HAc as novel binary solvent

Journal of Molecular Liquids 291 (2019) 111304

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Journal of Molecular Liquids journal homepage: www.elsevier.com/locate/molliq

Fabrication of highly concentrated collagens using cooled urea/HAc as novel binary solvent Qili Yang a, Chenchen Guo a, Feng Deng a, Cuicui Ding b, Junhui Yang a, Hui Wu a, Yonghao Ni a, Liulian Huang a, Lihui Chen a, Min Zhang a,⁎ a b

College of Materials Engineering, Fujian Agriculture and Forestry University, Fuzhou 350002, PR China College of Ecological Environment and Urban Construction, Fujian University of Technology, Fuzhou 350108, PR China

a r t i c l e

i n f o

Article history: Received 25 December 2018 Received in revised form 27 May 2019 Accepted 2 July 2019 Available online 03 July 2019 Keywords: Concentrated collagen Liquid crystalline Denaturation Urea Dissolution Rheological property

a b s t r a c t It is difficult to prepare homogeneous concentrated collagen with concentration N 40 mg/mL due to its extremely high viscosity. Herein, cooled (−12 °C) urea/HAc solutions was employed as novel solvent to prepare collagen samples with concentrations varied from 40 to 120 mg/mL. Fourier transform infrared spectroscopy and sodium dodecyl sulfonate-polyacrylamide gel electrophoresis demonstrated that the concentrated collagen maintained the intact triple-helical structure. As reflected by differential scanning calorimetry, collagen with acetic acid as solvent displayed two thermal transition peaks when concentration ≥ 60 mg/mL, which could be attributed to the denaturation of dissolved collagen and un-dissolved collagen. Whereas, collagen prepared in cooled urea/ HAc just exhibited one thermal denaturation peak other than samples with concentration ≥ 100 mg/mL. Images from polarizing optical microscopy displayed that the cholesteric band grew more pronounced upon increasing collagen concentration. Field-emission scanning electron microscopy exhibited more aligned topographical features of the collagen sponges when increasing concentration from 10 to 100 mg/mL, nevertheless both of the aligned structure and anomalous structure could be captured when collagen concentration further reached 120 mg/mL. Rheological properties were found to be dependent on the collagen concentration, additionally, compared with collagen in acetic acid, a weaker entanglement network and lower viscosity for collagen with the same concentration using cooled urea/HAc as solvent could be reflected by the rheological measurements. Overall, this method presents a simple mean for generating homogeneous concentrated collagens, which can be applied to wider fields such as wet spin and biomimetic mineralization. © 2019 Elsevier B.V. All rights reserved.

1. Introduction Collagen, which is the major protein in animals, accounting for approximately 25% of the total dry protein and distributes in connective tissues such as skins, bone, cartilage, tendons, vitreous and sclera [1]. Up to now, at least 28 different types of collagen, named type I– XXVIII, have been discovered and reported by researchers [2]. The most abundant collagen among those is type I collagen, which is a right-handed triple superhelical rod consisting of three polypeptide chains [3]. Owing to its excellent biodegradability, weak antigenicity and superior biocompatibility, type I collagen has been widely applied in fields including cosmetics, medicines, foods and chemical industries [4–7]. In some situations, collagen processing involves liquid aqueous preparations. For instance collagen were used for medical injection for

⁎ Corresponding author. E-mail address: [email protected] (M. Zhang).

https://doi.org/10.1016/j.molliq.2019.111304 0167-7322/© 2019 Elsevier B.V. All rights reserved.

the repair of dermatological defects or used to be drug carriers [8]. More often, collagen could also be transferred into products in the formats of gels, sheets, tubes, fibers, powders, sponges and membranes [9]. It should be pointed out that in most cases collagen concentration is relatively low (lower than 20 mg/mL), due to the high viscosity of collagen solution in the traditional solvent, primarily non-strong acids such as dilute acetic acid (HAc) and phosphoric acid (H3PO4) [10–12]. This limits its application in some aspects such as wet spin and biomimetic mineralization [13,14], which require highly concentration of collagen to maintain the stability of process. In recent years, some methods have been proposed for the preparation of highly concentrated collagen. It was reported that using 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and 2,2,2-trifluoroethanol (TFE) as solvents increased the collagen concentration (the maximum concentration was 100 mg/mL) [15,16]. Nevertheless, HFIP and TFE are corrosive, resulting in a significant loss of collagen triple helix structure [17]. Ionic liquids were also considered as a potential solvent to obtain collagen with higher concentration. For instance, Meng et al. used 1-butyl-3-methylimidazolium chloride to dissolve collagen at 100 °C

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to achieve a solubility up to 6% (~60 mg/mL) [18], however, this method also overlooked the preservation of collagen triple helix under the high temperature [19]. Giraud-Guille's group proposed that highly concentrated collagen could be obtained by slowly evaporating the ultrasonicated collagen solution under vacuum [20–22], but this method resulted in uneven collagen concentration at different locations from the exterior to the interior, and it was time-consuming and difficult to operate. As a whole, many efforts trying to obtain concentrated collagen in various solvents have been made, but to develop an appropriate method by which high concentration of collagen could be achieved without damaging the native collagen structure is still a crucial issue [23]. The facile solvent pre-cooled NaOH/urea for cellulose dissolution brought forward by Zhang's group sheds some light on the preparation of concentrated collagens. As reported in the relevant works [24,25], urea forms hydrogen bonding with the hydroxyl group of cellulose molecules, reducing the self-association of cellulose chains; meanwhile the application of low temperature (−5 to −20 °C) strengthens the hydrogen bonding between urea and cellulose [24]. As a consequence, the enhanced hydrogen bond breaks the intra-/intramolecular hydrogen bonds of the cellulose, promoting the complete dissolution of the cellulose, which is insoluble in the neat NaOH solution. As is well known, the mechanism of collagen dissolution in acetic acid is that hydrogen bonding between the hydrophilic groups such as –NH2, -COOH, and -OH on the side chains of collagen macromolecules was destroyed. Therefore, inspired by this dissolving mechanism of cellulose, we adopted cooled urea/acetic acid as a novel solvent for collagen in this study. Herein collagen was dissolved with cooled urea/acetic acid aqueous solution to reach concentrations varied from 40 to 120 mg/mL. Fourier transform infrared spectroscopy (FTIR), sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE), differential scanning calorimetry (DSC), polarizing optical microscopy (POM), fieldemission scanning electron microscopy (FESEM) and rheological measurements were performed to characterize the concentrated collagens. Based on these results, both of the integrity of collagen structure and the uniformity of the concentrated collagens were evaluated. Moreover, the possible mechanism by which homogeneous concentrated collagen was generated was speculated. 2. Experimental 2.1. Materials Acetic acid (HAc) (AR, N99.5% purity), HCl (AR, 36%~38% purity), urea (AR, 99% purity) and pepsin (1:3000) were purchased from Aladdin Co., Ltd (China). Protein marker (6.6–200 kDa) was purchased from Sigma-Aldrich Co., Ltd (China). Collagen was prepared from calf skins by the method described previously [26]. Briefly, the supernatants extracted from the delimed and neutralized bovine split pieces with 0.5 mol/L acetic acid containing 3 wt% pepsin were collected by refrigerated centrifugation at 9000 rpm, and then the supernatants were salted out by adding NaCl to the final concentration of 0.7 mol/L. The precipitate was dissolved and dialyzed against 0.1 mol/L acetic acid for 3 days. Finally, pepsin-soluble collagen solution was lyophilized using a freeze dryer (Boyikang, FD-1A-50, China) at −50 °C and stored at 4 °C until used. 2.2. Preparation of highly concentrated collagens The lyophilized pepsin-soluble collagen sponges were added to 0.1 mol/L acetic acid (HAc) to reach the content of 40 to 120 mg/mL with continuous stirring at 4 °C for 24 h. After that, urea was added at a concentration of 10 mg/mL, and then it was placed in a refrigerator (BOSCH BCD-610WCKAN92VO2TI, Germany) at −12 °C for 12 h. The samples were stirred continuously for 4 h as soon as taken out from the refrigerator until no discernible collagen sponge could be observed. The obtained collagen samples were dialyzed against 0.1 mol/L acetic

acid solution for 3 days and change of solution once per day to remove urea. Finally highly concentrated collagen solutions with concentration ranging from 40 to 120 mg/mL were obtained. For brevity, collagen gels of 40, 60, 80, 100, and 120 mg/mL prepared by cooled urea/HAc solutions as solvent were named as UHAc-COL(40), UHAc-COL(60), UHAcCOL(80), UHAc-COL(100), and UHAc-COL(120), respectively. To prepare a series of controls, 0.1 mol/L HAc, 1.148 × 10−3 mol/L HCl (pH = 2.88, equal to that of 0.1 mol/L HAc), cooled urea/HCl (1.148 × 10−3 mol/L HCl with 10 mg/mL urea), as well as pristine urea solution (10 mg/mL) were used as the solvents for collagen sponges. The samples with various collagen contents were prepared according to the operations as aforementioned. Likewise, samples prepared with pristine HAc solution were abbreviated as HAc-COL(10), HAc-COL(40), HAc-COL(60), HAc-COL(80) and HAc-COL(100), respectively; samples prepared with pristine HCl solution were named as HCl-COL(10), HCl-COL(40), HCl-COL(60), HCl-COL(80) and HCl-COL (100), respectively. Similarly, samples prepared with cooled urea/HCl solutions were named as UHCl-COL(40), UHCl-COL(60), UHCl-COL (80), UHCl-COL(100), and UHCl-COL(120), respectively; while samples prepared with pristine urea solution were abbreviated as Urea-COL(10), Urea-COL(40), Urea-COL(60), Urea-COL(80) and Urea-COL(100), respectively. With regard to these control samples, differential scanning calorimetry (DSC) and oscillatory rheological measurements would be carried out in this work. Part of the samples were lyophilized in a freeze dryer (Boyikang, FD-1A-50, China) at −40 °C for 3 days, stored in a desiccators until used. 2.3. Fourier transform-infrared spectroscopy (FTIR) FTIR of the lyophilized concentrated collagens were carried out using Thermo Fisher Scientific instrument (Nicolet380, U.S.). The measurements were performed at a data acquisition rate of 4 cm−1 per point in the range of 500 to 4000 cm−1. A total of 64 scans were performed for each sample. 2.4. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDSPAGE) The SDS-PAGE patterns of the concentrated collagens were performed according to the method of Laemmli [27]. All collagen samples were mixed with 0.5 mol/L Tris-HCl buffer (pH =6.8) solution containing 25% glycerol, 2% SDS and 0.01% bromophenol blue to reach a final collagen concentration of 1 mg/mL, and the mixed solutions were boiled for 10 min. Samples were loaded onto a polyacrylamide gel made of 7.5% running gel and 2.5% stacking gel, and then subjected to electrophoresis at a constant current of 12 mA, using a Mini protein II unit. After electrophoresis, the gel was stained for 45 min in the presence of 0.05% (w/v) coomassie brilliant blue R-250 in 50% (v/v) methanol and 10% (v/v) acetic acid, and then distained with 5% (v/v) methanol and 7.5% (v/v) acetic acid. Protein marker (6.6–200 kDa) was used as standard. 2.5. Differential scanning calorimetry (DSC) The thermal stability of the concentrated collagens was evaluated by differential scanning calorimetry (Netzsch DSC 214 PC, Germany). The samples was sealed in an aluminum pans, and then conducted in the temperature ranging from 20 to 60 °C with a constant heating rate of 3 °C/min under nitrogen flow. Liquid nitrogen was used as a cooling medium, and an aluminum pan with 0.1 mol/L acetic acid solution was used as the reference. 2.6. Polarizing optical microscopy (POM) The texture of the concentrated collagen gels (collagen samples were laid on glass slide, and then covered with cover glass) were

Q. Yang et al. / Journal of Molecular Liquids 291 (2019) 111304

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observed with a polarizing optical microscopy (Nikon, LV100POL, Japan). The samples were put onto the objective stage, which was rotated at different angles and a pressure or shear force was applied to the film. Images were captured with a video camera. 2.7. Field-emission scanning electron microscopy (FESEM) The surface morphological characteristics of the lyophilized concentrated collagens were observed using a field-emission electron microscopy (FEI Nova NanoSEM 230, U.S.), operated at an accelerating voltage of 5 kV. Lyophilized collagen sponges were mounted on stubs, sputter-coated with gold and then observed at various magnifications. 2.8. Rheological characterization of concentrated collagens 2.8.1. Oscillatory rheological measurements The concentrated collagens were tested on a Rheometer System (MARS III, Germany) using a stainless steel cone/plate geometry (2° cone angle, 35 mm cone diameter) with the gap set at 150 μm. Dynamic frequency sweeps for samples were performed from 0.1 to 10 Hz at 25 °C at a constant strain of 5%. The storage modulus (G′), complex viscosity (η⁎) and loss tangent (tanδ = G″/G′) were recorded. 2.8.2. Steady shear study Steady shear study was also performed on the same Rheometer System (MARS III, Germany) equipped with a stainless steel cone/plate geometry. Steady state viscosity was recorded as a function of shear rate from 0.05 to 100 s−1, under the controlled-stress (CS) mode and at a constant temperature of 25 °C. The obtained flow curves were fitted with the Ostwald-de Waele model, Cross model, and Carreau model to further analyze the relationship between shear viscosity and shear rate of systems. Ostwald-de Waele model [28]: η ¼ Kγn−1 where η is shear viscosity (Pa·s), γ is shear rate (s−1), K is viscosity index (Pa·sn) and n is flow index (dimensionless), which is determined by the type of materials. (For pseudoplastic fluid, nb1, for dilatants fluid, nN1, for Newtonian fluid, n = 1). Cross model [29]: η ¼ η∞ þ

η0 −η∞ 1 þ K1 γd

where η0 and η∞ (Pa·s) are the zero-shear viscosity and infinite viscosity(Pa·s), respectively, K1 is a characteristic relaxation time (s) and d is a dimensionless constant corresponding to the material. Carreau model [30]: h iðm−1Þ=2 η−η∞ ¼ 1 þ ðλγÞ2 η0 −η∞ where η0 is the zero-shear viscosity (Pa·s), referring to the constant viscosity in the first Newtonian plateau (γ → 0), η∞ is the infinite viscosity (Pa·s), representing the constant viscosity in the second Newtonian plateau (γ → ∞), λ is a characteristic time (s) and m is a dimensionless constant corresponding to the flow index (n). 3. Results and discussion 3.1. FTIR spectra of concentrated collagens The FTIR spectra of concentrated collagens are shown in Fig. 1, in which typical absorption peaks of collagen could be observed. The amide I (1600–1660 cm−1) band is associated with stretching vibrations of carbonyl groups, the amide II (~1550 cm−1) band is associated

Fig. 1. FTIR spectra of HAc-COL(10) and the concentrated collagens.

with N\\H bending and C\\N stretching, and the amide III (1220–1230 cm−1) band is related to C\\N and N\\H stretching, and involved with the integrity of triple helical structure of collagen [31–33]. The amide A and B bands at 3299 and 3083 cm−1 respectively, are mainly related to the stretching vibrations of N\\H groups [34]. The spectra of all the concentrated collagens were similar to that of HAc-COL(10). In addition, the absorption ratios of amide III to 1450 cm−1, expressed as AIII/A1450 are also considered as a measurement of preservation of integrity of the modified collagens [35]. All of the concentrated collagens as concentration from 40 to 100 mg/mL exhibited the calculated AIII/A1450 values in the range of 0.87–0.92, which was close to that of the control sample HAc-COL(10). Moreover, it has been reported that the ratio of gelatin, which was a denatured products of collagen, was only about 0.59 [36]. Hence, the results of FTIR showed that collagen dissolved with cooled urea/HAc maintained its intact triple helical structure (Table 1). 3.2. SDS-PAGE patterns of concentrated collagens The subunit composition of concentrated collagens was examined by electrophoresis pattern (Fig. 2). Type I collagen comprised two different α chains (α1 and α2) and their dimers (β chain) and trimmers (γ chain), and the intensity of α1 chain was about double than that of α2 chain [37]. Two bands (α1 and α2) with molecular weight (MW) of ~100 kDa were clearly shown on the SDS-PAGE pattern of HAc-COL (10). Moreover, a great amount of β-chains (~200 kDa for the dimers of α chains) and few γ-chains (the trimers of α chains) were also observed in the pattern. The features were consistent with the typical electrophoretic bands for type I collagen reported previously [38]. Compared with that of HAc-COL(10), the SDS-PAGE patterns of the samples prepared by cooled urea/HAc solutions did not show any significant differences, namely, all four polypeptide chains (α1, α2, β, and γ chains) were all retained. As reported in previously work which employed the evaporation/ultrasonication method to prepare Table 1 The AIII/A1450 values of HAc-COL(10) and concentrated collagens. Sample

AIII/A1450

HAc-COL(10) UHAc-COL(40) HAc-COL(60) UHAc-COL(60) UHAc-COL(80) UHAc-COL(100)

0.96 0.92 0.87 0.90 0.90 0.87

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3.3. DSC thermograms of concentrated collagens

Fig. 2. SDS-PAGE patterns of HAc-COL(10) and the concentrated collagens. Lane 1: protein marker, Lane 2: HAc-COL(10), Lane 3: UHAc-COL(40), Lane 4: UHAc-COL(60), Lane 5: HAc-COL(60), Lane 6: UHAc-COL(80), Lane 7: UHAc-COL(100), Lane 8: UHAc-COL(120).

concentrated collagen, smaller fragments appeared on the gels under the α1 and α2 bands, corresponding to molecular masses ranging from 100 to 30 kDa, and their intensity increasing upon the increase of sonication time [39]. In the present work, no bands of degradation product with lower molecular weight (b100 kDa) were observed on the gels. Thereby, the results of SDS-PAGE analysis revealed that the primary structure of collagen remained intact after dissolved in the cooled urea/HAc solutions [40], further confirmed that the addition of urea did not destroy the structure of collagen.

In DSC thermograms, thermal denaturation temperature (Td) and denaturation enthalpy (ΔH) could be determined. Td represents the thermal stability of collagen, while ΔH reflects the hydrophobicity/hydrophilicity and degree of aggregation of collagen molecules, namely, the energy required for the destruction of the hydrogen bonds which maintain the collagen triple helixes [41,42]. The temperature dependence of enthalpy of collagen is given in Fig. 3, and the values of Td and ΔH are summarized in Table 2. As could be seen from Fig. 3a, HAc-COL(10) and HAc-COL(40) exhibited individual endothermic peaks at 40.1 and 40.5 °C, respectively, due to the thermal denaturation of collagen. In comparison, except for the thermal denaturation peak located at ~40 °C, one or more peaks located at higher temperatures were observed for samples with higher collagen content including HAc-COL(60), HAc-COL(80), and HAc-COL(100). The emerging peaks at temperatures higher than the Td of collagen indicated the existence of collagen components that were not completely dissolved in the systems, in view of the highly aggregated collagens typically in the format of fiber [43], collagen sponge [38], film [44], and hydrogel [45] possessed Td of 72.6, 68.1, 52.2 and 45.2 °C, respectively, which were higher than the Td of collagen in aqueous solution (~40 °C). It was previously suggested that the increased temperature stability of collagen could be caused by the reduced hydration, drawing the collagen molecules closer together [46,47]. Therefore, the emerging peaks at temperatures higher than ~40 °C could be ascribed to the higher degree of aggregation of collagen molecules.

Fig. 3. DSC thermograms of concentrated collagens prepared with (a) HAc solution; (b) cooled urea/HAc solution; (c) HCl solution; (d) cooled urea/HCl solution.

Q. Yang et al. / Journal of Molecular Liquids 291 (2019) 111304 Table 2 Thermal denaturation temperature and thermal denaturation enthalpy of concentrated collagens. HAc systems HAc-COL(10) HAc-COL(40) UHAc-COL(40) HAc-COL(60) UHAc-COL(60) HAc-COL(80)

UHAc-COL(80) HAc-COL(100)

UHAc-COL(100)

Td (°C)

ΔH (cal/g)

40.1 40.5 41.0 41.3 44.7 41.1 41.1

0.8996 4.258 3.262 7.606 0.1952 6.132 10.76

55.7

0.1552

40.9 41.9 44.1 44.7 40.9 45.6

6.988 17.41

UHCl-COL(80) HCl-COL(100)

9.159 0.2858

UHCl-COL(100)

HCl systems HCl-COL(10) HCl-COL(40) UHCl-COL(40) HCl-COL(60) UHCl-COL(60) HCl-COL(80)

Td (°C)

ΔH (cal/g)

42.4 42.4 43.1 42.8 45.6 43.2 43.2 43.5 53.9 45.2 43.4 49.7

1.393 8.384 7.046 12.37

45.9 49.0

10.03 12.84 0.057 11.84 17.95 0.352 12.16

In contrast, as reflected from Fig. 3b, DSC thermograms of UHAc-COL (40), UHAc-COL(60) and UHAc-COL(80) exhibited just one endothermic peak at ~40 °C, suggesting that these dissolution systems were quite homogeneous without highly aggregated collagens. Nevertheless it should be pointed out that the emerging peaks at temperatures higher than 40 °C were generated for UHAc-COL(100) and UHAc-COL(120). Referring to Fig. 3a and b, it could be clearly found that the critical concentration to form a homogeneous collagen gel was increased when the novel solvent was applied. That is, the critical collagen concentration to be homogeneous in HAc might be located at around 40 mg/mL, while it could be 80 mg/mL in the case of using cooled urea/HAc solution as solvent. As exhibited in Fig. 3c and d, similar trends on the features of DSC thermograms could be observed by comparing collagens in HCl solution with cooled urea/HCl solution, which possessed pH values close to those of HAc and cooled urea/HAc solution, respectively. Therefore, it seemed that urea played a critical role in promoting the dissolution of collagen upon using either urea/HAc or urea/HCl as solvent. To further confirm this effect, we carried out the DSC test for collagens with pristine urea as solvent. As can be seen from Fig. 1s, all of the samples with solid content in the range of 10–100 mg/mL, using pristine urea as solvent, displayed at least two endothermic peaks, even three peaks could be observed for samples with content of 80 and 100 mg/mL (the Td values were summarized in Table 1s). This suggested that collagen could not be dissolved well even at a concentration as low as 10 mg/mL in pristine urea solution. Nevertheless, the endothermic peak at ~40 °C appeared in all of the samples demonstrated that collagen could be dissolved partially in pristine urea aqueous solution in the mixtures cause by the destruction of hydrogen bond. More information could be obtained by the comparison of Td and ΔH values of collagen samples with HAc (HCl), or cooled urea/HAc (HCl) as solvents (as listed in Table 2). On one hand, it was interesting to see that Td values of collagens dissolved in cooled urea/HAc were slightly lower by 0.2–1.0 °C than those of counter samples in HAc. On the other, in regard to the same concentration of collagens, the application of cooled urea/HAc slightly declined the values of ΔH. As is known, there are abundant non-covalent bonds primarily including hydrogen bonds between collagen α chains forming triple helix structure. Some researchers have confirmed the bridging effect of water molecules, that is, hydroxyproline in collagen provides hydroxyl groups, and water molecules participate in the formation of hydrogen bonds in a bridging manner [48,49]. These water-mediated hydrogen bonds enhanced the triple helix in regions lacking proline and contributed to collagen stability [42,50]. Thus, the declined ΔH was probably due to the effect of cooled urea molecules which partially replaced the hydrogen bond between water molecules and collagen. Consequently, the energy

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required to destroy the remaining hydrogen bonds (ΔH) was reduced, accompanied by the slight decrease in the Td values of collagen. With regard to the systems with HCl or cooled urea/HCl as solvents, the change trend in ΔH values was similar to that with HAc or cooled urea/HAc as solvents. However, Td of the samples increased when urea was involved in the cooled solvent, contrary to that with HAc or cooled urea/HAc as solvents. In addition, it was worth pointing out that all of the samples possessed Td value ~2 °C higher than the corresponding samples with HAc or cooled urea/HAc as solvents. To the best of our knowledge, the differences between HAc and HCl as collagen solvent aforementioned were hardly ever illuminated previously, thereby, this issue will be focused on in our next work. 3.4. Polarizing microscopy observation of concentrated collagens In animal tissues, collagen fibrils exhibit anisotropic packing at a suprafibrillar level, while three-dimensional organizations in extracellular matrices forms regular birefringent patterns within tissue sections [51]. The liquid crystal phase of collagen has been studied by Giraud Guille et al., who suggested that highly concentrated collagen in acid solution could be gathered spontaneously in liquid crystalline phases through ultrasonic vibration [22]. As the collagen concentration increased, collagen molecules tended to attract each other due to the enhanced hydrophobic effect [52]. Meanwhile, collagen molecules had a positive charge in acidic conditions where repulsions between positively charged residues were large enough to prevent aggregation [51]. Therefore, the balance between the two forces makes the liquid crystalline phases formed [20]. Fig. 4 shows polarizing microscopy images observed with polarizing microscopy (POM) of collagen dissolved in cooled urea/HAc with concentrations from 40 to 120 mg/mL. As can be seen, nearly only dark field was observed for UHAc-COL(40) and UHAc-COL(60), regardless of how the stage was rotated, which means that the prepared collagen samples as concentrations below 60 mg/mL hardly had any liquid crystal structure (Fig. 4a–b). As the collagen concentration was increased to 80 or 100 mg/mL, the cholesteric band began to appear but was not distinct, suggesting that collagen molecules tended to be oriented but still not form liquid crystal structure fully (Fig. 4c–d). The alignment of the molecular orientations became quite obvious and the alternate cross cholesteric bandings became much clearer, which reflected that the birefringent effect was pronounced in UHAc-COL(120) (Fig. 4e). It has been reported that the cholesteric banding became obvious when the concentration of collagen reached 40 mg/mL [39,51,53], which was lower than that of 80 mg/mL as detected in the present work. However, it should be pointed out that in these studies, ultra-sonication was used as an indispensable treatment to generate the twisted liquid crystalline phase well recognizable in polarizing microscopy by disentangling the aggregated collagen molecules [22]; nevertheless, the degradation of collagen during the process could not be neglected. Overall, the images observed using POM demonstrated that the concentrated collagens upon the addition of cooled urea/HAc as solvent possessed cholesteric texture similar to that concentrated through evaporation/ultra-sonication method without being degraded. These promising results were beneficial to the production of organized gels based on the native collagen molecules with the aim of mimicking extracellular scaffolding. 3.5. FESEM observation of concentrated collagens The FESEM images of freeze-dried HAc-COL(10) and collagens prepared with cooled urea/HAc are shown in Fig. 5. As expected, HAc-COL (10) displayed a network with interconnected three-dimensional porous structure. With the collagen concentration further increased to 40 or 60 mg/mL, the internal microstructure of the sponges grew denser and more uniform. Interestingly, when collagen concentration reached 80–100 mg/mL, the cross section of this sample no longer exhibited

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Fig. 4. POM photographs of concentrated collagen films at different concentrations of (a) UHAc-COL(40); (b) UHAc-COL(60); (c) UHAc-COL(80); (d) UHAc-COL(100); (e) UHAc-COL(120).

the porous structure, instead a layer by layer morphology resembled the corrugated shape could be observed. The corrugated shape with a smaller spacing between the adjacent layers was observed as the content of collagen further increased to 120 mg/mL. However, it should be pointed out that the morphology of UHAc-COL(120) was not uniform everywhere in the sample, that is, both of the corrugated (Fig. 2s-a) and haphazard morphologies (Fig. 2s-b) could be captured in the cross section of sponge. Combining with the results of DSC, it could be deduced that the heterogeneity of UHAc-COL(120) was ascribed to the co-existence of dissolved and undissolved collagen components in the system. 3.6. Rheological measurements of concentrated collagens 3.6.1. Dynamic rheological tests As a natural macromolecule, collagen is prone to entangle and aggregate in aqueous solution, so collagen solutions exhibit remarkable viscoelastic behavior [54]. The dependence of viscoelasticity on collagen concentration has been revealed previously [55,56], nevertheless, collagen concentration in these studies was relatively low (≤20 mg/mL). To our knowledge, the dynamic viscoelasticity of the highly concentrated collagen gels with concentration ≥ 40 mg/mL has hardly ever been reported. Fig. 6 shows the frequency dependence of G′, η* and tan δ for collagen samples over the frequency range of 0.1–10 Hz at 25 °C. G′ reflects

the elasticity of samples, while η* reflects shear-thinning flow behavior of samples [54,57]. As could be seen in Fig. 6(a–b), both of the G′ and η* increased upon increasing collagen concentration. As expected, the similar dependence of G′ and η* on collagen concentration could also be observed from samples with pristine urea as solvent in Fig. 3s(a, b), as well as from samples with HCl or cooled urea/HCl as solvents as reflected in Fig. 4s(a, b), respectively. More importantly, G′ and η* of UHAc-COL(60) were lower than HAc-COL(60), indicating that the network of collagen sample prepared with cooled urea/HAc was liable to deform and the flowability was improved. The ratio G″/G′, which is defined as the loss tangent (tanδ), crossed the threshold (tanδ = 1) from solid-like to liquid-like behavior [58]. The tanδ value of samples were decreased with increasing the concentration of collagen, indicated that collagen tended to exhibit a solid-like behavior (Fig. 6c) [28]. Tanδ N1 as the concentration of collagen = 10 mg/mL, while tanδ b1 as the concentration of collagen ≥40 mg/mL, reflecting that concentrated collagens possessed behaviors different from those with low concentration (thus the concentrated collagen should be regarded as a gel but not a solution). Furthermore, it should be noted that tanδ of HAc-COL(60) was smaller than that of the UHAc-COL(60), which coincided with the trend of G′ and η* mentioned above. Likewise, compared with the pristine HCl, the increase in tanδ, as well as decrease in both of G′ and η*, could be observed clearly from Fig. 4s(c) and s(d) in the case of using cooled urea/HCl as solvent, which further confirmed the weakening effect on hydrogen bonding by adding urea.

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Fig. 5. The FESEM images of (a) HAc-COL(10); (b) UHAc-COL(40); (c) UHAc-COL(60); (d) UHAc-COL(80); (e) UHAc-COL(100); (f) UHAc-COL(120).

Fig. 6. The dynamic modulus of concentrated collagens of (a) storage modulus (G′); (b) complex viscosity (η*); (c) loss tangent (tanδ); (d) comparison of G′ and η* of HAc-COL(60) and UHAc-COL(60).

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shear thinning [67]. The λ value of UHAc-COL(60) was smaller than that of HAc-COL(60), demonstrating that the shear thinning behavior was delayed to a higher critical shear rate. This trend indicated a decreased dependence on shear rate due to the more pronounced shear thinning behavior of UHAc-COL (60) [54]. 3.7. The possible dissolution mechanism of collagen in cooled urea/HAc solvent system

Fig. 7. Experimental data from concentrated collagens fitted using the Carreau model.

3.6.2. Flow behavior in steady shear tests Steady shear tests are commonly used to describe the flow behavior of polymer solution [59]. Fig. 7 shows the flow curves of collagen samples as a function of collagen concentration. All samples showed shear-thinning behavior, that is, the apparent viscosity decreased with increasing shear rate, demonstrating that all samples exhibited a typical non-Newtonian shear thinning behavior [60,61]. At the same shear rate, the apparent viscosity of collagen sample increased with increasing collagen concentration. As expected, reconfirming that the network of collagen sample treated with cooled urea/HAc was liable to deform and its flowability was improved (the inset in Fig. 7). In addition, Ostwald-de Waele model, Cross model and Carreau model were used to simulate the fluid flow curves of the concentrated collagens [54,62,63]. As summarized in Table 3, Carreau model was accurately fit the steady shear curves of concentrated collagens since the regression coefficients (R2) were almost equivalent to 1.0. The η0 value of samples HAcCOL(60) and UHAc-COL(60) were 1.511 and 1.363 Pa·s, respectively. The friction from the molecular chains, termed as internal friction has been found out to affect the dynamics of long chain molecules [64]. It is believed that weak interactions such as hydrogen bond contribute to internal friction [65], which is still operative even in solutions with a very low solvent viscosity [66]. Therefore, the declined collagen viscosity under shear measurements was attributed to the reduced internal/intermolecular friction forces in collagen gels prepared with cooled urea/HAc [54], which broke the hydrogen bonds of collagen aggregates. The time constant λ, the reciprocal of the critical shear rate, intersects the constant viscosity region and the Newtonian region in the flow curve, providing a useful indicator of the onset shear rate for

HAc is the preferred mild acid as a traditional solvent for natural insoluble collagen. As illustrated in Fig. 8(a). In the acidic environment, the –NH+ 3 concentration on the surface of collagen molecules was increased owing to the protonation of amino groups. As a consequence, electrostatic charge repulsion between the electropositive collagen chains results in disaggregation of collagen aggregates into single collagen molecules [6,68]. As displayed in Table 2s, it was found that for systems with HAc (HCl) or cooled urea/HAc (HCl) as solvents, pH displayed a similar dependence on collagen concentration, that is, pH slightly increased upon increasing collagen content, which reflected that more H+ was involved in the dissolution of collagen through bonding with amino groups. However, as the collagen concentration was above 40 mg/mL, the resistance of mass transfer of HAc into the interior of collagen aggregates enhanced, leading to the suppression on the dissolving capacity of collagen. A schematic describing the dissolution of the collagen with cooled urea/HAc is presented in Fig. 8(b). The addition of urea to HAc solution disrupted both intra- and inter-molecular hydrogen bonds via forming hydrogen bonds with carboxyl groups and hydroxyl groups of collagen, and this effect could be enhanced by cooling to −12 °C [69]. Thereby, it seemed that there was a synergistic effect between urea and HAc on the dissolution of collagen in the novel binary solvent system. In detail, as soon as collagen in solid format was exposed to the cooled urea/HAc aqueous solution, it was moisturized through hydration on the surface, and then mutual repulsion between some of the positively charged collagen chains occurred, nevertheless, the intra- and inter-molecular hydrogen bonds between collagens could not be destroyed completely by electrostatic repulsion when collagen content was too high, leaving the un-dissolved collagen components or highly aggregated collagen in the system. Urea, as an excellent hydrogen bond breaker, plays a key role in disrupting the hydrogen bonds among collagen molecules. More amino groups of collagen would be exposed to HAc and then protonated, enhancing the electrostatic repulsion in turn. Finally, more collagen molecules could be separated in the binary solvent. As shown in Table 2s, the addition of 10 mg/mL urea slightly increased the pH values of HAc or HCl (from 2.88 to 2.90 and 2.99, respectively), suggesting that the reaction between HAc and urea was insignificant, even if it occurred. Furthermore, by comparing the data of HAc and Urea/HAc, or those of HCl and Urea/HCl, it was surprising to find that the addition of urea obviously reduced the pH for collagens

Table 3 Parameters from Ostwald-de Waele model, Cross model and Carreau model for concentrated collagens. Parameters Ostwald-de Waele

Cross

Carreau

K n R2 η∞ η0 K1 d R2 η∞ η0 λ m R2

HAc-COL(10)

UHAc-COL(40)

HAc-COL(60)

UHAc-COL(60)

UHAc-COL(80)

UHAc-COL(100)

UHAc-COL(120)

0.011 0.531 0.955 0.001 0.0413 3.304 1.182 0.997 0.981 1.019 5.333 0.226 0.999

0.053 0.378 0.980 0.004 0.367 8.809 1.197 0.998 0.842 1.158 9.332 0.107 0.999

0.139 0.343 0.988 0.006 1.322 11.112 1.059 0.999 0.489 1.511 12.431 0.141 1.000

0.117 0378 0.983 0.008 0.864 8.970 1.138 0.999 0.637 1.363 10.178 0.136 1.000

0.193 0.343 0.987 0.007 1.713 9.982 1.076 0.999 0.326 1.674 11.123 0.129 1.000

0.221 0.344 0.988 0.009 1.968 9.823 1.055 0.999 0.237 1.763 11.304 0.149 1.000

0.500 0.310 0.994 0.0026 6.532 13.499 0.947 1.000 −1.183 3.183 15.625 0.176 1.000

Q. Yang et al. / Journal of Molecular Liquids 291 (2019) 111304

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Fig. 8. Dissolution mechanism of HAc-COL(60) (a) and UHAc-COL(60) (b).

with all the concentrations employed. Therefore, it could be speculated that urea assisted in the dissolution of collagen through hydrogen bonding destructive action, thus reducing in the consumption of H+ in the mixtures. As a whole, the results of pH for different systems provided side evidence to illustrate the unique effect of urea on collagen dissolution. The solubility enhancement could be verified intuitively using the phenomenon of “Tyndall effect”, as shown on the left of Fig. 8. Under the irradiation of laser, the light beam in the HAc-COL(60) was reflected, exhibited a short beam path, which could be ascribed to the reflection by the large particles in the sample; whereas, the light beam in UHAcCOL(60) displayed a longer and brighter channel, reflecting a better “Tyndall effect”. This comparison result further illustrated that a more uniform highly concentrated collagens could be produced with the cooled urea/HAc as solvent. 4. Conclusion In this study, the cooled urea/HAc binary system was employed as novel solvent to prepare collagen gels with high concentrations from 40 to 120 mg/mL. With pristine HAc, HCl (pH equal to that of HAc), urea, and cooled urea/HAc as solvents to prepare a series of control systems, some conclusions could be drawn in this work. The addition of urea disrupted both intra- and inter-molecular hydrogen bonds among collagens, which could be further enhanced by cooling. Therefore, a synergistic effect could be created between urea and HAc in the cooled solvent, which improved effectively the dissolution capacity of collagen. In brief, homogeneous collagen sample with concentration of 80 mg/mL could be produced in the binary solvent, which was higher than that of 40 mg/mL in HAc. What's more valuable is that the triple helical structural integrity of collagen was maintained after treated with cooled urea/HAc. Consequently, the obtained information

improved our understanding on the solubilization of collagen, and this novel solvent opened up a facile way to prepare highly concentrated collagen-based materials keeping the native structure. Moreover, the differences between HAc and HCl as collagen solvents would be studied systematically in future work. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (Grant Nos. 21606046 and 21306024), the Natural Science Foundation of Fujian Province (Grant No. 2016J01208), the Outstanding Young Scientific Research Talents in Universities of Fujian Province (Grant No. KLA18064A), and the Special Fund for Science and Technology Innovation of Fujian Agriculture and Forestry University (Grant Nos. KFA17217A and KFA17476A). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.molliq.2019.111304. References [1] A. Jongjareonrak, S. Benjakul, W. Visessanguan, T. Nagai, M. Tanaka, Isolation and characterisation of acid and pepsin-solubilised collagens from the skin of Brownstripe red snapper (Lutjanus vitta), Food Chem. 93 (2005) 475–484. [2] M.J. Mienaltowski, D.E. Birk, Structure, physiology, and biochemistry of collagens, Progress in Heritable Soft Connective Tissue Diseases, vol. 802, 2014, pp. 5–29. [3] J.H. Muyonga, C.G.B. Cole, K.G. Duodu, Extraction and physico-chemical characterisation of Nile perch (Lates niloticus) skin and bone gelatin, Food Hydrocoll. 18 (2004) 581–592. [4] N. Muhammad, G. Gonfa, A. Rahim, P. Ahmad, F. Iqbal, F. Sharif, et al., Investigation of ionic liquids as a pretreatment solvent for extraction of collagen biopolymer from waste fish scales using COSMO-RS and experiment, J. Mol. Liq. 232 (2017) 258–264.

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