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Bio-thermoplastics from grafted chicken feathers for potential biomedical applications
Colloids and Surfaces B: Biointerfaces 110 (2013) 51–58
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Bio-thermoplastics from grafted chicken feathers for potential biomedical applications Narendra Reddy a , Qiuran Jiang a , Enqi Jin a,b , Zhen Shi a,b , Xiuliang Hou b , Yiqi Yang a,b,c,d,∗ a
Department of Textiles, Merchandising & Fashion Design, 234, HECO Building, University of Nebraska-Lincoln, Lincoln, NE 68583-0802, United States Key Laboratory of Eco-Textiles Ministry of Education, College of Textiles and Garments, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China Department of Biological Systems Engineering, 234, HECO Building, University of Nebraska-Lincoln, Lincoln, NE 68583-0802, United States d Nebraska Center for Materials and Nanoscience, 234, HECO Building, University of Nebraska-Lincoln, Lincoln, NE 68583-0802, United States b c
a r t i c l e
i n f o
Article history: Received 6 September 2012 Received in revised form 18 April 2013 Accepted 22 April 2013 Available online 28 April 2013 Keywords: Feathers Proteins Graft polymerization Films Tissue engineering scaffolds
a b s t r a c t This research demonstrated the feasibility of using bio-thermoplastics developed from chicken feathers grafted with acrylates and methacrylates as scaffolds for tissue engineering. Keratin, the major protein in feathers, is a highly crosslinked biopolymer that has been reported to be biocompatible. However, it is difficult to break the disulfide bonds and make keratin soluble to develop materials for tissue engineering and other medical applications. Previously, keratin extracted from feathers using alkaline hydrolysis has been made into scaffolds but with poor water stability and mechanical properties. In this study, thermoplastic films were compression molded from chicken feathers grafted with 6 different acrylate monomers. The influence of the concentration and structures of grafted monomers on grafting parameters and the tensile strength, water stability and cytocompatibility of grafted feathers compression molded into films were investigated. It was found that the grafted feather films were water stable and had good strength and better supported cell growth than poly(lactic acid) films. Grafted feathers demonstrated the potential to be used for fabrication of biomaterials for various biomedical applications. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Protein-based biomaterials are preferred for tissue engineering due to their similarity to native extracellular matrix and ability to be made into various shapes [1,2]. Both plant (soyprotein, wheat gluten, zein) and animal proteins (keratin, silk, collagen) have been made into fibers, films, hydrogels and nano and micro particles for medical applications [3,4]. However, scaffolds made from most proteins have poor mechanical properties especially when wet and lack the stability required for medical applications [5]. Crosslinking or blending with water stable polymers is done to enhance the strength and water stability of protein scaffolds. However, these modifications decrease biodegradability and cytocompatibility of scaffolds [6,7]. Unlike the common proteins such as collagen used for medical applications, keratins found in wool, feather, hooves, horns, hair and nails [8] are water stable proteins. A large amount of interand intramolecular disulfide bonds provide extensive crosslinking and therefore high strength and water stability [9]. However, this
∗ Corresponding author at: Department of Textiles, Merchandising & Fashion Design, 234, HECO Building, University of Nebraska-Lincoln, Lincoln, NE 685830802, United States. Tel.: +1 402 472 5197; fax: +1 402 472 0640. E-mail address: [email protected] (Y. Yang). 0927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2013.04.019
highly crosslinked structure makes it difficult to dissolve keratin and develop biomaterials [10,11]. Currently, keratin-based scaffolds are mainly made by extracting keratin using reduction and oxidation methods which cause excessive cleavage of disulfide bonds and decrease in molecular weight resulting in scaffolds with poor strength and water stability [8,12]. Although chemical and physical modifications such as crosslinking are used to improve the properties of the scaffolds [8,13], the high strength and water stability of raw keratins cannot be realized. Therefore, alternative approaches to develop keratin biomaterials, which can retain the unique properties of native keratins, are desirable. Films are one of the most common types of materials developed from plant and animal proteins and have been used as scaffolds for tissue engineering. Compression molding and solvent casting are the two most common methods of producing films. Solvent casting is more commonly used than compression molding because most biopolymers are non-thermoplastic. However, compression molded materials have higher tensile strength and stability than those made by dissolution using the same polymer and many biopolymers do not dissolve in common solvents [14]. Attempts have been made to develop films from feathers using solution casting and compression molding. Since feathers are nonthermoplastic, chemical modifications and/or plasticizers are used to produce compression molded materials [15–17]. Acetylation, etherification and grafting are some of the common approaches
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used to make feathers thermoplastic [15–17]. It has also been shown that alkali hydrolyzed and citric acid crosslinked feathers can be made thermoplastic with good tensile strength and water stability [18]. Grafting of synthetic polymers has been widely used to make biopolymers thermoplastic. Unlike acetylation or etherification, grafting uses mild conditions, preserves the main structure of the polymer and therefore provides better properties to the materials developed. Feathers have been grafted with methacrylates [19] and the properties of the grafted feathers were studied. However, the water stability of the grafted feathers has not been reported and the suitability of using the feather films as scaffolds for tissue engineering has not been studied. Although several types of acrylate monomers have been grafted onto feathers, the effects of molecular structures on the performances of grafted feathers have also not been investigated. Molecular structure of the monomer also affect the cost, thermoplasticity, water stability, mechanical properties and biocompatibility of grafted feathers. In the present investigation, feathers were grafted with three acrylates (methyl/ethyl/butyl) and three methacrylates (methyl/ethyl/butyl) and thermoplastic films were developed from the grafted feathers. The effects of monomer concentration on molar grafting ratio and % monomer conversion were investigated. The influences of the length of alkyl ester group and ␣-methyl group on molar grafting ratio and % monomer conversion were researched. In addition, the ability of the feather-g-polyacrylates and feather-g-polymethacrylates with similar molar grafting ratios to form thermoplastics was investigated. The tensile strength, water stability and cytocompatibility of films developed from the grafted feathers were evaluated. Potential of the grafted feather films to be used as substrates for tissue engineering were also studied. 2. Materials and methods Native chicken feathers were supplied by Feather Fiber Corporation (Nixa, MO). The whole feathers containing quills and barbs were cleaned with ethanol and acetone and ground in a Wiley mill. The monomers, three acrylates (methyl acrylate (MA), ethyl acrylate (EA), butyl acrylate (BA)) and three methacrylates (methyl methacrylate (MMA), ethyl methacrylates (EMA) and butyl methacrylate (BMA)) were purchased from Alfa Aesar. Paradioxybenzene (99%) used as a terminator was also purchased from Alfa Aesar. Potassium persulfate as an oxidant (99%) and sodium bisulfite as a reductant (99%) were supplied by Spectrum and J.T. Baker, respectively. Polylactic acid (PLA) was obtained from Cargill Dow LLC. 2.1. Graft polymerization Native chicken feathers were grafted with the acrylates and methacrylates according to our previous work [16]. Three acrylates and three methacrylates were selected as monomers to investigate the influence of monomer structure (the length of alkyl esters and the ␣-methyl group of monomers) on grafting, thermoplasticity, water stability and cytocompatibility of the grafted products. About 5 g of feathers were used for each grafting reaction and the concentration of the monomers was varied between 10 and 60 wt.%, based on the weight of feathers. After deoxygenating by passing nitrogen gas for 30 min, the graft polymerization was initiated by adding the oxidant (potassium persulfate ((K2 S2 O8 ), 5 wt.% of feather), and the reductant (sodium bisulfite (NaHSO3 ), 1.92 wt.% of feather). The grafting reaction was performed at pH 5.5 and 60 ◦ C for 4 h. Grafting of the polymers was terminated by adding 1 ml of 2 wt.% paradioxybenzene. After neutralizing to pH 7.0, the grafted product was
filtered, washed and dried at 105 ◦ C. Grafting parameters such as % monomer conversion, molar grafting ratio and grafting efficiencies were calculated as reported in our previous work [16]. Samples with similar molar grafting ratios (2.4–2.8 mmol/g) were used in the following tests to characterize the properties of the grafted feathers. In previous studies, weight grafting ratio denoting the weight ratio of the monomers grafted onto substrate to the substrate was used to evaluate the grafting [19–22]. However, weight grafting ratio is not an appropriate parameter to compare the properties of feathers grafted by different monomers because of the difference in molecular weights of monomers. Two samples may have the same weight grafting ratio but their molar grafting ratio would be different if the molecular weights of the monomers were different. Hence, it is necessary to use samples with similar molar grafting ratios to study the impact of molecular structures of monomers on properties of grafted feathers. 2.2. Characterization 2.2.1. 1 H–NMR Since feathers do not dissolve in common solvents used for NMR analysis, feathers were first hydrolyzed using alkali (0.25%, 20:1 ratio of alkali solution to feather, 70 ◦ C, and 1 h) to obtain keratin and the hydrolyzed keratin was grafted using the same procedure to graft the feathers. Later, the ungrafted and grafted feather keratins obtained were characterized by proton nuclear magnetic resonance (1 H–NMR) to confirm the grafting. Before characterization, the grafted samples were extracted in acetone for 24 h to remove homopolymers. Samples were dissolved in DMSO d6 and measured in an Avance 600 MHz Digital NMR spectrometer (Bruker Co. Ltd., Switzerland). A basic proton pulse sequence (zg30) was used with a relaxation delay of 1s and acquisition time of 3s. Sixty four scans were acquired to obtain adequate signal to noise ratio. The concentration of each sample was about 1 wt.% in the solvent. 2.2.2. Thermal behavior Thermal behaviors of unmodified and grafted feathers were studied using differential scanning calorimetry (DSC). Homopolymers were extracted by acetone for 24 h before the thermal analysis. A Mettler Toledo DSC 822 e thermal analyzer was used to obtain the DSC thermograms and determine melting temperature (Tm). Samples were dried at 105 ◦ C for 4 h to remove moisture before testing. DSC measurement was conducted by heating the samples from 25 ◦ C to 220 ◦ C with a heating rate of 20 ◦ C/min under nitrogen atmosphere. 2.3. Preparation of films Films were made from the grafted feathers by compression molding in a Carver Press (Carver Inc., Wabash, IN) at 170 ◦ C for 8–12 min at a pressure of 40,000 pounds. Before compression molding, the homopolymers in the grafted feathers were removed by refluxing with acetone for 24 h. Glycerol (20% based on weight of feathers) was used as a plasticizer and mixed with the grafted samples before compression molding. To compare the cytocompatibility of the grafted feathers, films were also made from PLA, a FDA approved and widely used thermoplastic biomaterial, using casting method according to our previous research [23]. 2.4. Water stability The water stability of the washed and sterilized feather films was investigated based on weight loss after incubation in phosphate buffered saline (1× PBS, pH 7.4) at 37 ◦ C for up to 15 days.
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Fig. 1. Effects of types and concentrations of monomers (wt.% based on feathers) on % monomer conversion. Grafting was performed at 60 ◦ C and pH 5.5 for 4 h. The molar ratio of NaHSO3 /K2 S2 O8 was 1.0 and the concentration of K2 S2 O8 was 10 mmol/L. Within the same type of monomer, data points with different alphabets indicate statistically significant difference.
Samples were collected at the desired days and measured for weight loss.
2.5. Tensile test Feather films were cut into strips (80 mm × 15 mm) and conditioned at 65% R.H. and 21 ◦ C for 24 h before testing. Tensile strength of the films was measured on a MTS (Model Q Test 10; MTS Corporation, Eden Prairie, MN) tensile tester according to ASTM Standard D 882. Five samples were tested for each condition. The wet tensile properties were determined by testing immediately after immersing films in distilled water for 30 min at 21 ◦ C.
2.6. Cytocompatibility To evaluate the potential of using the grafted feather films for biomedical applications, the cytocompatibility of feather films were investigated in comparison to PLA films. The films (10 mm in diameter) made from grafted feathers without any homopolymers were first washed with distilled water and ethanol. Later the films were sterilized at 120 ◦ C for 1 h. PLA films were autoclaved in 75% ethanol for 1 h. Later, both feather and PLA films were sterilized under UV light for 8 h, and placed into 48-well culture plates. NIH 3T3 mouse fibroblast cells in Dulbecco’s modified low-glucose Eagle’s medium (DMEM, containing 10% calf serum, 1.4 vol.% 200 mM L-glutamine and 0.1 mg/ml penicillinstreptomycin) were seeded on the films (2 × 105 cells/well). Cells were cultured in an incubator at 37 ◦ C with a humidified 5% CO2 atmosphere. After culture for the desired time (4 h or 4 days), samples were washed in PBS to remove the loosely attached cells. Cell viability was evaluated by MTS assays [23], and the optical densities were normalized based on the size of the wet samples. The spreading of cells stained with Hoechst 33342 (10 g ml−1 , cell nuclei) and eosin-phloxine (cytoplasm) was observed under a confocal laser scanning microscope (CLSM, Olympus IX 81).
2.7. Statistical analysis A one-way analysis of variance with Tukey’s pairwise multiple comparison was used to analyze the data. The confidence interval was set at 95% and a p value lesser than 0.05 was considered to be a statistically significant difference. In the results, data labeled with the same characters were not significantly different from each other. The error bars shown in figures represent standard deviations. 3. Results and discussion 3.1. Effects of concentrations and molecular structures of monomers on % monomer conversion Fig. 1 shows the effects of monomer concentration and molecular structure (i.e. the length of alkyl ester and ␣-methyl group) of acrylates on % monomer conversion and the amount of monomers converted into grafted branches and homopolymers, respectively. At low monomer concentration (10%), the % monomer conversion for MA was relatively low (75%) compared to 94% and 91% for EA and BA, respectively. Irrespective of the type of monomer, the monomer conversion initially increased and then leveled off after monomer concentration reached a certain level (e.g. 20% for EA and BA). MA had considerably higher number of grafted branches (66–79 wt.%, to total the monomers used) and therefore less homopolymer (7–21 wt.%, to total monomers used) compared to EA and BA as seen from Fig. 1. EA and BA had considerably lower % grafted branches compared to MA, especially at low monomer concentrations. The amount of homopolymers was always higher than that of the grafted branches for BA at all monomer concentrations studied. The highest amount of grafted branches on the feathers was only 33% for BA whereas 54 and 79% for EA and MA, respectively. The methacrylates had different behaviors to the corresponding acrylates in terms of % grafted branches and homopolymers as seen from Fig. 1. The total monomer conversions were very similar for MMA and EMA at all the monomer concentrations studied
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except 10% but BMA had slightly higher conversion especially at higher (40, 60%) monomer concentrations. Unlike MA, MMA produced much higher amounts of homopolymers, especially at low monomer concentrations. Increasing monomer concentration increased the % monomer conversion, especially for MA and the methacrylates. At low monomer concentrations, a large proportion of the monomers were absorbed by the feathers limiting the number of free monomers available for the polymerization. When monomer concentration increased, the sorption of the monomers by feathers gradually reached saturation and a higher number of monomers were available for polymerization. Therefore the % monomer conversion increased initially and then leveled off. However, the monomer conversion was different for the different monomers even at the same monomer concentration. For example, the conversion for MA was lower than that of EA/BA, especially at low monomer concentrations. This phenomenon should be due to the monomer radicals reacting with each other and forming pre-polymerized radicals that were much larger in size than single monomers. The prepolymerized radicals of EA and BA were much larger than MA pre-polymerized radicals. Therefore, it is difficult for EA and BA to penetrate into the feathers. Most of EA and BA react with themselves forming higher amount of homopolymers and consequently leading to higher % monomer conversion than MA monomers which were more easily absorbed by feather fibers and became immobile. The high % monomer conversion of EA and BA compared to EMA and BMA, respectively, should also be due to the larger size of the monomers and pre-polymerized radicals formed by EMA and BMA that decreased the sorption of the monomers by the feathers. It has been reported that, due to the effect of methyl substitution, EMA and BMA had much lower potential for homopolymerization than EA and BA [19]. Therefore EA and BA formed higher amounts of homopolymers and had higher % monomer conversion than EMA and BMA at low monomer concentrations (10, 20%). 3.2. Effects of monomer concentration and length of alkyl ester of acrylates on molar grafting ratio Fig. 2a shows the effects of monomer concentration and length of alkyl esters of acrylates on molar grafting ratio. The grafting ratio increased almost linearly with the increase in monomer concentration irrespective of the type of acrylate monomer when molar concentration was in a range of 0.5–8.0 mmol/g. However, molar grafting ratio decreased with increasing length of alkyl ester of acrylates. For instance, at a molar concentration of 5.5 mmol/g, grafting ratios for MA, EA and BA were about 4.1 mmol/g, 2.8 mmol/g and 1.8 mmol/g, respectively. MA had higher grafting ratio than EA and EA had higher grafting ratio than BA. The differences between the monomers were more prominent especially at high monomer concentrations. MA had higher grafting ratio than EA and BA mainly because of the higher grafted branches and lower homopolymers as seen from Fig. 2a. Since size of single molecules and the pre-polymerized radicals of BA were larger than those of MA and EA, it is difficult for BA to penetrate into the feathers. BA reacted with itself more readily and formed higher homopolymers than grafted branches. In addition, due to higher steric effect, it was more difficult for BA which had much longer alkyl ester to be grafted onto the feather fibers than MA and EA. Therefore, BA had relatively low molar grafting ratio compared to MA and EA under similar grafting conditions. 3.3. Effects of monomer concentration and length of alkyl ester of methacrylates on molar grafting ratio Increasing the length of the alkyl ester also decreased the molar grafting ratio for the methacrylates as seen from Fig. 2b. However,
Fig. 2. Effects of monomer concentration and molecular structure on molar grafting ratio of acrylates (a) and methacrylates (b). Units of molar grafting ratio and molar concentration of the monomers were both mmol/g. Grafting was performed at 60 ◦ C and pH 5.5 for 4 h. The molar ratio of NaHSO3 /K2 S2 O8 was 1.0 and the concentration of K2 S2 O8 was 10 mmol/L. On each curve, data points with different letters indicate statistically significant difference.
the differences in the grafting ratios between the three methacrylates were much smaller than those between the three acrylates. Comparing the individual acrylates and methacrylates, MA had higher grafting than MMA, whereas EMA and BMA had slightly and considerably higher grafting ratios than EA and BA, respectively. The primary reactive groups of acrylate and methacrylate are hydrogen atom and methyl, respectively. Hydrogen atom takes up much less space than methyl and the length of alkyl ester of acrylates presents substantial steric hindrance than that of methacrylates. Therefore, increasing the length of alkyl ester decreased the molar grafting ratio of acrylates more remarkably than that of methacrylates. MMA had lower grafting ratio than MA mainly because of the lower penetration of MMA into feathers and consequent formation of high amounts of homopolymers of MMA as seen from Fig. 1. It is difficult for EA/EMA and BA/BMA to penetrate the feather fibers due to their larger size, especially for BA/BMA. EA and BA should have higher monomer conversion than EMA and BMA due to their
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smaller sizes. However, the low steric effect of EA and BA compared to EMA and BMA and the high ability of EA and BA to self-react lead to an increase in the amount of EA and BA homopolymers and fewer EA and BA monomers that were available for the grafting onto feathers. Thus, EA and BA had lower molar grafting ratios than EMA and BMA, respectively. The high molar grafting ratio of EA and EMA compared to BA and BMA, respectively, should be due to the relatively large sizes of the monomers and pre-polymerized radicals of BA and BMA. 3.4.
1 H–NMR
analysis
1 H–NMR spectra of ungrafted and grafted feathers are shown in Fig. 3. The existence of grafted branches on feathers was confirmed by 1 H–NMR. Compared to the spectrum of unmodified feather (Fig. 3a), new chemical linkages were found in grafted feathers. In Fig. 3b and c, the protons of methyl ester ( OCH3 ) appeared at 3.5 ppm confirming the grafting of MA and MMA [24,25]. A new peak belonging to the protons of COOCH3 appeared at about 4.0 ppm in Fig. 3d and e indicating that EA and EMA were grafted on to the feathers. Similarly, the protons of COOCH2 appeared at about 4.0 ppm in both Fig. 3f and g, which indicated successful grafting of BA and BMA. After grafting, the vinylic protons on MA, EA and BA became the ester protons on CH(R ) COOR, which displayed at around 2.2 ppm in Fig. 3b, d and f, respectively. The allylic protons on MMA, EMA and BMA changed to primary protons shown at around 0.9 ppm. However, all of the spectrums for the methacrylates also displayed peaks at 0.9 ppm that belong to the primary protons from the grafted polymer and the amino acid residues in the feather proteins.
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Heat during compression molding realigned protein molecules and the presence of plasticizers helps the proteins to move relatively freely. The synthetic polymer grafted onto the feathers facilitated melting of feathers and the formation of the films. 3.7. Water stability The effect of monomer structures on water stability (% weight loss in PBS at 37 ◦ C) of grafted feather films with similar molar grafting ratios of monomers is shown in Fig. 6. The unmodified but compressed feathers were unstable and disintegrated when immersed in the aqueous media at 37 ◦ C. In contrast, samples produced from grafted feathers maintained their film structures. Films modified by larger monomers had lower weight losses, indicating better water stability. For instance, BA and BMA grafted feather films had lower weight loss than MA and MMA grafted films. MA grafted feathers had the highest weight loss among all the four monomers studied. Inherent differences in hydrophilicity of the films should be responsible for the differences in water stability. MA grafted feathers did not melt completely during compression molding and the shorter length of MA polymers would make the films more vulnerable to water than the films containing the other monomers. Hence, faster degradation and release of hydrolyzed products could be observed from MA grafted films. 3.8. Tensile strength
DSC thermograms of ungrafted and keratin grafted with the three acrylates and methacrylates are shown in Fig. 4. Unlike the unmodified feather which did not show any melting peak, the grafted feathers displayed obvious melting peaks indicating the thermoplasticity of the grafted samples. Endothermic melting peaks of the feathers grafted with MA, EA and BA appeared at 162 ◦ C, 170 ◦ C and 156 ◦ C and that of MMA, EMA and BMA appeared at 159 ◦ C, 156 ◦ C and 135 ◦ C, respectively. As seen from Fig. 4, polymers with the largest size of the carbon chain (BA and BMA) representing the acrylates and methacrylates had the lowest melting temperature. When the respective acrylates were compared with methacrylates, the methacrylates showed lower melting temperature for all the three monomers studied indicating that feathers grafted with methacrylates would melt at lower temperatures and may also provide thermoplastics with better properties than the corresponding acrylates.
Table 1 provides the results of dry and wet tensile strengths of grafted feather films with similar molar grafting ratios of monomers. The films made from feathers grafted with methacrylates had higher dry and wet strengths than those modified with corresponding acrylates (MMA vs. MA and BMA vs. BA), because the ␣-methyl groups on the backbone of polymethacrylate formed steric hindrance to the movement of molecular chains and the existence of the short branch increased rigidity of the macromolecules. However, the dry and wet strengths of films produced from feather-g-poly(butyl (meth)acrylate) were substantially lower than the strengths of films from feather-g-poly(methyl (meth)acrylate). Larger amounts of homopolymers present in the films modified with butyl (meth)acrylates than those grafted with methyl (meth)acrylate. The tensile strength decreased probably due to the reduced interactions among the keratin molecules by homopolymers and long carbochains, though more homopolymers and longer carbochain of alkly ester groups could improve water resistance of films. Except for the BA grafted films, other samples possessed higher strengths than the unmodified films made from extracted keratins (5.14 ± 0.27 MPa) and the TGase-mediated crosslinked keratin films (6.22 ± 0.11 MPa) that were developed previously [26].
3.6. Thermoplastic feather films
3.9. Cytocompatibility
Fig. 5 displays the unmodified and uncompressed feathers and the thermoplastics developed from the feathers after grafting with various acrylates and methacrylates. Fig. 5a shows the unmodified feathers before compression molding and Fig. 5b displays the compression molded unmodified feathers after adding 20% glycerol. As seen from Fig. 5b, the ungrafted feathers were not thermoplastic and could not form films. On the contrary, all the grafted feathers could melt during compression molding and formed films. However, MMA grafted films (Fig. 5d) appeared to have better thermoplasticity than MA grafted feathers (Fig. 5c) due to the higher melting enthalpy and lower melting temperature than the MA grafted feathers. Feathers modified with BA (Fig. 5e) and BMA (Fig. 5f) did not show major differences in melting temperature or melting enthalpy and produced homogenous and isotropic films.
Fig. 7shows the effect of monomer structures on the cytocompatibility of films from grafted feathers. The result of Methanethiosulfonate (MTS) assay (Fig. 7A and B) revealed that cell viabilities on all the grafted feather films were higher than that on the PLA film. In neutral conditions, PLA carries more negative charges than keratin (PLA: −20 mV [23]; keratin: −4.5 mV). In addition, the grafting processes could decrease the negative charges carried by feather keratin, since carboxylic groups on keratins were one of the reactive sites for grafting [27]. Due to the difference in charges, grafted feathers attracted more cells on to their surface than PLA resulting in higher cell attachment on grafted feathers (Fig. 7A). BA and BMA modified films displayed better support to cell attachment and proliferation than MA and MMA grafted films most likely due to their better water stability. Fig. 7C and D show that
3.5. DSC analysis
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Fig. 3. 1 H–NMR spectra of unmodified feather (a) and the feathers grafted with MA (b), MMA (c), EA (d), EMA (e), BA (f) and BMA (g). Molar grafting ratios of the feathers grafted with MA, MMA, EA, EMA, BA, and BMA were 2.7, 2.8, 2.4, 2.5, 2.9, and 2.7 mmol/g, respectively.
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Fig. 4. DSC spectra of unmodified and feathers grafted with the three acrylates and methacrylates. Molar grafting ratios of the feathers grafted with MA, MMA, EA, EMA, BA, and BMA were 2.7, 2.8, 2.4, 2.5, 2.9, and 2.7 mmol/g, respectively.
the cells have spread and reached confluence on the BA and BMA grafted films after four days of culture, indicating the suitability of the films for tissue engineering and other medical applications. Previous studies on using wool keratin as tissue engineering scaffolds have reported that wool keratins contain RGD and LDV adhesion sequences found in several extracellular matrix such as fibronectin
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Fig. 6. Effect of molecular structure of monomers on the water stability of grafted feather films (molar grafting ratios of the feathers grafted with MA, MMA, BA, and BMA were 2.7, 2.8, 2.9, and 2.7 mmol/g, respectively). The grafted films were incubated in PBS at 37 ◦ C for up to 15 days. Significant differences among data are denoted by different letters.
that make keratin an excellent biomaterial for growing cells [28]. Feather keratin should also have similar sequences that provide support to cell attachment and growth and make feathers suitable as tissue engineering scaffolds.
Fig. 5. Digital pictures of raw feathers (A), feathers compression molded with 20% glycerol (B), feathers grafted with MA (C), MMA (D), BA (E) and BMA (F) with similar grafting ratios.
Table 1 Tensile strengths of films from grafted feathers after compression molding at 170 ◦ C for 12 min with 20% glycerol as plasticizer. Type and molar grafting ratio of monomers MA (2.7 mmol/g) [0.075]
Tensile strength (MPa)
MMA (2.8 mmol/g) [0.111]
BA (2.9 mmol/g) [0.079]
BMA (2.7 mmol/g) [0.195]
Dry
Wet
Dry
Wet
Dry
Wet
Dry
Wet
6.80 ± 0.80
0.69 ± 0.12
18.00 ± 0.8
4.31 ± 0.42
1.40 ± 0.09
0.37 ± 0.09
10.30 ± 0.32
2.17 ± 0.50
* The numbers in [] indicate the weight ratios of homopolymers to grafted feathers.
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length of alkyl ester of monomers. The films produced from feathers modified by monomers with ␣-methyl groups containing shorter alkly ester groups possessed higher strengths. However, better water stability and cytocompatibility were observed on feathers modified by monomers with ␣-methyl groups having longer carbochain of alkly ester groups. Films with the properties desired for biomedical applications can be developed by selecting appropriate monomers and grafting conditions. Grafting appears to be most convenient method to produce biomaterials from the cytocompatible and unique feather proteins. Acknowledgments This research was financially supported by Agricultural Research Division at the University of Nebraska-Lincoln, USDA Hatch Act, Multistate Research Project S1054 (NEB 37-037), AATCC student research grant and Program for Changjiang Scholars and Innovative Research Team in Jiangnan University IRT1135, Scientific Support Program of Jiangsu Province (BE2011404), the Graduate student innovation plan of Jiangsu Province (CX10B 222Z), the Doctor Candidate Foundation of Jiangnan University (JUDCF10004) and the China Scholarship Council. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] Fig. 7. Effect of molecular structures of monomers on cytocompatibility of grafted feather films. The attachment (4 h, (A)) and proliferation (4 days, (B)) of NIH 3T3 mouse fibroblast cells on feather films were evaluated by MTS assay and compared with PLA films. Cell spreading on the BA (C) and BMA (D) grafted films was observed by a confocal laser scanning microscope (cell nuclei: blue; cytoplasm: red). Significant differences among data were denoted by different letters. (For interpretation of the references to color in figure legend, the reader is referred to the web version of the article.)
[13] [14] [15] [16] [17] [18] [19] [20]
4. Conclusions Grafting of acrylic monomers is a viable approach to develop thermoplastics from feathers with the water stability and biocompatibility required for medical applications. The concentration and molecular structure of acrylic monomers could play a major role in determining the % monomer conversion and molar grafting ratio. Molar grafting ratio increased with the decrease in the
[21] [22] [23] [24] [25] [26] [27] [28]
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Bio-thermoplastics from grafted chicken feathers for potential biomedical applications Narendra Reddy a , Qiuran Jiang a , Enqi Jin a,b , Zhen Shi a,b , Xiuliang Hou b , Yiqi Yang a,b,c,d,∗ a
Department of Textiles, Merchandising & Fashion Design, 234, HECO Building, University of Nebraska-Lincoln, Lincoln, NE 68583-0802, United States Key Laboratory of Eco-Textiles Ministry of Education, College of Textiles and Garments, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China Department of Biological Systems Engineering, 234, HECO Building, University of Nebraska-Lincoln, Lincoln, NE 68583-0802, United States d Nebraska Center for Materials and Nanoscience, 234, HECO Building, University of Nebraska-Lincoln, Lincoln, NE 68583-0802, United States b c
a r t i c l e
i n f o
Article history: Received 6 September 2012 Received in revised form 18 April 2013 Accepted 22 April 2013 Available online 28 April 2013 Keywords: Feathers Proteins Graft polymerization Films Tissue engineering scaffolds
a b s t r a c t This research demonstrated the feasibility of using bio-thermoplastics developed from chicken feathers grafted with acrylates and methacrylates as scaffolds for tissue engineering. Keratin, the major protein in feathers, is a highly crosslinked biopolymer that has been reported to be biocompatible. However, it is difficult to break the disulfide bonds and make keratin soluble to develop materials for tissue engineering and other medical applications. Previously, keratin extracted from feathers using alkaline hydrolysis has been made into scaffolds but with poor water stability and mechanical properties. In this study, thermoplastic films were compression molded from chicken feathers grafted with 6 different acrylate monomers. The influence of the concentration and structures of grafted monomers on grafting parameters and the tensile strength, water stability and cytocompatibility of grafted feathers compression molded into films were investigated. It was found that the grafted feather films were water stable and had good strength and better supported cell growth than poly(lactic acid) films. Grafted feathers demonstrated the potential to be used for fabrication of biomaterials for various biomedical applications. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Protein-based biomaterials are preferred for tissue engineering due to their similarity to native extracellular matrix and ability to be made into various shapes [1,2]. Both plant (soyprotein, wheat gluten, zein) and animal proteins (keratin, silk, collagen) have been made into fibers, films, hydrogels and nano and micro particles for medical applications [3,4]. However, scaffolds made from most proteins have poor mechanical properties especially when wet and lack the stability required for medical applications [5]. Crosslinking or blending with water stable polymers is done to enhance the strength and water stability of protein scaffolds. However, these modifications decrease biodegradability and cytocompatibility of scaffolds [6,7]. Unlike the common proteins such as collagen used for medical applications, keratins found in wool, feather, hooves, horns, hair and nails [8] are water stable proteins. A large amount of interand intramolecular disulfide bonds provide extensive crosslinking and therefore high strength and water stability [9]. However, this
∗ Corresponding author at: Department of Textiles, Merchandising & Fashion Design, 234, HECO Building, University of Nebraska-Lincoln, Lincoln, NE 685830802, United States. Tel.: +1 402 472 5197; fax: +1 402 472 0640. E-mail address: [email protected] (Y. Yang). 0927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2013.04.019
highly crosslinked structure makes it difficult to dissolve keratin and develop biomaterials [10,11]. Currently, keratin-based scaffolds are mainly made by extracting keratin using reduction and oxidation methods which cause excessive cleavage of disulfide bonds and decrease in molecular weight resulting in scaffolds with poor strength and water stability [8,12]. Although chemical and physical modifications such as crosslinking are used to improve the properties of the scaffolds [8,13], the high strength and water stability of raw keratins cannot be realized. Therefore, alternative approaches to develop keratin biomaterials, which can retain the unique properties of native keratins, are desirable. Films are one of the most common types of materials developed from plant and animal proteins and have been used as scaffolds for tissue engineering. Compression molding and solvent casting are the two most common methods of producing films. Solvent casting is more commonly used than compression molding because most biopolymers are non-thermoplastic. However, compression molded materials have higher tensile strength and stability than those made by dissolution using the same polymer and many biopolymers do not dissolve in common solvents [14]. Attempts have been made to develop films from feathers using solution casting and compression molding. Since feathers are nonthermoplastic, chemical modifications and/or plasticizers are used to produce compression molded materials [15–17]. Acetylation, etherification and grafting are some of the common approaches
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used to make feathers thermoplastic [15–17]. It has also been shown that alkali hydrolyzed and citric acid crosslinked feathers can be made thermoplastic with good tensile strength and water stability [18]. Grafting of synthetic polymers has been widely used to make biopolymers thermoplastic. Unlike acetylation or etherification, grafting uses mild conditions, preserves the main structure of the polymer and therefore provides better properties to the materials developed. Feathers have been grafted with methacrylates [19] and the properties of the grafted feathers were studied. However, the water stability of the grafted feathers has not been reported and the suitability of using the feather films as scaffolds for tissue engineering has not been studied. Although several types of acrylate monomers have been grafted onto feathers, the effects of molecular structures on the performances of grafted feathers have also not been investigated. Molecular structure of the monomer also affect the cost, thermoplasticity, water stability, mechanical properties and biocompatibility of grafted feathers. In the present investigation, feathers were grafted with three acrylates (methyl/ethyl/butyl) and three methacrylates (methyl/ethyl/butyl) and thermoplastic films were developed from the grafted feathers. The effects of monomer concentration on molar grafting ratio and % monomer conversion were investigated. The influences of the length of alkyl ester group and ␣-methyl group on molar grafting ratio and % monomer conversion were researched. In addition, the ability of the feather-g-polyacrylates and feather-g-polymethacrylates with similar molar grafting ratios to form thermoplastics was investigated. The tensile strength, water stability and cytocompatibility of films developed from the grafted feathers were evaluated. Potential of the grafted feather films to be used as substrates for tissue engineering were also studied. 2. Materials and methods Native chicken feathers were supplied by Feather Fiber Corporation (Nixa, MO). The whole feathers containing quills and barbs were cleaned with ethanol and acetone and ground in a Wiley mill. The monomers, three acrylates (methyl acrylate (MA), ethyl acrylate (EA), butyl acrylate (BA)) and three methacrylates (methyl methacrylate (MMA), ethyl methacrylates (EMA) and butyl methacrylate (BMA)) were purchased from Alfa Aesar. Paradioxybenzene (99%) used as a terminator was also purchased from Alfa Aesar. Potassium persulfate as an oxidant (99%) and sodium bisulfite as a reductant (99%) were supplied by Spectrum and J.T. Baker, respectively. Polylactic acid (PLA) was obtained from Cargill Dow LLC. 2.1. Graft polymerization Native chicken feathers were grafted with the acrylates and methacrylates according to our previous work [16]. Three acrylates and three methacrylates were selected as monomers to investigate the influence of monomer structure (the length of alkyl esters and the ␣-methyl group of monomers) on grafting, thermoplasticity, water stability and cytocompatibility of the grafted products. About 5 g of feathers were used for each grafting reaction and the concentration of the monomers was varied between 10 and 60 wt.%, based on the weight of feathers. After deoxygenating by passing nitrogen gas for 30 min, the graft polymerization was initiated by adding the oxidant (potassium persulfate ((K2 S2 O8 ), 5 wt.% of feather), and the reductant (sodium bisulfite (NaHSO3 ), 1.92 wt.% of feather). The grafting reaction was performed at pH 5.5 and 60 ◦ C for 4 h. Grafting of the polymers was terminated by adding 1 ml of 2 wt.% paradioxybenzene. After neutralizing to pH 7.0, the grafted product was
filtered, washed and dried at 105 ◦ C. Grafting parameters such as % monomer conversion, molar grafting ratio and grafting efficiencies were calculated as reported in our previous work [16]. Samples with similar molar grafting ratios (2.4–2.8 mmol/g) were used in the following tests to characterize the properties of the grafted feathers. In previous studies, weight grafting ratio denoting the weight ratio of the monomers grafted onto substrate to the substrate was used to evaluate the grafting [19–22]. However, weight grafting ratio is not an appropriate parameter to compare the properties of feathers grafted by different monomers because of the difference in molecular weights of monomers. Two samples may have the same weight grafting ratio but their molar grafting ratio would be different if the molecular weights of the monomers were different. Hence, it is necessary to use samples with similar molar grafting ratios to study the impact of molecular structures of monomers on properties of grafted feathers. 2.2. Characterization 2.2.1. 1 H–NMR Since feathers do not dissolve in common solvents used for NMR analysis, feathers were first hydrolyzed using alkali (0.25%, 20:1 ratio of alkali solution to feather, 70 ◦ C, and 1 h) to obtain keratin and the hydrolyzed keratin was grafted using the same procedure to graft the feathers. Later, the ungrafted and grafted feather keratins obtained were characterized by proton nuclear magnetic resonance (1 H–NMR) to confirm the grafting. Before characterization, the grafted samples were extracted in acetone for 24 h to remove homopolymers. Samples were dissolved in DMSO d6 and measured in an Avance 600 MHz Digital NMR spectrometer (Bruker Co. Ltd., Switzerland). A basic proton pulse sequence (zg30) was used with a relaxation delay of 1s and acquisition time of 3s. Sixty four scans were acquired to obtain adequate signal to noise ratio. The concentration of each sample was about 1 wt.% in the solvent. 2.2.2. Thermal behavior Thermal behaviors of unmodified and grafted feathers were studied using differential scanning calorimetry (DSC). Homopolymers were extracted by acetone for 24 h before the thermal analysis. A Mettler Toledo DSC 822 e thermal analyzer was used to obtain the DSC thermograms and determine melting temperature (Tm). Samples were dried at 105 ◦ C for 4 h to remove moisture before testing. DSC measurement was conducted by heating the samples from 25 ◦ C to 220 ◦ C with a heating rate of 20 ◦ C/min under nitrogen atmosphere. 2.3. Preparation of films Films were made from the grafted feathers by compression molding in a Carver Press (Carver Inc., Wabash, IN) at 170 ◦ C for 8–12 min at a pressure of 40,000 pounds. Before compression molding, the homopolymers in the grafted feathers were removed by refluxing with acetone for 24 h. Glycerol (20% based on weight of feathers) was used as a plasticizer and mixed with the grafted samples before compression molding. To compare the cytocompatibility of the grafted feathers, films were also made from PLA, a FDA approved and widely used thermoplastic biomaterial, using casting method according to our previous research [23]. 2.4. Water stability The water stability of the washed and sterilized feather films was investigated based on weight loss after incubation in phosphate buffered saline (1× PBS, pH 7.4) at 37 ◦ C for up to 15 days.
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Fig. 1. Effects of types and concentrations of monomers (wt.% based on feathers) on % monomer conversion. Grafting was performed at 60 ◦ C and pH 5.5 for 4 h. The molar ratio of NaHSO3 /K2 S2 O8 was 1.0 and the concentration of K2 S2 O8 was 10 mmol/L. Within the same type of monomer, data points with different alphabets indicate statistically significant difference.
Samples were collected at the desired days and measured for weight loss.
2.5. Tensile test Feather films were cut into strips (80 mm × 15 mm) and conditioned at 65% R.H. and 21 ◦ C for 24 h before testing. Tensile strength of the films was measured on a MTS (Model Q Test 10; MTS Corporation, Eden Prairie, MN) tensile tester according to ASTM Standard D 882. Five samples were tested for each condition. The wet tensile properties were determined by testing immediately after immersing films in distilled water for 30 min at 21 ◦ C.
2.6. Cytocompatibility To evaluate the potential of using the grafted feather films for biomedical applications, the cytocompatibility of feather films were investigated in comparison to PLA films. The films (10 mm in diameter) made from grafted feathers without any homopolymers were first washed with distilled water and ethanol. Later the films were sterilized at 120 ◦ C for 1 h. PLA films were autoclaved in 75% ethanol for 1 h. Later, both feather and PLA films were sterilized under UV light for 8 h, and placed into 48-well culture plates. NIH 3T3 mouse fibroblast cells in Dulbecco’s modified low-glucose Eagle’s medium (DMEM, containing 10% calf serum, 1.4 vol.% 200 mM L-glutamine and 0.1 mg/ml penicillinstreptomycin) were seeded on the films (2 × 105 cells/well). Cells were cultured in an incubator at 37 ◦ C with a humidified 5% CO2 atmosphere. After culture for the desired time (4 h or 4 days), samples were washed in PBS to remove the loosely attached cells. Cell viability was evaluated by MTS assays [23], and the optical densities were normalized based on the size of the wet samples. The spreading of cells stained with Hoechst 33342 (10 g ml−1 , cell nuclei) and eosin-phloxine (cytoplasm) was observed under a confocal laser scanning microscope (CLSM, Olympus IX 81).
2.7. Statistical analysis A one-way analysis of variance with Tukey’s pairwise multiple comparison was used to analyze the data. The confidence interval was set at 95% and a p value lesser than 0.05 was considered to be a statistically significant difference. In the results, data labeled with the same characters were not significantly different from each other. The error bars shown in figures represent standard deviations. 3. Results and discussion 3.1. Effects of concentrations and molecular structures of monomers on % monomer conversion Fig. 1 shows the effects of monomer concentration and molecular structure (i.e. the length of alkyl ester and ␣-methyl group) of acrylates on % monomer conversion and the amount of monomers converted into grafted branches and homopolymers, respectively. At low monomer concentration (10%), the % monomer conversion for MA was relatively low (75%) compared to 94% and 91% for EA and BA, respectively. Irrespective of the type of monomer, the monomer conversion initially increased and then leveled off after monomer concentration reached a certain level (e.g. 20% for EA and BA). MA had considerably higher number of grafted branches (66–79 wt.%, to total the monomers used) and therefore less homopolymer (7–21 wt.%, to total monomers used) compared to EA and BA as seen from Fig. 1. EA and BA had considerably lower % grafted branches compared to MA, especially at low monomer concentrations. The amount of homopolymers was always higher than that of the grafted branches for BA at all monomer concentrations studied. The highest amount of grafted branches on the feathers was only 33% for BA whereas 54 and 79% for EA and MA, respectively. The methacrylates had different behaviors to the corresponding acrylates in terms of % grafted branches and homopolymers as seen from Fig. 1. The total monomer conversions were very similar for MMA and EMA at all the monomer concentrations studied
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except 10% but BMA had slightly higher conversion especially at higher (40, 60%) monomer concentrations. Unlike MA, MMA produced much higher amounts of homopolymers, especially at low monomer concentrations. Increasing monomer concentration increased the % monomer conversion, especially for MA and the methacrylates. At low monomer concentrations, a large proportion of the monomers were absorbed by the feathers limiting the number of free monomers available for the polymerization. When monomer concentration increased, the sorption of the monomers by feathers gradually reached saturation and a higher number of monomers were available for polymerization. Therefore the % monomer conversion increased initially and then leveled off. However, the monomer conversion was different for the different monomers even at the same monomer concentration. For example, the conversion for MA was lower than that of EA/BA, especially at low monomer concentrations. This phenomenon should be due to the monomer radicals reacting with each other and forming pre-polymerized radicals that were much larger in size than single monomers. The prepolymerized radicals of EA and BA were much larger than MA pre-polymerized radicals. Therefore, it is difficult for EA and BA to penetrate into the feathers. Most of EA and BA react with themselves forming higher amount of homopolymers and consequently leading to higher % monomer conversion than MA monomers which were more easily absorbed by feather fibers and became immobile. The high % monomer conversion of EA and BA compared to EMA and BMA, respectively, should also be due to the larger size of the monomers and pre-polymerized radicals formed by EMA and BMA that decreased the sorption of the monomers by the feathers. It has been reported that, due to the effect of methyl substitution, EMA and BMA had much lower potential for homopolymerization than EA and BA [19]. Therefore EA and BA formed higher amounts of homopolymers and had higher % monomer conversion than EMA and BMA at low monomer concentrations (10, 20%). 3.2. Effects of monomer concentration and length of alkyl ester of acrylates on molar grafting ratio Fig. 2a shows the effects of monomer concentration and length of alkyl esters of acrylates on molar grafting ratio. The grafting ratio increased almost linearly with the increase in monomer concentration irrespective of the type of acrylate monomer when molar concentration was in a range of 0.5–8.0 mmol/g. However, molar grafting ratio decreased with increasing length of alkyl ester of acrylates. For instance, at a molar concentration of 5.5 mmol/g, grafting ratios for MA, EA and BA were about 4.1 mmol/g, 2.8 mmol/g and 1.8 mmol/g, respectively. MA had higher grafting ratio than EA and EA had higher grafting ratio than BA. The differences between the monomers were more prominent especially at high monomer concentrations. MA had higher grafting ratio than EA and BA mainly because of the higher grafted branches and lower homopolymers as seen from Fig. 2a. Since size of single molecules and the pre-polymerized radicals of BA were larger than those of MA and EA, it is difficult for BA to penetrate into the feathers. BA reacted with itself more readily and formed higher homopolymers than grafted branches. In addition, due to higher steric effect, it was more difficult for BA which had much longer alkyl ester to be grafted onto the feather fibers than MA and EA. Therefore, BA had relatively low molar grafting ratio compared to MA and EA under similar grafting conditions. 3.3. Effects of monomer concentration and length of alkyl ester of methacrylates on molar grafting ratio Increasing the length of the alkyl ester also decreased the molar grafting ratio for the methacrylates as seen from Fig. 2b. However,
Fig. 2. Effects of monomer concentration and molecular structure on molar grafting ratio of acrylates (a) and methacrylates (b). Units of molar grafting ratio and molar concentration of the monomers were both mmol/g. Grafting was performed at 60 ◦ C and pH 5.5 for 4 h. The molar ratio of NaHSO3 /K2 S2 O8 was 1.0 and the concentration of K2 S2 O8 was 10 mmol/L. On each curve, data points with different letters indicate statistically significant difference.
the differences in the grafting ratios between the three methacrylates were much smaller than those between the three acrylates. Comparing the individual acrylates and methacrylates, MA had higher grafting than MMA, whereas EMA and BMA had slightly and considerably higher grafting ratios than EA and BA, respectively. The primary reactive groups of acrylate and methacrylate are hydrogen atom and methyl, respectively. Hydrogen atom takes up much less space than methyl and the length of alkyl ester of acrylates presents substantial steric hindrance than that of methacrylates. Therefore, increasing the length of alkyl ester decreased the molar grafting ratio of acrylates more remarkably than that of methacrylates. MMA had lower grafting ratio than MA mainly because of the lower penetration of MMA into feathers and consequent formation of high amounts of homopolymers of MMA as seen from Fig. 1. It is difficult for EA/EMA and BA/BMA to penetrate the feather fibers due to their larger size, especially for BA/BMA. EA and BA should have higher monomer conversion than EMA and BMA due to their
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smaller sizes. However, the low steric effect of EA and BA compared to EMA and BMA and the high ability of EA and BA to self-react lead to an increase in the amount of EA and BA homopolymers and fewer EA and BA monomers that were available for the grafting onto feathers. Thus, EA and BA had lower molar grafting ratios than EMA and BMA, respectively. The high molar grafting ratio of EA and EMA compared to BA and BMA, respectively, should be due to the relatively large sizes of the monomers and pre-polymerized radicals of BA and BMA. 3.4.
1 H–NMR
analysis
1 H–NMR spectra of ungrafted and grafted feathers are shown in Fig. 3. The existence of grafted branches on feathers was confirmed by 1 H–NMR. Compared to the spectrum of unmodified feather (Fig. 3a), new chemical linkages were found in grafted feathers. In Fig. 3b and c, the protons of methyl ester ( OCH3 ) appeared at 3.5 ppm confirming the grafting of MA and MMA [24,25]. A new peak belonging to the protons of COOCH3 appeared at about 4.0 ppm in Fig. 3d and e indicating that EA and EMA were grafted on to the feathers. Similarly, the protons of COOCH2 appeared at about 4.0 ppm in both Fig. 3f and g, which indicated successful grafting of BA and BMA. After grafting, the vinylic protons on MA, EA and BA became the ester protons on CH(R ) COOR, which displayed at around 2.2 ppm in Fig. 3b, d and f, respectively. The allylic protons on MMA, EMA and BMA changed to primary protons shown at around 0.9 ppm. However, all of the spectrums for the methacrylates also displayed peaks at 0.9 ppm that belong to the primary protons from the grafted polymer and the amino acid residues in the feather proteins.
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Heat during compression molding realigned protein molecules and the presence of plasticizers helps the proteins to move relatively freely. The synthetic polymer grafted onto the feathers facilitated melting of feathers and the formation of the films. 3.7. Water stability The effect of monomer structures on water stability (% weight loss in PBS at 37 ◦ C) of grafted feather films with similar molar grafting ratios of monomers is shown in Fig. 6. The unmodified but compressed feathers were unstable and disintegrated when immersed in the aqueous media at 37 ◦ C. In contrast, samples produced from grafted feathers maintained their film structures. Films modified by larger monomers had lower weight losses, indicating better water stability. For instance, BA and BMA grafted feather films had lower weight loss than MA and MMA grafted films. MA grafted feathers had the highest weight loss among all the four monomers studied. Inherent differences in hydrophilicity of the films should be responsible for the differences in water stability. MA grafted feathers did not melt completely during compression molding and the shorter length of MA polymers would make the films more vulnerable to water than the films containing the other monomers. Hence, faster degradation and release of hydrolyzed products could be observed from MA grafted films. 3.8. Tensile strength
DSC thermograms of ungrafted and keratin grafted with the three acrylates and methacrylates are shown in Fig. 4. Unlike the unmodified feather which did not show any melting peak, the grafted feathers displayed obvious melting peaks indicating the thermoplasticity of the grafted samples. Endothermic melting peaks of the feathers grafted with MA, EA and BA appeared at 162 ◦ C, 170 ◦ C and 156 ◦ C and that of MMA, EMA and BMA appeared at 159 ◦ C, 156 ◦ C and 135 ◦ C, respectively. As seen from Fig. 4, polymers with the largest size of the carbon chain (BA and BMA) representing the acrylates and methacrylates had the lowest melting temperature. When the respective acrylates were compared with methacrylates, the methacrylates showed lower melting temperature for all the three monomers studied indicating that feathers grafted with methacrylates would melt at lower temperatures and may also provide thermoplastics with better properties than the corresponding acrylates.
Table 1 provides the results of dry and wet tensile strengths of grafted feather films with similar molar grafting ratios of monomers. The films made from feathers grafted with methacrylates had higher dry and wet strengths than those modified with corresponding acrylates (MMA vs. MA and BMA vs. BA), because the ␣-methyl groups on the backbone of polymethacrylate formed steric hindrance to the movement of molecular chains and the existence of the short branch increased rigidity of the macromolecules. However, the dry and wet strengths of films produced from feather-g-poly(butyl (meth)acrylate) were substantially lower than the strengths of films from feather-g-poly(methyl (meth)acrylate). Larger amounts of homopolymers present in the films modified with butyl (meth)acrylates than those grafted with methyl (meth)acrylate. The tensile strength decreased probably due to the reduced interactions among the keratin molecules by homopolymers and long carbochains, though more homopolymers and longer carbochain of alkly ester groups could improve water resistance of films. Except for the BA grafted films, other samples possessed higher strengths than the unmodified films made from extracted keratins (5.14 ± 0.27 MPa) and the TGase-mediated crosslinked keratin films (6.22 ± 0.11 MPa) that were developed previously [26].
3.6. Thermoplastic feather films
3.9. Cytocompatibility
Fig. 5 displays the unmodified and uncompressed feathers and the thermoplastics developed from the feathers after grafting with various acrylates and methacrylates. Fig. 5a shows the unmodified feathers before compression molding and Fig. 5b displays the compression molded unmodified feathers after adding 20% glycerol. As seen from Fig. 5b, the ungrafted feathers were not thermoplastic and could not form films. On the contrary, all the grafted feathers could melt during compression molding and formed films. However, MMA grafted films (Fig. 5d) appeared to have better thermoplasticity than MA grafted feathers (Fig. 5c) due to the higher melting enthalpy and lower melting temperature than the MA grafted feathers. Feathers modified with BA (Fig. 5e) and BMA (Fig. 5f) did not show major differences in melting temperature or melting enthalpy and produced homogenous and isotropic films.
Fig. 7shows the effect of monomer structures on the cytocompatibility of films from grafted feathers. The result of Methanethiosulfonate (MTS) assay (Fig. 7A and B) revealed that cell viabilities on all the grafted feather films were higher than that on the PLA film. In neutral conditions, PLA carries more negative charges than keratin (PLA: −20 mV [23]; keratin: −4.5 mV). In addition, the grafting processes could decrease the negative charges carried by feather keratin, since carboxylic groups on keratins were one of the reactive sites for grafting [27]. Due to the difference in charges, grafted feathers attracted more cells on to their surface than PLA resulting in higher cell attachment on grafted feathers (Fig. 7A). BA and BMA modified films displayed better support to cell attachment and proliferation than MA and MMA grafted films most likely due to their better water stability. Fig. 7C and D show that
3.5. DSC analysis
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Fig. 3. 1 H–NMR spectra of unmodified feather (a) and the feathers grafted with MA (b), MMA (c), EA (d), EMA (e), BA (f) and BMA (g). Molar grafting ratios of the feathers grafted with MA, MMA, EA, EMA, BA, and BMA were 2.7, 2.8, 2.4, 2.5, 2.9, and 2.7 mmol/g, respectively.
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Fig. 4. DSC spectra of unmodified and feathers grafted with the three acrylates and methacrylates. Molar grafting ratios of the feathers grafted with MA, MMA, EA, EMA, BA, and BMA were 2.7, 2.8, 2.4, 2.5, 2.9, and 2.7 mmol/g, respectively.
the cells have spread and reached confluence on the BA and BMA grafted films after four days of culture, indicating the suitability of the films for tissue engineering and other medical applications. Previous studies on using wool keratin as tissue engineering scaffolds have reported that wool keratins contain RGD and LDV adhesion sequences found in several extracellular matrix such as fibronectin
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Fig. 6. Effect of molecular structure of monomers on the water stability of grafted feather films (molar grafting ratios of the feathers grafted with MA, MMA, BA, and BMA were 2.7, 2.8, 2.9, and 2.7 mmol/g, respectively). The grafted films were incubated in PBS at 37 ◦ C for up to 15 days. Significant differences among data are denoted by different letters.
that make keratin an excellent biomaterial for growing cells [28]. Feather keratin should also have similar sequences that provide support to cell attachment and growth and make feathers suitable as tissue engineering scaffolds.
Fig. 5. Digital pictures of raw feathers (A), feathers compression molded with 20% glycerol (B), feathers grafted with MA (C), MMA (D), BA (E) and BMA (F) with similar grafting ratios.
Table 1 Tensile strengths of films from grafted feathers after compression molding at 170 ◦ C for 12 min with 20% glycerol as plasticizer. Type and molar grafting ratio of monomers MA (2.7 mmol/g) [0.075]
Tensile strength (MPa)
MMA (2.8 mmol/g) [0.111]
BA (2.9 mmol/g) [0.079]
BMA (2.7 mmol/g) [0.195]
Dry
Wet
Dry
Wet
Dry
Wet
Dry
Wet
6.80 ± 0.80
0.69 ± 0.12
18.00 ± 0.8
4.31 ± 0.42
1.40 ± 0.09
0.37 ± 0.09
10.30 ± 0.32
2.17 ± 0.50
* The numbers in [] indicate the weight ratios of homopolymers to grafted feathers.
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length of alkyl ester of monomers. The films produced from feathers modified by monomers with ␣-methyl groups containing shorter alkly ester groups possessed higher strengths. However, better water stability and cytocompatibility were observed on feathers modified by monomers with ␣-methyl groups having longer carbochain of alkly ester groups. Films with the properties desired for biomedical applications can be developed by selecting appropriate monomers and grafting conditions. Grafting appears to be most convenient method to produce biomaterials from the cytocompatible and unique feather proteins. Acknowledgments This research was financially supported by Agricultural Research Division at the University of Nebraska-Lincoln, USDA Hatch Act, Multistate Research Project S1054 (NEB 37-037), AATCC student research grant and Program for Changjiang Scholars and Innovative Research Team in Jiangnan University IRT1135, Scientific Support Program of Jiangsu Province (BE2011404), the Graduate student innovation plan of Jiangsu Province (CX10B 222Z), the Doctor Candidate Foundation of Jiangnan University (JUDCF10004) and the China Scholarship Council. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] Fig. 7. Effect of molecular structures of monomers on cytocompatibility of grafted feather films. The attachment (4 h, (A)) and proliferation (4 days, (B)) of NIH 3T3 mouse fibroblast cells on feather films were evaluated by MTS assay and compared with PLA films. Cell spreading on the BA (C) and BMA (D) grafted films was observed by a confocal laser scanning microscope (cell nuclei: blue; cytoplasm: red). Significant differences among data were denoted by different letters. (For interpretation of the references to color in figure legend, the reader is referred to the web version of the article.)
[13] [14] [15] [16] [17] [18] [19] [20]
4. Conclusions Grafting of acrylic monomers is a viable approach to develop thermoplastics from feathers with the water stability and biocompatibility required for medical applications. The concentration and molecular structure of acrylic monomers could play a major role in determining the % monomer conversion and molar grafting ratio. Molar grafting ratio increased with the decrease in the
[21] [22] [23] [24] [25] [26] [27] [28]
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