some of the bands for the co-extracted cellulose were split and shifted. The new component at 3549 cm 1 corresponds to the m (OHO) vibration of the non-bonded hydroxyl group. The other components correspond to the components for the control fibers as follows:
3.2. Infra-red spectrophotometry of PHB isolated from transgenic plants The infra-red spectrum of PHB extracted from M50 transgenic plants was compared to the spectrum of bacterial PHB (Fig. 3). The spectrum of PHB from the transgenic fibers overlapped with both the spectrum of bacterial PHB and the spectrum of fibers from the control plants [18,19]. The broad contour from 3000 to 3700 cm 1 corresponds to free OH groups of cellulose and OH groups involved in intra-molecular and inter-molecular hydrogen bonds [9]. This indicates that some cellulose was extracted along with the PHB. This suggests that the PHB was strongly bound to the cellulose of the fibers. On the other hand, this contour is more complex and shifted lower for
1630
1536
Absorbance
1735
1662
M50
1800
1750
1700
165 0
1600
1550
1500
1600
1550
1500
-1
wavenumber [cm ]
Bacterial
1800
1750
1724
1740
Absorbance
PHB
1700
165 0 -1
wavenumber [cm ] Fig. 4. Lorentzian components in the region from 1500 to 1800 cm
1
for PHB from M50 transgenic plants and bacterial PHB.
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3470 cm 1 = 3466 cm 1; 3388 cm 1 = 3410 cm 1; 3297 cm 1 = 3353 cm 1; and 3201 and 3115 cm 1 = 3301 cm
1
.
The large shift observed in the component at 3466 cm 1 indicates that the inter-molecular hydrogen bonds between the PHB and cellulose are very strong. This may explain why chloroform extracted some cellulose together with PHB from the fibers of the transgenic plants. In the region from 1500 to 1800 cm 1, the spectrum of bacterial PHB consisted of two Lorentzian components at 1740 and 1724 cm 1. On the other hand, the spectrum for PHB isolated from M50 transgenic plants consisted of four Lorentzian components at 1735, 1662, 1630 and 1536 cm 1 (Fig. 4). This indicates that there are four types of carboxyl groups in PHB from the transgenic plants. The component at 1735 cm 1 corresponds to the component at 1724 cm 1 in bacterial PHB. This component corresponds to COOH terminal groups of PHB that are not bound to the cellulose of the fibers. The fact that this component is shifted about 11 cm 1 higher indicates that the C@O bond of the carboxyl group is shorter in PHB from the transgenic plants and that the
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–CH(CH3)–CH2–CO– units of the polymer are more strongly bound by C–O bridge bonds. The component at 1662 cm 1 corresponds to ionized COO groups. The fact that it is shifted lower indicates that the C@O groups in PHB from the transgenic plants are bound to cellulose by C@O H–O hydrogen bonds. The component at 1630 cm 1 has no counterpart in the spectrum of bacterial PHB. This component may correspond to COO– groups bound to cellulose by both C–O–C covalent bonds and C@O H–O hydrogen bonds. The component at 1536 cm 1 corresponds to carboxyl groups of residual lignin bound to the cellulose of the fiber. 3.3. Scanning electron microscopy of fibers treated with NaOH and chloroform Scanning electron microscopy of fibers from both the transgenic and control plants revealed that the retting process had not completely finished (Fig. 5a). The elementary fibers still adhered to each other, and fragments of cuticle and non-fibrous tissue were still present. Fibers from transgenic plants, however, showed lower
Fig. 5. Scanning electron microscopy of flax fibers from transgenic (M50) and control plants, either untreated or treated with NaOH or chloroform. The microscopy was performed as described in Section 2. Bar represents 100 lm (a and c) and 50 lm (b).
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plants in comparison to untreated fibers. Fibers from the wild-type plants showed no changes after chloroform treating (Fig. 5c).
Young's modulus 3.5 3.0
3.4. Mechanical testing of composites sheets
GPa
2.5 2.0 1.5 1.0 0.5 0.0
PP
PP+20%WT+PHB PP+20%WT
PP+20% M50
Tensile strength
35 30
MPa
25 20 15 10 5 0
PP
PP+20%WT+PHB PP+20%WT
PP+20% M50
Fig. 6. Strength and Young’s modulus of composites containing fibers from M50 transgenic plants, fibers from the control plants, or fibers from the control plants and bacterial PHB in a matrix of polypropylene. Sheets of pure polypropylene served as the reference. The measurements were conducted as indicated in Section 2. The mean value (n = 7–10) ± SD is presented.
amounts of residual substances (pectins, parenchyma, fats and waxes) on their surface. The method and place of retting was the same for transformed and untransformed, control plants, thus observed lower level of substances (e.g. pectins, which are degraded during retting process) on the surface of M50 fibers may demonstrate that the retting was more effective for transgenic plants in comparison to control plants. In fact earlier data showed that transgenic plants from M50 line were almost 2-fold easier retted than control plants [20]. Fiber bundles were treated overnight at room temperature with 5% NaOH in order to remove the residual cuticle and non-fibrous tissue [21]. The process was completely effective with fibers from transgenic plants, and no traces of residual tissue were detected (Fig. 5b). The fiber bundles were then treated overnight at room temperature with chloroform, which dissolves PHB but not cellulose or lignin. Scanning electron microscopy revealed shrinkage (almost 250% in diameter) in the elementary fibers from the transgenic
Mechanical tests showed that introduction of the flax fibers as a reinforcing element to PP matrix improved markedly stiffness, however, declined the tensile strength (Fig. 6). All tested composites had more than 2.5-fold higher effective Young’s modulus in comparison to pure polypropylene. The highest value of the parameter showed the composite, which contained fibers from M50 transgenic plants (Fig. 6). The ultimate strength of tested composites was decreased in comparison to the pure polypropylene, however, the declination of the parameter differed in dependence on the fiber origin. Fibers from control plants reduced the strength of composite by about 50%, while M50 transgenic plants only by 23%. In the composite containing fibers from the control plants and bacterial PHB, tensile strength was about 40% lower than in the reference. 3.5. Scanning electron microscopy of composite sheets Images of the fractured surface of composite sheets are presented in Fig. 7. Examination of the composite containing fibers from the control plants revealed the presence of gaps where the fibers were pulled out of the matrix rather than broken [15]. These gaps were absent in the composite containing fibers from the transgenic plants. 3.6. Platelet aggregation Scanning electron microscopy images of the surfaces of composite sheets incubated together with blood platelets are presented in Fig. 8. Almost no platelet aggregation was detected on the composite containing fibers from transgenic plants. On the other hand, the aggregation of those cells was observed on the composite containing fibers from the control plants and on the composite containing fibers from the control plants and bacterial PHB. Platelet aggregation was also noticed on pure polypropylene. 4. Discussion All of the infra-red spectra had a broad band at 3400 cm 1. This band can be attributed to free OH groups of cellulose and OH groups involved in intra-molecular and inter-molecular hydrogen
Fig. 7. Scanning electron microscopy of fractured composite sheets containing fibers from M50 transgenic plants or the control plants. The microscopy was performed as described in Section 2. Bar represents 200 lm.
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Fig. 8. Platelets aggregation on composites containing fibers from M50 transgenic plants, fibers from the control plants, or fibers from the control plants and bacterial PHB in a matrix of polypropylene. Sheets of pure polypropylene served as the reference. The analysis of the platelets aggregation was performed as described in the Section 2. Bar represents 50 lm.
bonds [9]. In fibers from both transgenic plants and control plants, the broad band could be resolved into four Lorentzian components. The shape of the broad band and its Lorentzian components indicates that the cellulose content was about the same in the transgenic and control plants, which agrees well with the results for biochemical testing [10]. On the other hand, the amplitude of the broad band at 3400 cm 1 was higher in fibers from the transgenic plants. This indicates that the cellulose in fibers from the transgenic plants was more highly structured than in fibers from the control plants. Another parameter that can be used to describe the level of structure in cellulose is the Crystallinity Index [22]. This index is calculated by dividing the amplitude of the band at 1370 cm 1 by the amplitude of the band at 2900 cm 1. The band at 1370 cm 1 represents –CH– groups, and the band at 2900 cm 1 represents both –CH2– and –CH– groups. The Crystallinity Index correlates with the degree of crystallinity obtained using X-ray diffraction [23]. The Crystallinity Index ranged from 0.71 to 0.75 in fibers from all of the transgenic lines, as compared to 0.62 in fibers from the control plants or 0.64 in purified cotton cellulose. It was also higher than in flax fibers that had been treated with surfactants or with enzymes that free the fibers from lignin, pectin, fats and waxes [22]. This indicates that PHB increased the level of structure in the cellulose in fibers from the transgenic plants. The infra-red spectrum of PHB isolated from the transgenic plants contains a broad contour from 3000 to 3700 cm 1. This contour is more complex and shifted lower for PHB from the transgenic plants than for fibers from the control plants. The differences in the spectra of the PHB isolated from the transgenic plants and bacterial PHB indicate that the PHB was strongly bound to the cellulose in the fibers by covalent and hydrogen bonds. Retting progressed more rapidly in transgenic plants than in the control plants. Treatment with NaOH was also more effective in removing cuticle and non-fibrous tissue from fibers of transgenic plants. This suggests that the PHB reduces the binding of pectin and hemicellulose to the fibers, which facilitates retting. The shrinkage observed in technical fibers from transgenic plants after treatment with chloroform indicates that some of
the cellulose of the fiber was co-extracted with the PHB. This showed that the PHB was strongly bound to the cellulose of the fiber, which confirms the results obtained using infra-red spectroscopy. All of the composites tested had a lower maximum tensile strength than pure polypropylene. The composite containing fibers from transgenic plants were, however, significantly stronger than the composites prepared with fibers from the control plants. All of the composites tested had a higher Young’s modulus than pure polypropylene. This was particularly true for the composite containing fibers from transgenic plants. Therefore, the composite containing fibers from transgenic plants was significantly superior in terms of mechanical properties than the composites containing fibers from the control plants. This indicated that the fibers from the transgenic plants were bonded more strongly to the matrix than fibers from the control plants. The absence of gaps on the surface of fractured sheets of the composite containing fibers from transgenic plants also shows that these fibers adhered to the polypropylene matrix significantly stronger than fibers from the control plants. The composite containing fibers from transgenic plants induced almost no platelet aggregation. Further research is however needed to elucidate the exact molecular mechanism involved in this process. Composites containing fibers from transgenic plants expressing polymers may therefore be useful in the construction of biomedical devices that come in contact with blood. To the best of our knowledge, this is the first report on the structural analysis of PHB produced by transgenic plants. It is also the first report on the use of genetic techniques to modify natural fibers in order to improve the compatibility between flax fibers and the matrix in composite materials.
Acknowledgments This study was supported by the Grants Polish Ministry of Education and Sciences (Grant Numbers POLPOST-DOC II Nr PBZ/ MEiN/01/2006/17, NN 302061834, 2P06A 02029, 2P04B 01528, N R12 0009 06 and PBZ-MNiI-2/1/2005).
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[13] Williams SF, Martin PD. (2002) Applications of PHAs in medicine and pharmacy. in: Doi Y, Steinbuchel A, editors. Biopolymers, Polyesters III, vol. 4. Weinheim: Wiley-VCH. [14] Misra SK, Valappil SP, Roy I, Boccaccini AR. Polyhydroxyalkanoate (PHA)/ inorganic phase composites for tissue engineering applications. Biomacromolecules 2006;7:2249–58. [15] Hong J, Nilsson-Ekdahl K, Reynolds H, Larsson R, Nilsson B. A new in vitro model to study interaction between whole blood and biomaterials. Studies of platelet and coagulation activation and the effect of aspirin. Biomaterials 1999;20:603–11. [16] Aksamit A, Korobczak A, Skala J, Lukaszewicz M, Szopa J. The 14-3-3 gene expression specificity in response to stress is promoter-dependent. Plant Cell Physiol 2005;46:1635–45. [17] Okroj W, Kaminska M, Klimek L, Szymanski W, Walkowiak B. Blood platelets in contact with nanocrystalline diamond surfaces. Diam Relat Mater 2006;15:1535–9. [18] Zhang JSH, Tsuji H, Noda I, Ozaki Y. Differences in the CH3 O@C interactions among poly(L-lactide), poly(L-lactide/poly(D-lactide) stereocomplex, and poly(3-hydroxybutyrate) studied by infrared spectroscopy). J Mol Struct 2006:735–6. [19] Sato HDJ, Murakami R, Noda I, Ozaki Y. Infrared and Raman spectroscopy and quantum chemistry calculation studies of C–H O hydrogen bondings and thermal behavior of biodegradable polyhydroxyalkanoate. J Mol Struct 2005:744–7. [20] Wróbel-Kwiatkowska M, Skórkowska-Telichowska K, Dymin´ska L, Ma˛czka M, Hanuza J, Szopa J. Biochemical, mechanical and spectroscopic analyses of genetically engineered flax fibres producing bioplastic (poly-bhydroxybutyrate). Biotech Progr 2009; in press. doi:10.1002/btpr.194. [21] Jahn A, Schroder WM, Futing M, Schenzel K, Diepenbrock W. Characterization of alkali treated flax fibres by means of FT Raman spectroscopy and environmental scanning electron microscopy. Spectrochim Acta Part A 2002;58:2271–9. [22] Shamolina IIBAM, Zabivalova NM, Vlasova EN, Volchek BZ, Sinitsin AP. Biochemical and physicochemical treatment of flax fibers. Russ J Appl Chem 2004;77:1729–32. [23] Bikales NM, Segel L. Cellulose and cellulose derivatives. New York: WileyInterscience; 1971.
Contents lists available at ScienceDirect
Composites Science and Technology journal homepage: www.elsevier.com/locate/compscitech
Chemical composition and molecular structure of fibers from transgenic flax producing polyhydroxybutyrate, and mechanical properties and platelet aggregation of composite materials containing these fibers Jan Szopa a,*, Magdalena Wróbel-Kwiatkowska a,b, Anna Kulma a, Magdalena Zuk a, Katarzyna Skórkowska-Telichowska c, Lucyna Dymin´ska d, Mirosław Ma˛czka e, Jerzy Hanuza d,e, Jacek Zebrowski f, Marta Preisner a a
Faculty of Biotechnology, University of Wrocław, Poland Laboratory of Polymers, Institute of Mechanical Engineering and Automation, Wrocław University of Technology, Wrocław, Poland c Department of Endocrinology, 4th Military Hospital, Wrocław, Poland d Department of Bioorganic Chemistry, Institute of Chemistry and Food Technology, Faculty of Economics and Engineering, University of Economics, Wrocław, Poland e Institute of Low Temperatures and Structure Research, Polish Academy of Sciences, Wrocław, Poland f Faculty of Biotechnology, University of Rzeszow, Poland b
a r t i c l e
i n f o
Article history: Received 22 October 2008 Received in revised form 15 June 2009 Accepted 20 June 2009 Available online 26 June 2009 Keywords: A. Flax fiber polypropylene composite B. Fiber/matrix bond B. Mechanical properties D. Infra-red (IR) spectroscopy D. Scanning electron microscopy (SEM)
a b s t r a c t In order to improve the properties of flax fibers so that they interact better with the matrix material in composites, several lines of transgenic flax were produced over-expressing the bacterial polyhydroxybutyrate (PHB) synthesis genes. Infra-red spectrophotometry revealed that the cellulose in fibers from the transgenic plants was more highly structured than in fibers from the control plants and PHB was strongly bound to the cellulose of the fibers by covalent ester and hydrogen bonds. The composite containing fibers from transgenic plants was significantly stronger and stiffer than the composites containing fibers from the control plants. Scanning electron microscopy of the fracture surface of composite sheets indicated that fibers from transgenic plants adhered to the polypropylene matrix significantly better. The composite containing fibers from transgenic plants induced almost no platelet aggregation and so may therefore be useful in the construction of biomedical devices that come in contact with blood. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction There has recently been a surge of interest in the industrial and biomedical uses of composites containing bio-fibers reinforced with polymers. The bio-fibers most often used for industrial purposes are cellulose fibers, which may be used to replace fiberglass in plastics used to produce a variety of products in many industries. For example, composites reinforced with bio-fibers have been used in the manufacture of automobile parts such as door panels, seat backs and glove compartments. The main advantage of these fibers is that they are more biodegradable and less reliant on petrochemical resources than most man-made polymers [1]. A valuable source of high-quality ligno-cellulose fibers is flax. The quality of flax fibers highly depends on the fine structure of the cellulose molecules, the growing conditions, and the retting method. * Corresponding author. E-mail address: [email protected] (J. Szopa). 0266-3538/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.compscitech.2009.06.017
During retting, bast fiber bundles are separated from the core, epidermis and cuticle [2]. This is brought about by enzymatic degradation of pectin and hemicellulose in the flax cell-wall by various microorganisms, including filamentous fungi [3]. Flax fibers contain 70% cellulose, which is a polymer of D-glucopyranosyl units strongly linked by intra-molecular and intermolecular hydrogen bonds. Because of these bonds, cellulose does not melt before it undergoes thermal degradation. One of the main problems in using bio-fibers such as flax fibers in composite materials is that natural fibers are less strong than currently used materials such as fiberglass (the tensile strength of glass fiber is 2400 106 N/m2 and of flax fiber, which is the best among natural fibers, only 800–1500 106 N/m2 [1]). Also components made from unmodified polypropylene or polyethylene and natural fibers are less strong that polypropylene on its own [4,5]. In composite materials, bio-fibers adhere poorly to hydrophobic matrices, often to the point that the composite is mechanically inferior to either the bio-fibers or the matrix material on their own. On the other hand, when the fibers adhere well to the matrix, which may be achieved via fiber or matrix modification,
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mechanical stress is transferred from the matrix to the fibers, which improves the mechanical properties of the composite [5,6]. Polypropylene is a thermoplastic polymer used in a wide variety of products including textiles and plastic items for medical or laboratory use [7]. It is often used as a matrix in composites that include natural fibers. However, polypropylene is hydrophobic, and therefore does not bind strongly to hydrophilic natural fibers. Modification of polypropylene such as malonylation can help to overcome this effect [8]. Proper adhesion of the fibers to the matrix is essential for ensuring optimal mechanical properties in the composite. Better contact between the fibers and the matrix also enhances the hydrophobicity of the composite. Additionally several approaches have been used to improve the properties of cellulose fibers so that they interact better with the matrix material in composites. Chemical, physical and enzymatic treatment changes the solubility of the cellulose in the fiber mainly by polymorphically transforming cellulose I into cellulose II [9]. Other methods include surface fibrillation and electric discharge of the fibers, de-waxing, alkali treatment, acetylation and grafting. Coupling agents such as silanes and dihydric phenols can also be used to improve adhesion. These agents chemically react with the fibers and the matrix, promoting the formation of covalent and hydrogen bonds between them [6]. Unfortunately, all of these methods complicate the production of industrial fiber by introducing additional steps to the process. Therefore, genetic engineering is currently being evaluated as a safe and relatively inexpensive alternative method for modifying and improving fiber quality. Polyhydroxybutyrate (PHB) is a biodegradable bacterial polymer which has been extensively studied because of its potential biomedical applications. The polymer itself is fragile, which severely limits its usefulness. Its mechanical properties are significantly improved when it is incorporated into bio-fibers. Furthermore, PHB may improve adhesion between flax fibers and the matrix in composite materials. In previous studies, we produced several lines of transgenic flax that over-express the bacterial genes responsible for PHB synthesis and evaluated the biochemical and mechanical properties of stems and fibers from field grown specimens [10,11]. The transgenic plants had a normal phenotype and produced reasonable amounts of PHB. Fibers from the transgenic plants did not significantly differ from those of the control plants in terms of cellulose, lignin and pectin content. This indicates that the synthesis of these substances was not affected by the synthesis of PHB. Fibers from transgenic plants were stronger and stiffer than fibers from untransformed flax. They also contained more phenolic acids, which are potent anti-oxidants. This may be significant when these fibers are used for biomedical purposes. The improvement in fiber quality was attributed to changes either in the chemical composition of the fibers or in the molecular structure of the cellulose in the fibers. The hydroxyl group of PHB can participate in intra-molecular and inter-molecular hydrogen bonds. The arrangement of these bonds may affect the fine structure of flax fibers. Changes in the chemical composition of flax fibers can be detected by infra-red spectroscopy. This method can also be used to detect changes in molecular structure [12]. Composites containing bio-fibers also have properties that may be useful in several fields of medicine [13]. They may serve as an artificial scaffold in tissue engineering. They may also be useful in constructing drug-release systems, cardiovascular patches and nerve cuffs [14]. Materials that induce platelet aggregation on their surface cannot be used in devices that are to be implanted such as heart valves and stents. On contact with blood, these materials adsorb plasma proteins that induce platelet aggregation. This can lead to serious
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thrombo-embolic complications. On the other hand, these materials also may be useful in some applications because they promote the formation of bone tissue [15]. The aim of the present study was to use infra-red spectroscopy to evaluate the chemical composition and molecular structure of fibers from flax plants that have been genetically modified to produce PHB. Composites containing flax fibers from transgenic and control plants in a polypropylene matrix were also compared in terms of their mechanical properties and their ability to induce blood platelet aggregation.
2. Materials and methods 2.1. Transgenic plants Flax plants (Linum usitatissimum L., fibrous cultivar Nike) were genetically modified to over-express the three bacterial genes that code for all of the enzymes required for the synthesis of PHB [10]. The plants were transformed using the binary vector pBinARHygABC 14-3-3 containing three cDNA sequences from Ralstonia eutropha: phbA (EMBL/GenBank accession number J04987) encoding bketothiolase; phbB (EMBL/GenBank accession number J04987) encoding acetoacetyl-CoA reductase; and phbC (EMBL/GenBank accession number J05003) encoding the last enzyme of the route i.e. PHB synthase. The genes were placed under the control of a stem specific promoter of the 16R isoform of the gene for 14-3-3 protein of Solanum tuberosum [16] and the plastid directing sequence of the Rubisco gene. The vector was introduced into flax explants using Agrobacterium tumefaciens. The inoculated explants were then grown on callus induction medium and shoot regeneration medium. Transgenic plants were preselected using PCR (polymerase chain reaction), and selected using northern blotting [10]. Three lines of transgenic plants were used in this study: M13, M42 and M50. They differed in the concentration of PHB and they accumulated, respectively, 1.33 lg PHB/g of a fresh weight (FW), 4.35 lg/g FW and 4.62 lg/g FW. 2.2. Retting Transgenic and control plants were grown in the same field near Wrocław. The plants were harvested at the same time: 4 months after planting and were retted using the dew method [11]. Briefly, the harvested plants were spread out in an open field and left exposed for at least 40 days. The plants were turned every 2 weeks. During this time, bacteria and fungi grew on the plants and degraded the cell-wall polysaccharide and middle lamella, thereby releasing fibers from the stem matrix. 2.3. Infra-red spectrophotometry of fibers from transgenic and control plants In order to determine the chemical composition and molecular structure of fibers from transgenic and control plants, fibers were analyzed using infra-red spectrometry. Spectra were measured at room temperature using a Biorad 575C FT–IR spectrometer. Data were collected over a spectral range from 50 to 4000 cm 1 with a resolution of 2 cm 1. In the mid infra-red part of this range, samples were prepared in a KBr pellet. In the far infra-red part of this range, samples were suspended in Nujol.
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2.4. Isolation of PHB from transgenic plants PHB was isolated as described in a previous paper [10]. Samples of transgenic plants (25–250 mg) were extracted for 1 h at 55 °C with 1 ml of methanol per gram of plant material, and then extracted twice for 1 h at 55 °C with 1 ml of ethanol per gram of plant material. This ensured the dissolution and removal of lipids and 3hydroxybutyrate monomers. The samples were then suspended in 1 ml of chloroform per gram of plant material. 3-Hydroxy-valeratemethyl ester was added as an internal standard. The samples were then esterified by adding 17 ml ethanol and 0.2 ml 1 N HCl per gram of plant material. The samples were incubated for 4 h at 100 °C. The extracts were filtered and washed with 0.9 N NaCl. The organic phase was removed and analyzed using infra-red spectrophotometry.
of a Merlin software and used to calculate E from a formulae E = (F/x) l3/4ah3, where l – the distance between the supports, a – the specimen width and h – the specimen thickness. Representative load–displacement curves for tensile and bending tests are presented in Fig. 1. Seven to ten samples were evaluated in each test for each composite. Sheets of pure polypropylene were used as a reference. 2.7. Scanning electron microscopy In order to determine the degree of adhesion between the fibers and the matrix, the surface morphology of fractured composite sheets was examined using a JEOL JSM-5800LV scanning electron microscope. Samples were coated with graphite before analysis. 2.8. Aggregation of platelets on the surface of composite sheets
2.5. Preparation of composites Three kinds of composite material were prepared using a matrix of commercially available polypropylene granules (HP 548E). The first was made with fibers derived from M50 transgenic plants, the second with fibers from control plants, and the third with fibers from control plants supplemented with bacterial PHB (Sigma–Aldrich). In all cases, the ratio of fiber to polypropylene was 1:4 (weight ratio). Finally pulverized combed flax fibers were mixed with polypropylene granules at 170 °C. The mixture was mechanically pressed into sheets. These were cut into smaller pieces and heat-pressed between Teflon sheets for 75 s at 175 °C under 20 tons of pressure to produce finished composite sheets that were 1 mm thick. 2.6. Mechanical testing of composite sheets The tensile strength and the effective Young’s modulus in bending of the composite sheets were measured using a computer-driven Instron 5542 system (High Wycombe, UK) as described in a previous paper [11]. In a tensile test, 25 mm long sections of composite sheet cut into a ‘‘dog bone” shape were fixed between clamps at the initial distance of 5 mm. The samples were stretched at a rate of 20 mm/min up to failure and the maximum load, Fmax, was recorded. The ultimate tensile strength, rmax, was calculated as rmax = Fmax/ah, where a – the actual width of the specimen at the cross section location where failure occurred and h – the sheet thickness. To determine the effective Young’s modulus, E, a threepoint flexure test was performed at the loading rate of 20 mm/min. Maximum slope of the load–displacement curve inclination to the displacement axis, (F/x), was automatically determined by means
The aggregation of platelets on composite sheets was examined using scanning electron microscopy as described by Okroj et al. [17]. Sheets of pure polypropylene were used as a reference. Venous blood was collected from healthy volunteers in tubes containing 3.8% sodium citrate. The tubes were centrifuged for 10 min at room temperature at 1000g, after which the plasma was drawn off. Samples of composite were incubated in the plasma for 12 h at 4 °C, after which the samples were rinsed with phosphobuffered saline. The samples were then incubated with citrated whole blood for 1 h with gentle agitation, after which they were washed with phospho-buffered saline. The samples were then fixed for 1 h at 4 °C with 3% glutaraldehyde in phospho-buffered saline. The fixed samples were serially dehydrated in ethanol, air-dried, and coated with a 20 nm thick layer of metallic gold in a Jeol JEE-4X sputtering apparatus. Platelets that adhered to the surface of the composite sheets were visualized using a Hitachi S-3000 N scanning electron microscope. 3. Results 3.1. Infra-red spectrophotometry of fibers from transgenic and control plants The infra-red spectra of fibers from transgenic and control plants are presented in Fig. 2. All of the spectra had a broad band at 3400 cm 1, which corresponds to free OH groups on cellulose molecules and OH groups involved in intra-molecular and intermolecular hydrogen bonds [9]. The bands for fibers from both the transgenic and control plants contained four Lorentzian components at 3466, 3410, 3353, and 3301 cm 1 (Fig. 2, inserts).
Fig. 1. Representative load–displacement curves for bending (a) and tensile (b) tests. The mechanical tests were conducted as specified in Section 2.
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wt
37 00
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-1
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3388
3297 3115
3201
2942 2678
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1724
Fig. 2. Infra-red spectra of fibers from the transgenic and control plants. Inserts show the Lorentzian components in the region from 3000 to 3700 cm transgenic plants and the control plants.
2000
2500
3000
3500
4000
-1
2935 2976 2999
1539
1653
1734
wavenumber [cm ]
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bacterial PHB 1500
2000
2500
3000
3500
4000
-1
wavenumber [cm ] Fig. 3. Infra-red spectra of PHB from M50 transgenic plants and bacterial PHB. Inserts show the Lorentzian components in the region from 2500 to 3700 cm
1
.
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The amplitude of the broad band, on the other hand, was significantly higher in fibers from the transgenic plants than in fibers from the control plants. The amplitude was highest in fibers from M42 and M13 transgenic plants.
PHB in fibers from the transgenic plants than in fibers from the control plants (Fig. 3). This indicates that the PHB interacts strongly with the cellulose of the fibers, forming a stable and strong complex. In fibers from the control plants, the broad contour consisted of four Lorentzian components at 3466, 3410, 3353 and 3301 cm 1 (Fig. 2, insert). In fibers from M50 transgenic plants, these four components were also present. The first three components correspond to inter-molecular hydrogen bonds of the type m (OH O) in the cellulose molecule. The fourth component corresponds to inter-molecular hydrogen bonds [9]. In PHB isolated from the transgenic plants, the broad contour contained six Lorentzian components at 3549, 3470, 3388, 3297, 3201 and 3115 cm 1 (Fig. 3, insert). This indicates that some of the bands for the co-extracted cellulose were split and shifted. The new component at 3549 cm 1 corresponds to the m (OHO) vibration of the non-bonded hydroxyl group. The other components correspond to the components for the control fibers as follows:
3.2. Infra-red spectrophotometry of PHB isolated from transgenic plants The infra-red spectrum of PHB extracted from M50 transgenic plants was compared to the spectrum of bacterial PHB (Fig. 3). The spectrum of PHB from the transgenic fibers overlapped with both the spectrum of bacterial PHB and the spectrum of fibers from the control plants [18,19]. The broad contour from 3000 to 3700 cm 1 corresponds to free OH groups of cellulose and OH groups involved in intra-molecular and inter-molecular hydrogen bonds [9]. This indicates that some cellulose was extracted along with the PHB. This suggests that the PHB was strongly bound to the cellulose of the fibers. On the other hand, this contour is more complex and shifted lower for
1630
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1662
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-1
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wavenumber [cm ] Fig. 4. Lorentzian components in the region from 1500 to 1800 cm
1
for PHB from M50 transgenic plants and bacterial PHB.
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3470 cm 1 = 3466 cm 1; 3388 cm 1 = 3410 cm 1; 3297 cm 1 = 3353 cm 1; and 3201 and 3115 cm 1 = 3301 cm
1
.
The large shift observed in the component at 3466 cm 1 indicates that the inter-molecular hydrogen bonds between the PHB and cellulose are very strong. This may explain why chloroform extracted some cellulose together with PHB from the fibers of the transgenic plants. In the region from 1500 to 1800 cm 1, the spectrum of bacterial PHB consisted of two Lorentzian components at 1740 and 1724 cm 1. On the other hand, the spectrum for PHB isolated from M50 transgenic plants consisted of four Lorentzian components at 1735, 1662, 1630 and 1536 cm 1 (Fig. 4). This indicates that there are four types of carboxyl groups in PHB from the transgenic plants. The component at 1735 cm 1 corresponds to the component at 1724 cm 1 in bacterial PHB. This component corresponds to COOH terminal groups of PHB that are not bound to the cellulose of the fibers. The fact that this component is shifted about 11 cm 1 higher indicates that the C@O bond of the carboxyl group is shorter in PHB from the transgenic plants and that the
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–CH(CH3)–CH2–CO– units of the polymer are more strongly bound by C–O bridge bonds. The component at 1662 cm 1 corresponds to ionized COO groups. The fact that it is shifted lower indicates that the C@O groups in PHB from the transgenic plants are bound to cellulose by C@O H–O hydrogen bonds. The component at 1630 cm 1 has no counterpart in the spectrum of bacterial PHB. This component may correspond to COO– groups bound to cellulose by both C–O–C covalent bonds and C@O H–O hydrogen bonds. The component at 1536 cm 1 corresponds to carboxyl groups of residual lignin bound to the cellulose of the fiber. 3.3. Scanning electron microscopy of fibers treated with NaOH and chloroform Scanning electron microscopy of fibers from both the transgenic and control plants revealed that the retting process had not completely finished (Fig. 5a). The elementary fibers still adhered to each other, and fragments of cuticle and non-fibrous tissue were still present. Fibers from transgenic plants, however, showed lower
Fig. 5. Scanning electron microscopy of flax fibers from transgenic (M50) and control plants, either untreated or treated with NaOH or chloroform. The microscopy was performed as described in Section 2. Bar represents 100 lm (a and c) and 50 lm (b).
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plants in comparison to untreated fibers. Fibers from the wild-type plants showed no changes after chloroform treating (Fig. 5c).
Young's modulus 3.5 3.0
3.4. Mechanical testing of composites sheets
GPa
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35 30
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25 20 15 10 5 0
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PP+20%WT+PHB PP+20%WT
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Fig. 6. Strength and Young’s modulus of composites containing fibers from M50 transgenic plants, fibers from the control plants, or fibers from the control plants and bacterial PHB in a matrix of polypropylene. Sheets of pure polypropylene served as the reference. The measurements were conducted as indicated in Section 2. The mean value (n = 7–10) ± SD is presented.
amounts of residual substances (pectins, parenchyma, fats and waxes) on their surface. The method and place of retting was the same for transformed and untransformed, control plants, thus observed lower level of substances (e.g. pectins, which are degraded during retting process) on the surface of M50 fibers may demonstrate that the retting was more effective for transgenic plants in comparison to control plants. In fact earlier data showed that transgenic plants from M50 line were almost 2-fold easier retted than control plants [20]. Fiber bundles were treated overnight at room temperature with 5% NaOH in order to remove the residual cuticle and non-fibrous tissue [21]. The process was completely effective with fibers from transgenic plants, and no traces of residual tissue were detected (Fig. 5b). The fiber bundles were then treated overnight at room temperature with chloroform, which dissolves PHB but not cellulose or lignin. Scanning electron microscopy revealed shrinkage (almost 250% in diameter) in the elementary fibers from the transgenic
Mechanical tests showed that introduction of the flax fibers as a reinforcing element to PP matrix improved markedly stiffness, however, declined the tensile strength (Fig. 6). All tested composites had more than 2.5-fold higher effective Young’s modulus in comparison to pure polypropylene. The highest value of the parameter showed the composite, which contained fibers from M50 transgenic plants (Fig. 6). The ultimate strength of tested composites was decreased in comparison to the pure polypropylene, however, the declination of the parameter differed in dependence on the fiber origin. Fibers from control plants reduced the strength of composite by about 50%, while M50 transgenic plants only by 23%. In the composite containing fibers from the control plants and bacterial PHB, tensile strength was about 40% lower than in the reference. 3.5. Scanning electron microscopy of composite sheets Images of the fractured surface of composite sheets are presented in Fig. 7. Examination of the composite containing fibers from the control plants revealed the presence of gaps where the fibers were pulled out of the matrix rather than broken [15]. These gaps were absent in the composite containing fibers from the transgenic plants. 3.6. Platelet aggregation Scanning electron microscopy images of the surfaces of composite sheets incubated together with blood platelets are presented in Fig. 8. Almost no platelet aggregation was detected on the composite containing fibers from transgenic plants. On the other hand, the aggregation of those cells was observed on the composite containing fibers from the control plants and on the composite containing fibers from the control plants and bacterial PHB. Platelet aggregation was also noticed on pure polypropylene. 4. Discussion All of the infra-red spectra had a broad band at 3400 cm 1. This band can be attributed to free OH groups of cellulose and OH groups involved in intra-molecular and inter-molecular hydrogen
Fig. 7. Scanning electron microscopy of fractured composite sheets containing fibers from M50 transgenic plants or the control plants. The microscopy was performed as described in Section 2. Bar represents 200 lm.
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Fig. 8. Platelets aggregation on composites containing fibers from M50 transgenic plants, fibers from the control plants, or fibers from the control plants and bacterial PHB in a matrix of polypropylene. Sheets of pure polypropylene served as the reference. The analysis of the platelets aggregation was performed as described in the Section 2. Bar represents 50 lm.
bonds [9]. In fibers from both transgenic plants and control plants, the broad band could be resolved into four Lorentzian components. The shape of the broad band and its Lorentzian components indicates that the cellulose content was about the same in the transgenic and control plants, which agrees well with the results for biochemical testing [10]. On the other hand, the amplitude of the broad band at 3400 cm 1 was higher in fibers from the transgenic plants. This indicates that the cellulose in fibers from the transgenic plants was more highly structured than in fibers from the control plants. Another parameter that can be used to describe the level of structure in cellulose is the Crystallinity Index [22]. This index is calculated by dividing the amplitude of the band at 1370 cm 1 by the amplitude of the band at 2900 cm 1. The band at 1370 cm 1 represents –CH– groups, and the band at 2900 cm 1 represents both –CH2– and –CH– groups. The Crystallinity Index correlates with the degree of crystallinity obtained using X-ray diffraction [23]. The Crystallinity Index ranged from 0.71 to 0.75 in fibers from all of the transgenic lines, as compared to 0.62 in fibers from the control plants or 0.64 in purified cotton cellulose. It was also higher than in flax fibers that had been treated with surfactants or with enzymes that free the fibers from lignin, pectin, fats and waxes [22]. This indicates that PHB increased the level of structure in the cellulose in fibers from the transgenic plants. The infra-red spectrum of PHB isolated from the transgenic plants contains a broad contour from 3000 to 3700 cm 1. This contour is more complex and shifted lower for PHB from the transgenic plants than for fibers from the control plants. The differences in the spectra of the PHB isolated from the transgenic plants and bacterial PHB indicate that the PHB was strongly bound to the cellulose in the fibers by covalent and hydrogen bonds. Retting progressed more rapidly in transgenic plants than in the control plants. Treatment with NaOH was also more effective in removing cuticle and non-fibrous tissue from fibers of transgenic plants. This suggests that the PHB reduces the binding of pectin and hemicellulose to the fibers, which facilitates retting. The shrinkage observed in technical fibers from transgenic plants after treatment with chloroform indicates that some of
the cellulose of the fiber was co-extracted with the PHB. This showed that the PHB was strongly bound to the cellulose of the fiber, which confirms the results obtained using infra-red spectroscopy. All of the composites tested had a lower maximum tensile strength than pure polypropylene. The composite containing fibers from transgenic plants were, however, significantly stronger than the composites prepared with fibers from the control plants. All of the composites tested had a higher Young’s modulus than pure polypropylene. This was particularly true for the composite containing fibers from transgenic plants. Therefore, the composite containing fibers from transgenic plants was significantly superior in terms of mechanical properties than the composites containing fibers from the control plants. This indicated that the fibers from the transgenic plants were bonded more strongly to the matrix than fibers from the control plants. The absence of gaps on the surface of fractured sheets of the composite containing fibers from transgenic plants also shows that these fibers adhered to the polypropylene matrix significantly stronger than fibers from the control plants. The composite containing fibers from transgenic plants induced almost no platelet aggregation. Further research is however needed to elucidate the exact molecular mechanism involved in this process. Composites containing fibers from transgenic plants expressing polymers may therefore be useful in the construction of biomedical devices that come in contact with blood. To the best of our knowledge, this is the first report on the structural analysis of PHB produced by transgenic plants. It is also the first report on the use of genetic techniques to modify natural fibers in order to improve the compatibility between flax fibers and the matrix in composite materials.
Acknowledgments This study was supported by the Grants Polish Ministry of Education and Sciences (Grant Numbers POLPOST-DOC II Nr PBZ/ MEiN/01/2006/17, NN 302061834, 2P06A 02029, 2P04B 01528, N R12 0009 06 and PBZ-MNiI-2/1/2005).
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