International Journal of Biological Macromolecules 76 (2015) 70–79

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Dissolution and regeneration of hide powder/cellulose composite in Gemini imidazolium ionic liquid Guowei Wang a,∗,1 , Jiarong Guo b , Linghua Zhuang c,∗,1 , Yan Wang a , Bin Xu a a b c

College of Food Science and Light Industry, Nanjing Tech University, Jiangsu 211816, People’s Republic of China College of Biological and Pharmaceutical Engineering, Nanjing Tech University, Jiangsu 211816, People’s Republic of China College of Science, Nanjing Tech University, Jiangsu 211816, People’s Republic of China

a r t i c l e

i n f o

Article history: Received 27 November 2014 Received in revised form 17 February 2015 Accepted 20 February 2015 Available online 26 February 2015 Keywords: Gemini imidazolium ionic liquids White hide powder/cellulose Dissolution and regeneration Composite materials

a b s t r a c t Gemini imidazolium ionic liquid, 3,3 -[1,2-ethanediylbis (oxy-2,1-ethanediyl)]-bis[1-methylimidazolium]-dibromide ([C6 O2 (mim)2 ][Br]2 ), was used for the dissolution and regeneration of white hide powder (from pigskin), and blend white hide powder with cellulose for the easy production of white hide powder/cellulose composite. Dissolution performance of white hide powder in [C6 O2 (mim)2 ][Br]2 was studied. The native white hide powder and [C6 O2 (mim)2 ][Br]2 regenerated white hide powder were characterized by FT-IR, XRD, DSC–TG and FE-SEM. The results showed that [C6 O2 (mim)2 ][Br]2 was a good solvent to white hide powder. The dissolution time was 55 min when the white hide powder was 8% at 120 ◦ C. The dissolution time of [C6 O2 (mim)2 ][Br]2 for white hide powder was shorter than those of common ionic liquids. The triple helical structure of white hide powder was partly destroyed during [C6 O2 (mim)2 ][Br]2 dissolution. The possible mechanism of white hide powder dissolution in [C6 O2 (mim)2 ][Br]2 and the regeneration of white hide powder in methanol had been proposed. White hide powder/cellulose composites were successfully dissolved in [C6 O2 (mim)2 ][Br]2 . The performance of white hide powder/cellulose film was measured by FT-IR and TG. The tensile strength, and elongation at break of white hide powder/cellulose composite films were tested. This work demonstrated that the white hide powder/cellulose composite exhibited some potential in collagen-based tissue engineering. © 2015 Elsevier B.V. All rights reserved.

1. Introduction The increasing global awareness of environmental protection and societal concern, high rate of depletion of petroleum resources, sustainability concept, and ever more stringent environmental regulations have triggered the utilization of natural biopolymers such as cellulose, collagen and starch [1–3]. Collagen, the most abundant structural protein in nature, which accounts for approximately one-third of all proteins in all vertebrate bodies, has excited considerable research interest because of its unusual combination of excellent biological activity, nice biocompatibility, and lower immunogenicity [4–6]. Collagen is found exclusively in animals, especially in the flesh and biological

Abbreviations: XRD, X-ray diffraction; 1 HNMR, H nuclear magnetic resonance; FT-IR, Fourier transform infrared; FE-SEM, field emission scanning electron microscopy; DSC, differential scanning calorimetry; TG, thermogravimetry; [C6 O2 (mim)2 ][Br]2 , 3,3 -[1,2-ethanediylbis (oxy-2,1-ethanediyl)]-bis[1-methylimidazolium]-dibromide. ∗ Corresponding authors. Tel.: +86 25 5813 9426; fax: +86 25 5813 9426. E-mail address: [email protected] (G. Wang). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.ijbiomac.2015.02.029 0141-8130/© 2015 Elsevier B.V. All rights reserved.

tissues of mammals, including bone, tendon, teeth, skin, ligament and cartilage [7–9]. This fibrous, structural protein comprises a right-handed bundle of three parallel, left-handed helices [10]. Generally, collagen can be applied in the commercial fields of food [11], cosmetic [12], pharmaceutical and biomedical, and tissue engineering [13–16]. However, applications of collagen are limited because of the strong inter-molecular and intra-molecular hydrogen bonds, ionic bonds, van der Waals’ force and hydrophobic bonds between the polar and non-polar groups. The triple-helix conformation within collagen molecules, which is the prerequisite and foundation that supports collagen’s biological activity, also prevents it from being dissolved in ordinary solvents [17]. Furthermore, the solubility of collagen is limited in general solvents (acetic acid, 3 wt%, 1,1,1,3,3,3-hexafluoro-2-propanol, 8.3 wt%) [18,19]. Therefore, it is highly desirable to develop more friendly and more efficient solvent systems for collagen. Cellulose is the most abundant natural biopolymers on Earth and is known to have fascinating properties such as biocompatibility, desirable mechanical properties, and biodegradability [1,20]. Cellulose and its derivatives are extensively utilized in industrial fields such as textiles, plastics, coatings, cosmetics, environmentfriendly and biocompatible materials such as barrier membranes

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and scaffolds in biomedical fields [21]. The highly ordered structure of cellulose is responsible for its desirable mechanical properties but makes it a challenge to find suitable solvents for its dissolution. Some solvent systems have been developed for cellulose dissolution. In general, the traditional cellulose dissolution processes require relatively harsh conditions and the use of expensive and uncommon solvents such as N-methylmorpholine oxide (NMMO), dimethyl sulfoxide (DMSO)/tetrabutylammonium fluoride (TBAF), N,N-dimethylformamide/nitrous tetroxide (DMF/N2 O4) and N,Ndimethylacetamide/lithium chloride (DMAc/LiCl), which usually prevent them from wide industrial application[22–24]. Recently, collagen/cellulose composite has been prepared as promising biomaterials in biomedical field and biosorbent [25–27]. In the blending of collagen and cellulose, the homogeneity of collagen/cellulose composites is uncontrollable because the two biopolymers cannot dissolute easily in single co-dissolving process. The difficulty lies in the fact that a common solvent for collagen and cellulose is not easy to select, as cellulose is insoluble in water or conventional organic solvents, while collagen is instable and prone to denature in the solvent for cellulose such as N,N-dimethylacetamide/LiCl (DMAc/LiCl), NaOH/urea and N-methylmorpholine-N-oxide/H2 O (NMMO/H2 O) [23,28,29]. With the development of green chemistry and its emphasis on environmental protection, room temperature ionic liquids (ILs) have become a focus because of their unique properties such as good and tunable solubility properties, negligible vapor pressure and excellent thermal stability [30,31]. Room temperature ILs have been studied in electrochemical research [32], for catalysis and biocatalysis [33,34], as plasticizers [35], in dissolution and regeneration of some natural polymers such as cellulose fibers [22,36–42], silk fibroin fibers [43], wool keratin fibers [44], chitosan [45], starch [46,47] and collagen fibers [17,48–50]. Among these dissolution ionic liquids system, traditional single imidazolium ionic liquids such as 1-butyl-3-methylimidazolium chloride ([BMIM)]Cl), 1-ethyl-3-methylimidazolium chloride ([EMIM]Cl), and 1-allyl-3-methylimidazolium chloride ([AMIM]Cl) were used to dissolve and regenerate natural polymers [1,36,50]. There were only few papers concerning the dissolution and regeneration of collagen in ILs [17,48–50]. Geminal dicationic ILs possess superior physical properties in terms of thermal stability and volatility to those of conventional room temperature ionic liquids (ILs). Dicationic ILs are suitable for organic synthesis, ultrastable separation phases, novel lubricants and ramie cellulose pretreatment [51–53]. To the best of our knowledge, geminal dicationic ILs have not been applied for the dissolution and regeneration of collagen fibers from white hide powder. In this paper, a Gemini imidazolium ionic liquid, 3,3 -[1,2ethanediylbis (oxy-2,1-ethanediyl)]-bis[1-methylimidazolium]dichloride ([C6 O2 (mim)2 ][Br]2 ), was applied for the dissolution and regeneration of white hide powder, and blend white hide powder with cellulose for the easy production of white hide powder/cellulose composite. Dissolution performance of white hide powder in [C6 O2 (mim)2 ][Br]2 was determined. The raw and regenerated white hide powders were characterized by Fourier transform infrared (FT-IR) spectroscopy, X-ray diffraction (XRD), differential scanning calorimetry and thermogravimetry analyses (DSC–TG), and field emission scanning electron micrographs (FESEM). The possible mechanism of white hide powder dissolution in [C6 O2 (mim)2 ][Br]2 and the regeneration in precipitators have been proposed. Also, white hide powder/cellulose composite materials were made by [C6 O2 (mim)2 ][Br]2 and the performance of white hide powder/cellulose composite materials was measured by infrared spectroscopy (FT-IR), and thermogravimetric analysis (TG). Mechanical behavior (including tensile strength, and

71 e

e

e

b

N

H3C N c

a

O

d

a

d

O

e

N

N

CH3 c

f b

f

2 Br

Fig. 1. Chemical structure of [C6 O2 (mim)2 ][Br]2 .

elongation at break) of white hide powder/cellulose composite films was tested. 2. Experimental 2.1. Materials Chrome free white hide powder from pig skin, obtained from Institute of Chemical Industry of Forest Products (Nanjing Forestry University, Nanjing, Jiangsu), was used without any pretreatment. Microcrystalline cellulose (MCC) was purchased from Sinopharm Chemical Reagent Co., Ltd. Ethanol, methanol, and other organic and inorganic compounds (all purchased from Shanghai Aladdin Industrial Chemical Co. Ltd) were all A.R grade and used without further purification. Water used in this study was double distilled. [C6 O2 (mim)2 ][Br]2 (viscous light yellow oil), as shown in Fig. 1, was synthesized according to our previous papers with the Nmethylimidazole and triethylene glycol as raw materials [54–56]. 2.2. Dissolution of white hide powder in [C6 O2 (mim)2 ][Br]2 5 mL dried [C6 O2 (mim)2 ][Br]2 was added to a 20 mL test tube. Then, the tube was immersed in an oil bath (type DF-101S, Gongyi Instrument Factory, China) with initial temperatures of 60 ◦ C, 80 ◦ C, 100 ◦ C and 120 ◦ C. After that, the test tube was stirred magnetically for 10 min to ensure the designing temperature of the [C6 O2 (mim)2 ][Br]2 . Then the white hide powder specimen was added to the tube with the mass fraction of 2%, 4%, 6%, and 8%. The whole dissolving process of white hide powder in [C6 O2 (mim)2 ][Br]2 was determined under a polarization microscope (Nikon LV100N, Nikon, Japan). The dissolution time of white hide powder in [C6 O2 (mim)2 ][Br]2 at the given temperature (60, 80, 100, and 120 ◦ C) was determined. Solubility experiments were conducted at least three times to check the reproducibility, and the mean values were considered as the measured results. 2.3. Regeneration of white hide powder specimens After the complete dissolution of white hide powder in the [C6 O2 (mim)2 ][Br]2 , it was convenient to prepare the regenerated white hide powder specimens (names as Re-Colla) with methanol. The blended white hide powder/[C6 O2 (mim)2 ][Br]2 solutions were transferred to a 100 mL beaker containing 30 mL methanol. Subsequently, the beaker was stirred for 1 h at room temperature. After that, the precipitated white hide powder was separated by centrifugation. The Re-Colla white hide powder specimen was thoroughly washed with methanol and double distilled water at least three times to remove residual [C6 O2 (mim)2 ][Br]2 . Finally, the white hide powder specimen was dried in vacuo at 25 ◦ C for 6 h and the ReColla was obtained as powder. 2.4. Preparation of white hide powder/cellulose films 10 mL dried [C6 O2 (mim)2 ][Br]2 was added to a 20 mL test tube. Then, the tube was immersed in an oil bath (type DF-101S, Gongyi Instrument Factory, China) with initial temperature of 100 ◦ C. After

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that, the test tube was stirred magnetically for 10 min to ensure the designing temperature of the [C6 O2 (mim)2 ][Br]2 . Then the white hide powder/cellulose specimens were added to the tube with the mass fraction of 2%. The different ratio of added white hide powder/cellulose was 5:1, 2:1, 1:1, 1:2 and 1:5. The whole dissolving process of white hide powder/cellulose specimens in [C6 O2 (mim)2 ][Br]2 was determined under a polarization microscope (Nikon LV100N, Nikon, Japan). White hide powder/cellulose films were obtained by casting the white hide powder/cellulose/IL solution on a PTFE plate and soaking in methanol at a constant temperature 15 ◦ C. 2.5. Characterization of regenerated white hide powder specimens (Re-Colla) 2.5.1. FT-IR detection for white hide powder specimens FT-IR spectra of raw white hide powder and Re-Colla in KBr pellets were obtained with Nicolet AUV360 spectrometer (Thermo Fisher Scientific Inc. USA). Approximately 2 mg sample was mixed with 200 mg KBr (spectroscopic grade) and FT-IR spectra were recorded in the range of 400–4000 cm−1 at a resolution of 4 cm−1 with 32 scans per sample. The raw and regenerated white hide powder structure was mainly determined by the following typical bands spectra: amide A band (3300–3325 cm−1 ), amide B band (3080 cm−1 ), amide I band (1650 cm−1 ) and amide II band (1550 cm−1 ). 2.5.2. XRD detection for white hide powder specimens XRD patterns of raw white hide powder and Re-Colla were recorded from 2 = 5◦ to 50◦ with Bruker D8 Advance XRD diffractometer (Bruker Co. Ltd., Germany) equipped with a graphite monochromator and Cu K␣ radiation at  = 0.154 nm (50 kV, 40 mA). 2.5.3. DSC–TG detection for white hide powder specimens Differential scanning calorimetry (DSC) and thermogravimetry (TG) were performed on Netzsch STA 449F3 thermogravimetric analyzer (Netzsch Geratebam GmbH, Germany) over a temperature range of 40–500 ◦ C at a heating rate of 10 ◦ C/min in nitrogen atmosphere. 2.5.4. SEM detection for white hide powder specimens FE-SEM photomicrographs were recorded with Ultra 55 field emission scanning electron microscope (Carl Zeiss SMT Pte Ltd, Germany) at an accelerating voltage of 20 kV. The free surfaces of native white hide powder and Re-Colla were coated with thin layers of gold before observation. 2.5.5. Recycling of [C6 O2 (mim)2 ][Br]2 The residual [C6 O2 (mim)2 ][Br]2 was recovered by simply reducing the pressure and subsequently removing methanol and water. 2.6. Characterization of white hide powder/cellulose films 2.6.1. FT-IR detection for white hide powder/cellulose FT-IR spectra of white hide powder/cellulose films in KBr pellets were obtained with Nicolet AUV360 spectrometer (Thermo Fisher Scientific Inc., USA). Approximately 2 mg sample was mixed with 200 mg KBr (spectroscopic grade) and FT-IR spectra were recorded in the range of 400–4000 cm−1 at a resolution of 4 cm−1 with 32 scans per sample. 2.6.2. TG of white hide powder/cellulose Thermogravimetry (TG) was performed on Netzsch STA 449F3 thermogravimetric analyzer (Netzsch Geratebam GmbH, Germany)

Table 1 Dissolution time of white hide powder in the [C6 O2 (mim)2 ][Br]2 (min). Mass fraction 2% 4% 6% 8%

60 ◦ C

80 ◦ C

100 ◦ C

120 ◦ C

180 220 280 320

120 135 140 180

80 100 120 130

20 35 42 55

over a temperature range of 40–500 ◦ C at a heating rate of 10 ◦ C/min in nitrogen atmosphere. 2.6.3. Mechanical tests of white hide powder/cellulose The mechanical behavior (including tensile strength, and elongation at break) of white hide powder/cellulose composite films was evaluated using a universal materials testing machine (model 350-9511, Testometric, Rochdale, England) according to GB/T10402006 (Plastics – Determination of tensile properties for films and sheets). Standardized strip specimens (20 mm × 100 mm) were cut from the films and stretched at room temperature with a constant speed of 5 mm/min. The accurate width and thickness of each sample were determined using a slide caliber. The tensile strength (TS , MPa) was calculated by the following equation: TS =

F S

(1)

where F was the maximum tension energy (Newton) when the sample was broken; S was sample area (mm2 ). The elongation at break (E, %) was calculated by the following equation E (%) =

L − L0 × 100 L0

(2)

where L0 was the sample length (mm); L was the sample length (mm) when sample was broken. Each sample was tested five times and the average value was used. 3. Results and discussion 3.1. Dissolution performance of white hide powder in [C6 O2 (mim)2 ][Br]2 The dissolution time of white hide powder in [C6 O2 (mim)2 ][Br]2 was investigated at different temperatures (60, 80, 100, and 120 ◦ C). Table 1 shows that the dissolution time of white hide powder in the [C6 O2 (mim)2 ][Br]2 was affected by different white hide powder mass fraction. It can be seen that the dissolution time of white hide powder in the [C6 O2 (mim)2 ][Br]2 decreased with the increase of temperature and the decrease of the white hide powder mass fraction. Also at low temperature, white hide powder had lower dissolved rate, but the dissolved rate increased significantly at higher temperature. The reason was that when the temperature was low, the energy was low, the viscosity of ionic liquids was larger, and the solution of white hide powder and ionic liquids was difficult to spread evenly, so the intramolecular and intermolecular hydrogen bonds of white hide powder could not be broken fast. Thus white hide powder was unable to dissolve quickly in ionic liquids solution. When the temperature reached a certain degree, the energy was higher, so the ionic liquid was disintegrated into free zwitterion ion rapidly. In this dissolution system, the negative ion of [C6 O2 (mim)2 ][Br]2 acted as electron donor, which interacted with the hydrogen atom of N H group or CONH group in white hide powder, and the positive ion acted as electron acceptor which interacted with the oxygen atom in white hide powder ( CONH , or free carboxyl group). So the dissolution time greatly reduced. But

G. Wang et al. / International Journal of Biological Macromolecules 76 (2015) 70–79

Fig. 2. FT-IR spectra of white hide powder specimens. (a) Native white hide powder, (b) Re-Colla-60 ◦ C, (c) Re-Colla-80 ◦ C, (d) Re-Colla-100 ◦ C, and (e) Re-Colla-120 ◦ C.

the dissolving temperature cannot be higher than 140 ◦ C because the ionic liquid began to oxidize and the collagen was degraded when the temperature was higher than 140 ◦ C. The dissolution time of collagen fibers in common ionic liquids is listed in Table 2. From Tables 1 and 2, it can be seen that [C6 O2 (mim)2 ][Br]2 had a great advantage in dissolving white hide powder compared to common ionic liquids. The dissolving time was greatly decreased using [C6 O2 (mim)2 ][Br]2 . As we all know, the solubility of white hide powder mainly relied on the structure of the solvent. The two imidazolium rings and more C O C group in main chain of [C6 O2 (mim)2 ][Br]2 resulted in higher capacity for destroying intramolecular and intermolecular hydrogen bonds in white hide powder. 3.2. Structural characterization of white hide powder specimens 3.2.1. FT-IR of white hide powder specimens FT-IR spectra of native white hide powder and Re-Colla samples at different temperatures are shown in Fig. 2. The typical amide bands including amide A (3300–3325 cm−1 ), amide B (3080 cm−1 ), amide I (1650 cm−1 ), amide II (1550 cm−1 ) and amide III (1240 cm−1 ) of Re-Colla samples were consistent with those of native white hide powder [4,17,57,58]. The similar spectral profiles indicated similar structures of the native white hide powder and Re-Colla samples. However, on close examination of the spectra, differences were clearly identified. Amide A bands of Re-Colla samples (Fig. 2b–e) appeared to red shift and become smoother and broader comparing with that of native white hide powder sample. This might result from the partial disruption of hydrogen bonds within collagen molecules, resulting from the interaction between collagen and ionic liquid molecules. Also, FT-IR spectra of Re-Colla samples (amide I and amide II bands) became broad and flat comparing with those of native white hide powder. Deconvolution of the amide I and II regions (wavenumbers 1500–1700 cm−1 ) is often used to understand the qualitative and quantitative contributions of different structural components of collagen [4,17]. In order to assess the quantitative contribution of the different structures to amide I and II bands, eight components of the amide I and amide II bands were determined by peak deconvolution, as presented in Fig. 3. Native collagen skin fibers possessed these band components centered at 1512 cm−1 , 1540 cm−1 , 1564 cm−1 , 1594 cm−1 , 1606 cm−1 , 1626 cm−1 , 1660 cm−1 and 1694 cm−1 , respectively. The band position and assignment are listed in Table 3. The band areas of helical

73

I/coil I and helical (I + II)/coil (I + II) are summarized in Table 4. The native white hide powder possessed intensive bands at 1660 cm−1 (amide I in helical form, band area was 7.94) and 1564 cm−1 (amide II in helical form, band area was 2.35). The band areas of amide I in helical form and amide II in helical form of Re-Colla decreased dramatically when the treating temperature increased from 60 ◦ C to 120 ◦ C (from 7.94 to 0.71 for amide I helix, from 2.35 to 0.0032 for amide II helix). The decrease of band areas indicated that white hide powder might undergo degradation during dissolution of [C6 O2 (mim)2 ][Br]2 and methanol regeneration. As we know, the absorbance peak at 1240 cm−1 had an effect on the changes of the triple-helical structure of collagen. FT-IR absorption ratios of amide III (1240 cm−1 ) to 1450 cm−1 was denoted as A1240 /A1450 which could be used to quantify the denaturation of the white hide powder before and after dissolution in the IL solvent system [17,58–62]. Table 5 shows that the ratio of the native white hide powder and Re-Colla at different temperatures decreased slightly from 1.00 (native white hide powder) to 0.971 (Re-Colla-120 ◦ C). The results showed that the triple-helical conformation of Re-Colla was slightly destroyed during dissolution in the [C6 O2 (mim)2 ][Br]2 .

3.2.2. TG and DSC characterization of white hide powder specimens TG curves of native white hide powder and Re-Colla in different temperatures are shown in Fig. 4. As for native white hide powder (Fig. 4a), there were three visible thermal weight loss periods: 40–110 ◦ C, 210–240 ◦ C, and 280–350 ◦ C. The first stage mainly relied on the heat evaporation of water molecule and other low weight molecules. The weight loss was about 5.28% at 67 ◦ C. The weight loss was about 10.55% at 110 ◦ C. The weight loss became not so obvious, and appeared to be stable when the temperatures were continuously increased. White hide powder was likely to gelatinize during 210–240 ◦ C. The weight loss was about 12.61% at 231 ◦ C. Apparent weight loss appeared when temperature was increased to 280 ◦ C (the weight loss was 19.22%). There occurred the degradation of the peptide bonds at this stage. And the gradual degradation of peptide and amino acid eventually led to the deamination and dehydration. There existed no significant difference between TG curve of native white hide powder and Re-Colla curves (Fig. 4b–e). However, on close analysis of the curves, differences were clearly identified. Re-Colla (Fig. 4b–e) was more stable than native white hide powder at the first stage (40–110 ◦ C) and third stage (280–350 ◦ C). Their weight losses were lower than that of native white hide powder. DSC curves of native white hide powder and Re-Colla in different temperature are shown in Fig. 5. As for native white hide powder (Fig. 5a), there were three visible endothermic processes: 40–110 ◦ C, 210–240 ◦ C, and 280–350 ◦ C and three absorption peaks: 67 ◦ C, 231 ◦ C, and 287 ◦ C. The first endothermic process meant the evaporation of water molecule and other low weight molecules. Endothermic process from 210 ◦ C to 240 ◦ C was the gelatinization of white hide powder. Another strong endothermic process was around 280–350 ◦ C, which meant the decomposing of white hide powder molecules. The fracture of collagen peptide bonds and the gradual degradation of peptide and amino acid eventually led to the deamination and dehydration. On further analysis of the curves, differences of native white hide powder and Re-Colla were clearly identified. The endothermic energy of Re-Colla (Fig. 5b–e) was lower than native white hide powder at the first stage. And the first and third absorption peaks of Re-Colla samples shifted to the right. The phenomenon was consistent with TG analysis, which relied on the fact that collagen molecule chain was rebuilt after regeneration. The number and position of hydrogen bonds in collagen molecules were increased after regeneration.

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Table 2 Dissolution time of collagen fibers in other ionic liquids. Ionic liquids

Temperature (◦ C)

Mass fraction (%)

Dissolution time (min)

Refs.

[BMIM]Cl [EMIM]Ac [EMIM]Ac/Na2 HPO4 [EMIM]Cl [DMIM]Cl

100 45 45 35 35

6 7.4 10.5 Not mentioned Not mentioned

360 1440 1440 1440 1440

[17] [48] [48] [49] [49]

3.2.3. XRD characterization of collagen specimens The structure of the native white hide powder and Re-Colla at different temperature was examined with X-ray diffraction (XRD), as shown in Fig. 6.

The native white hide powder (Fig. 6a) showed three diffraction peaks (2 = 7.44◦ , 21.82◦ , 30.84◦ ). The first sharp peak at 2 = 7.44◦ indicated a lateral intermolecular packing distance between the white hide powder molecular chains. The second broad peak at

Fig. 3. FT-IR spectra (range amide I and amide II bands) of white hide powder specimens. (a) Native white hide powder, (b) Re-Colla-60 ◦ C, (c) Re-Colla-80 ◦ C, (d) Re-Colla100 ◦ C, and (e) Re-Colla-120 ◦ C.

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75

Table 3 FT-IR spectra peak position and assignments for native white hide powder and regenerated white hide powder. Region

Peak wave number (cm−1 ) White hide powder

Assignment Regenerated white hide powder (with methanol) 60 ◦ C

80 ◦ C

100 ◦ C

120 ◦ C

Amide I

1694 1660 1626 1606

1692 1660 1629 1608

1700 1649 1627 1605

1693 1649 1628 1605

1685 1641 1628 1607

C O Amide I in helical form Amide I in random coil form ␤-Turn

Amide II

1594 1564 1540 1512

– 1572 1544 1513

1565 1546 1532 1516

1564 1548 1536 1520

1570 1556 1540 1510

COO− Amide II in helical form Amide II in random coil form Tyrosine side chain

Amide III

1239

1237

1239

1232

1242

N H bending

Table 4 Changes of the helical structure of native white hide powder and regenerated white hide powder. Attribution

Area of band Native white hide powder

Helical I Coil I Helical II Coil II Helical I/coil I Helical (I + II)/coil (I + II)

7.94 2.15 2.35 3.16 3.69 1.94

Regenerated white hide powder (with methanol) 60 ◦ C

80 ◦ C

100 ◦ C

120 ◦ C

1.80 0.10 0.73 1.39 18.0 1.70

3.32 0.093 0.017 0.015 35.70 30.90

1.51 0.027 0.043 0.034 55.93 25.46

0.71 0.030 0.0032 1.42 23.67 0.49

Table 5 FT-IR absorption ratios of A1240 to A1450 for white hide powder specimens before and after dissolution in the [C6 O2 (mim)2 ][Br]2 solvent system.

A1240 /A1450

Native white hide powder

Re-Colla-60 ◦ C

Re-Colla-80 ◦ C

Re-Colla-100 ◦ C

Re-Colla-120 ◦ C

1.00

0.986

0.980

0.972

0.971

2 = 21.82◦ indicated that the amorphous scatter resulted from unordered components of white hide powder. And the third peak at 2 = 30.84◦ corresponded to the unit height, typical of the triple helical structure [63,64]. XRD of Re-Colla at different temperatures (60 ◦ C, 80 ◦ C, 100 ◦ C, and 120 ◦ C) is shown in Fig. 6b–e. Comparing to Fig. 6a, the peak at 2 = 7.44◦ and 2 = 30.84◦ of Fig. 6b–e disappeared. This resulted from the disruption of intermolecular hydrogen bonds between collagen helix, and also meant that the triple helical structure of collagen was partly broken into random

coil form during the [C6 O2 (mim)2 ][Br]2 dissolution and regeneration. Besides, the disappearance of the peak at 2 = 30.84◦ implied that the triple helical structure of collagen was disrupted during [C6 O2 (mim)2 ][Br]2 dissolution and regeneration.

Fig. 4. TG of white hide powder specimens. (a) Native white hide powder, (b) ReColla-60 ◦ C, (c) Re-Colla-80 ◦ C, (d) Re-Colla-100 ◦ C, and (e) Re-Colla-120 ◦ C.

Fig. 5. DSC of white hide powder specimens. (a) Native white hide powder, (b) Re-Colla-60 ◦ C, (c) Re-Colla-80 ◦ C, (d) Re-Colla-100 ◦ C, and (e) Re-Colla-120 ◦ C.

3.2.4. SEM surface morphology of white hide powder specimens SEM surface morphology of native white hide powder and ReColla at 80 ◦ C is shown in Fig. 7. White hide powder is precipitated from the [C6 O2 (mim)2 ][Br]2 solution by the addition of methanol.

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acted as the electron acceptor center. These two kinds of ions were completely free. In the system, the negative ion interacted with the hydrogen atom of N-H group in white hide powder, and the positive ion interacted with the oxygen atom of CONH group. So the intermolecular hydrogen bonds of white hide powder were destroyed, the white hide powder molecules chain spread, and eventually resulted in the dissolution of white hide powder in ILs. During regeneration process, the zwitterion ions of ionic liquid which combined with white hide powder were washed by precipitant, collagen peptide chain moved closely to each other, and the hydrogen bonds between collagen molecule chain were rebuilt. But after regeneration, the relative position of collagen molecule chain was changed; the number and position of hydrogen bonds were different from those of native white hide powder molecular chain. 3.4. Characterization of white hide powder/cellulose films 3.4.1. Photographs of white hide powder/cellulose films Photographs of different proportions of white hide powder/cellulose films are shown in Fig. 9. As we all know, collagen has good film-forming ability and lower hardness, and cellulose has better hardness and lower film-forming ability. So to make better white hide powder/cellulose films, the proportion of white hide powder and cellulose should be established. From Fig. 9 we could see that the white hide powder/cellulose composite materials were gradually strengthened with the increase of the content of white hide powder/cellulose, and when the content of cellulose was higher than that of white hide powder/cellulose, the white hide powder/cellulose composites had poor film-forming ability. In conclusion, when the compound ratio was 2:1 to 1:2 (white hide powder to cellulose), the properties of white hide powder/cellulose films were acceptable. Fig. 6. XRD diagrams of the white hide powder specimens. (a) Native white hide powder, (b) Re-Colla-60 ◦ C, (c) Re-Colla-80 ◦ C, (d) Re-Colla-100 ◦ C, and (e) Re-Colla120 ◦ C.

The native white hide powder (Fig. 7a) had a width of about 10–15 ␮m and length from hundreds of microns to millimeters. The film regenerated in methanol had smooth morphology (Fig. 7b). It was seen that the morphology of regenerated white hide powder films was very different from the native white hide powder. 3.3. Possible dissolution and regeneration mechanisms of white hide powder in [C6 O2 (mim)2 ][Br]2 Dissolution and regeneration mechanism of white hide powder in [C6 O2 (mim)2 ][Br]2 are shown in Fig. 8. The bromine ions in [C6 O2 (mim)2 ][Br]2 acted as the electron donor and [C6 O2 (mim)2 ]2+

3.4.2. FT-IR of white hide powder/cellulose films FT-IR spectra of native white hide powder, microcrystalline cellulose (MCC) and a series of white hide powder/cellulose films are shown in Fig. 10. As discussed in Section 3.2.1, native white hide powder (Fig. 10a) exhibited typical amide bands, including amide A, amide B, amide I, amide II and amide III. MCC (Fig. 10b) showed characteristic absorption peaks at 3420, 1640, 1429, 1316, 1056, and 1034 cm−1 , which arose from the aliphatic acid chain ascribed to C O and O C O stretching vibrations. Comparing with native white hide powder, MCC and white hide powder/cellulose films (Fig. 10c–g) presented higher transmittance. The band around 3300 cm−1 of white hide powder/cellulose films shifted to higher wavenumber (about 3420 cm−1 ), which were characteristic bands of MCC. Absorbance peaks of white hide powder/cellulose films increased with the rising content of MCC, especially for bands at 3300 cm−1 , 1056 cm−1 , and 1034 cm−1 . Comparing with the spectra

Fig. 7. SEM surface morphology of the white hide powder samples. (a) Native white hide powder and (b) Re-Colla-80 ◦ C.

G. Wang et al. / International Journal of Biological Macromolecules 76 (2015) 70–79

O O H

N

C

H

C

R

N

H

O

O

O

R

C

H

H

N

O

R

H

C

O [C6O 2(mim)2][Br]2 H

C

C

O

H

R

N C C

O

Heat

C

R Br-

N

H

R

C

H

H

N

H

H

C

R

O

C

O O H

Br

H

N

R

C

R

Regenerant

N

H

Rebuilt

O

C

O

H O

N

H

H

C

R

O

C

C C

2+

-

O

N

[C6O2(mim)2]2+ O

O [C6O2(mim)2] C

H O

C

77

C

O

R

C

H

H

N

O H

N

R

C C

H O

O

Fig. 8. Dissolution and regeneration mechanism of white hide powder in [C6 O2 (mim)2 ][Br]2 .

of white hide powder and CMC, the band of white hide powder/cellulose film (1:1, Fig. 10e) was shifted to 1658 cm−1 with stronger absorbance peak, and this shift might attribute to strong interactions between the hydroxyl groups of CMC and the carboxyl groups of white hide powder. Moreover, the spectrum of white hide powder exhibited characteristic amide I (1650 cm−1 ) and amide II (1550 cm−1 ), while just one peak was present at 1658 cm−1 in the white hide powder/cellulose films, which might ascribe to the strong intermolecular hydrogen bonds between the OH and the carbonyl groups, or peptide bonds of white hide powder, and the overlap of amide I and amide II bands. 3.4.3. TG of white hide powder/cellulose films TG of white hide powder/cellulose films with different proportion is shown in Fig. 11. As shown in Fig. 11, all samples exhibited two visible thermal weight loss periods: 50–130 ◦ C and 250–360 ◦ C. The first stage mainly relied on the heat evaporation of water molecule and other low weight molecules. The initial thermal degradation of the white hide powder occurred at a low temperature, whereas the onset degradation temperature for white hide

powder/cellulose composite films was significantly higher. Also, the weight loss of composite films increased with the rising content of CMC at this stage. When the temperature was less than 250 ◦ C, the white hide powder/cellulose composites were stable (the weight loss was about 5.16–12.3%). The TG curve continuously declined with the rising temperature. A second, sharper weight loss occurred from 250 ◦ C to 360 ◦ C, which corresponded to the thermal decomposition process of the respective films. At this stage, TG behaviors of composite films differed dramatically (Fig. 11a–e). The onset degradation temperature for white hide powder/cellulose composite films decreased with the increase of CMC content, while the weight loss of composite films decreased with the rising content of CMC. When the temperature was above 450 ◦ C, the weight loss became not so obvious and tended to be stable. As we know, white hide powder consisted of polypeptide chains (hydroxyproline). These chains contain many carboxyl groups, peptide bonds and other polar groups, which can form hydrogen bonds with OH groups in CMC. CMC have a large number of OH groups. The long chains of CMC can wind around the triple helix of

Fig. 9. Photographs of different proportions of white hide powder/cellulose films. (a) 5:1, (b) 2:1, (c) 1:1, (d) 1:2, and (e) 1:5

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Table 6 Mechanical strength of white hide powder/cellulose composite films. White hide powder

Tensile strength (MPa) Elongation at break (%)

1.62 150

White hide powder/cellulose 5:1

2:1

1:1

1:2

1:5

2.34 186

2.58 214

2.20 178

2.04 120

– –

Fig. 10. FT-IR of white hide powder/cellulose films with different proportions. (a) Native white hide powder, (b) MCC, (c) 5:1, (d) 2:1, (e) 1:1, (f) 1:2, and (g) 1:5.

The tensile strength of native white hide powder was 1.62 MPa and the tensile strength of white hide powder/cellulose composite films increased with the rising content of MCC (5:1 to 2:1, from 2.34 MPa to 2.58 MPa respectively). When MCC content continued to grow, the tensile strength decreased (1:1 to 1:2, from 2.20 to 2.04 respectively). When the content proportion of white hide powder to cellulose was 1:5, the film was brittle and could not form a film. Also, the elongation at break of white hide powder/cellulose composite films increased with the increase of MCC content (5:1 to 2:1, from 186% to 214% respectively). When MCC content continued to grow, the elongation at break decreased dramatically (1:1 to 1:2, from 178% to 120% respectively). And, when the content ratio of white hide powder to cellulose was 1:5, the film was brittle and could not form a film. White hide powder consisted of triple helix (including carboxyl groups, peptide bonds and other polar groups). MCC have a large number of OH groups. When white hide powder was mixed with MCC, the carboxyl groups, peptide bonds and other polar groups in triple helix will form large number of intermolecular hydrogen bonds with OH groups in MCC. The entanglement of two biomacromolecules may form a complex with much higher tensile strength and elongation ratio than either of the single components.

4. Conclusions

Fig. 11. TG of white hide powder/cellulose films. (a) 5:1, (b) 2:1, (c) 1:1, (d) 1:2, and (e) 1:5.

white hide powder, and the entanglement of two different macromolecules may form a rigid matrix with much greater strength, leading to space maintenance and improved thermal stability of the composite films. TG observations provide further evidence of interactions between white hide powder and CMC, and higher thermal stability of composite films. 3.4.4. Mechanical tests of white hide powder/cellulose Mechanical behavior (including tensile strength, and elongation at break) of white hide powder/cellulose composite films is listed in Table 6. As shown in Table 6, the tensile strength of white hide powder/cellulose composite films was higher than that of native white hide powder.

In this paper, a Gemini imidazolium ionic liquid, 3,3 -[1,2ethanediylbis (oxy-2,1-ethanediyl)]-bis[1-methyl-imidazolium]dibromide ([C6 O2 (mim)2 ][Br]2 ), was applied for the dissolution and regeneration of white hide powder (from pig skin), and blend white hide powder with cellulose for the production of white hide powder/cellulose composite. The native white hide powder and [C6 O2 (mim)2 ][Br]2 regenerated white hide powder were characterized by FT-IR, XRD, DSC–TG and FE-SEM. The results showed that [C6 O2 (mim)2 ][Br]2 was a good solvent to white hide powder. White hide powder could be regenerated from the [C6 O2 (mim)2 ][Br]2 solvent system by precipitator such as methanol. The dissolution time of white hide powder in [C6 O2 (mim)2 ][Br]2 solvent system was significantly affected by the temperature. The dissolution time was 55 min, when the white hide powder content was 8% at 120 ◦ C. The dissolution time of [C6 O2 (mim)2 ][Br]2 to white hide powder was shorter than those of common ionic liquids. The triple helical structure of white hide powder was partly destroyed during [C6 O2 (mim)2 ][Br]2 dissolution. The possible mechanisms of dissolving of white hide powder in [C6 O2 (mim)2 ][Br]2 and the regeneration of white hide powder by methanol were proposed. Also, white hide powder/cellulose composite materials were dissolved in [C6 O2 (mim)2 ][Br]2 . And performance of white hide powder/cellulose film was measured by FT-IR and TG. Mechanical behavior (including tensile strength, and elongation at break) of white hide powder/cellulose composite films was tested. The tensile strength and elongation at break of white hide powder/cellulose composite films increased with the rising content of MCC. This work demonstrated that the white hide powder/cellulose composite films exhibited some potential in the field of collagenbased tissue engineering.

G. Wang et al. / International Journal of Biological Macromolecules 76 (2015) 70–79

Acknowledgments This work was supported by Natural Science Foundation of Jiangsu Province (No. BK2011799 and No. 20140939) and the specialized research fund for the Doctoral Program of Higher Education of China (No. 20113221120006). The authors also gratefully appreciate the support from Jiangsu Overseas Research & Training Program for University Prominent Young & Middle-aged Teachers and Presidents and Jiangsu Students Innovation and Entrepreneurship Training Program (No. 2012JSSPITP3054 and No. 201413905004Y).

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