polymers Article
The Preparation of Nano-SiO2/Dialdehyde Cellulose Hybrid Materials as a Novel Cross-Linking Agent for Collagen Solutions Cuicui Ding 1 , Yang Zhang 1 , Binhan Yuan 1 , Xiaodong Yang 1 , Ronghui Shi 1 and Min Zhang 2, * 1
2
*
College of Ecological Environment and Urban Construction, Fujian University of Technology, Fuzhou 350108, China;
[email protected] (C.D.);
[email protected] (Y.Z.);
[email protected] (B.Y.);
[email protected] (X.Y.);
[email protected] (R.S.) College of Materials Engineering, Fujian Agriculture and Forestry University, Fuzhou 350002, China Correspondence:
[email protected]; Tel.: +86-0591-8371-5175
Received: 29 March 2018; Accepted: 15 May 2018; Published: 21 May 2018
Abstract: Nano-SiO2 was immobilized onto dialdehyde cellulose (DAC) to prepare SiO2 /DAC hybrid materials. Fourier transform infrared spectra (FTIR), thermogravimetric analysis and field emission scanning electron microscopy of SiO2 /DAC indicated that nano-SiO2 had been successfully hybridized with DAC. X-ray diffraction suggested that the structure of DAC was influenced by the nano-SiO2 . SiO2 /DAC was then used as the cross-linker of collagen solutions. Gel electrophoresis patterns and FTIR reflected that cross-linking occurred between DAC and collagen, but that collagen retained the native triple-helix, respectively. Differential scanning calorimetry indicated that the thermal stability of collagen could be effectively improved by SiO2 /DAC. Dynamic rheology tests revealed that the flowability of collagens cross-linked by SiO2 /DAC was superior to that of those cross-linked by DAC; meanwhile, collagens cross-linked by SiO2 /DAC possessed a more homogeneous morphology compared to those cross-linked by DAC. The hybridization of SiO2 /DAC as a cross-linker for collagen could effectively prevent the gelation caused by excessive cross-linking, and significantly improve the thermostability of collagen, which could be helpful for collagen being applied in fields including biomaterials, cosmetics, etc. Keywords: dialdehyde cellulose; nano-silicon dioxide (nano-SiO2 ); collagen; hybridization; flowability; thermal stability
1. Introduction Collagen, the major component of extracellular matrices, has become a popular biomaterial and has been widely used in pharmacy, food, cosmetics and tissue engineering due to its bioactivity, biocompatibility and biodegradability [1–3]. Usually, aqueous preparations of collagen can be used for medial injection [4] or used as drug carriers and so on [5,6]; additionally, collagen solutions may also be processed into a variety of formats, including sheets, fibers, sponges and packaging films [7–10]. During these applications and processes, the thermal stability of collagen in solutions plays an essential role in keeping collagen against denaturation, as the excellent characteristics of the native collagen would be lost if the collapse of the triple helix to a random coil occurred. The thermal stability of collagen in solutions has been studied widely, and all of these studies suggest that collagen has a relatively weak thermal stability, which limits its industrial and biological applications. Chemical cross-linking procedures are a useful method for reinforcing the collagen structure, and several cross-linkers, such as glutaraldehyde (GA) [11], formaldehyde [12] and diisocyanates [13], have been used. However, these cross-linkers usually show toxicity. Some researchers have developed cross-linking agents with low toxicity, such as carbodiimide [14], Polymers 2018, 10, 550; doi:10.3390/polym10050550
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but nevertheless, incomplete cross-linking under a sufficiently high carbodiimide concentration could be caused by side reaction of the carbodiimide [15]. Therefore, it was necessary to develop an alternative cross-linking agent with good biocompatibility and low cytotoxicity. Cellulose is the most abundant natural polymer and has some fascinating properties, such as biocompatibility, biodegradability and desirable mechanical properties, and thus a variety of cellulose derivatives that can be widely used in many fields have been produced by chemical modifications such as esterification, halogenation, oxidation and etherification [16], or by chemical grafting [17]. Amongst these derivatives, periodate oxidized cellulose is prepared by specific cleavage of the C2–C3 bond of the glucopyranoside ring, producing two aldehyde groups per unit [18]. Thus, it is named as dialdehyde cellulose (DAC), which is an open chain polymer containing aldehyde groups. Considering the functional aldehyde groups could produce cross-links with free amino groups, DAC can be used as a novel cross-linking reagent for collagen. Kanth et al. investigated the effect of DAC on cross-linking efficiency, hydrothermal and enzymatic stability of collagen fibers using DAC as a cross-linker. The results suggested that the hydrothermal stability of collagen fibers could be improved significantly [19]. However, as for collagen in solutions, a solid-like network structure would be gradually developed, and thus gelation is prone to occur when cross-linking reagent is added [20]. In particular, compared to cross-linkers with low molecular weight, the gelation of collagen solutions was more difficult to avoid, due to the presence of a large number of aldehyde groups in the long chain of DAC. Hence, DAC needs to be modified in order to maintain the flowability of the cross-linked collagen in solutions. SiO2 , which was formed by the hydrolysis of tetraethoxy silane, has been introduced into leather to improve the hydrothermal stability [21]. With collagen as a model of leather, it was revealed that hydrogen bonding between the silanol of the silica and the carboxyl hydroxyl, as well as amino groups of collagen, occurred between SiO2 and collagen [22]. Obviously, this effect was weaker than covalent cross-linking induced by DAC, so the gelation of collagen solutions could be expected to be inhibited if part of the aldehyde group of DAC were replaced by SiO2 ; meanwhile, the thermal stability of collagen solutions could still be improved by the effects from both DAC and SiO2 . Therefore, in the present work, we attempted to prepare SiO2 /DAC hybrid materials via organic/inorganic hybridization of nano-SiO2 and DAC to be a novel cross-linking agent for collagen. In the present study, the structure and morphology of the SiO2 /DAC hybrid materials were investigated by the methods of Fourier transform infrared (FTIR), X-ray diffraction (XRD) and thermogravimetric analysis (TGA). Then the hybrid materials were used as a novel cross-linking agent for collagen solutions. Subsequently, the modified collagens were characterized, and the effect of modification was evaluated via sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), differential scanning calorimetry (DSC), oscillatory rheological measurements and Field emission scanning electron microscopy (FESEM). New insights into and understanding of the mechanism of stabilization of collagen by SiO2 /DAC hybrid materials via organic/inorganic hybridization of nano-SiO2 and DAC were gained. 2. Materials and Methods 2.1. Materials Collagen was self-prepared from calf skins according to our previous work [23]. Briefly, the supernatants extracted from the delimed and neutralized bovine split pieces by 0.5 mol/L acetic acid containing 3% pepsin (1:3000) were collected by refrigerated centrifugation at 9000× g. After this step, the supernatants were salted out by the addition of NaCl to a final concentration of 0.7 mol/L, and then the precipitate was again dissolved in 0.5 mol/L acetic acid and dialyzed against 0.1 mol/L acetic acid for 3 days. Finally, pepsin-soluble collagen solution (about 3 mg/mL) was lyophilized by a freeze dryer (Labconco Freeze Dryer FreeZone 6 Liter, LABCONCO Co., Kansas City, KS, USA) at −50 ◦ C for 2 days and stored at 4 ◦ C until used.
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Microcrystalline cellulose (degree of polymerization = 200) was purchased from Aladdin Co., Ltd. (Shanghai, China). Sodium periodate (NaIO4 ), N-(β-aminoethyl)-γ-aminopropyl trimethoxy-silane (AEAPTS) and tetraethyl siloxane (TEOS) with analytical pure grade were purchased from National Pharmaceutical Group (Shanghai, China). 2.2. Preparation of SiO2 /DAC Hybrid Materials Dialdehyde cellulose (DAC) was prepared according to the methods reported earlier, with modifications as described [24]. Microcrystalline cellulose (25 g) was hydrolyzed in 5 mol/L hydrochloric acid for 12 h. The hydrolyzed cellulose was suspended in deionized water, and then 25 g of sodium periodate was added, stirring for 4 h with a magnetic stirrer. The pH of the solution was maintained at 3.5 during the reaction and the reaction was performed in the dark at 40 ◦ C. Ethylene glycol (5 mL) was added to terminate the reaction, and then the product was extracted with centrifugation in t-butyl alcohol for three times. Finally, the resultant was lyophilized by the freeze dryer at −50 ◦ C for 2 days. The obtained DAC were converted to nitrogen-containing compounds using the Schiff base reaction with hydroxylamine hydrochloride, according to the method of reported previously [25]. Elemental composition of nitrogen-containing derivatives was determined for C, H, and N by an elemental analyzer (Elementar Vario EL, Elementar Co., Hanau, Germany), and then the dialdehyde content was calculated to be 21.2%. TEOS (50 mL) was added to a 50% (v/v) ethanol solution (250 mL); after stirring at room temperature for 1 h, glacial acetic acid (2.5 mL) was added and then stirred at 75 ◦ C for 15 min. After this step, AEAPTS was dropwise added into the solution until a gel was formed. The gel was washed with 50% (v/v) ethanol solution for three times, and then SiO2 –NH2 was lyophilized by the freeze dryer at −50 ◦ C for 2 days. The SiO2 /DAC hybrid materials were then prepared as follows: a mixture was prepared by dispersed SiO2 -NH2 and DAC together in 50% (v/v) ethanol solution with the mass ratios (SiO2 /DAC) of 1/20, 1/10, 1/5 and 1/2, respectively. After stirring at 65 ◦ C for 18 h, the resultant was washed by deionized water three times, and then hybrid materials were obtained by lyophilization with the freeze dryer at −50 ◦ C for 2 days and were stored at 4 ◦ C until use. The hybrid materials were denoted as SiO2 /DAC (1/20), SiO2 /DAC (1/10), SiO2 /DAC (1/5) and SiO2 /DAC (1/2), respectively, according to the initial mass ratios of SiO2 /DAC. The nano-SiO2 in the following description was called SiO2 for short. 2.3. Characterization of SiO2 /DAC Hybrid Materials 2.3.1. Fourier Transform Infrared (FTIR) Spectra of SiO2 /DAC FTIR spectra of the SiO2 /DAC hybrid materials were recorded with a FTIR spectrophotometer (Thermo Scientific Nicolet IS10, Thermo Fisher Scientific Co., Waltham, MA, USA) using potassium bromide (KBr) pellets. The measurements were performed at a data acquisition rate of 2 cm−1 per point and in the range from 400 to 4000 cm−1 . To obtain a nice signal-to-noise ratio, 48 scans were performed for each sample. 2.3.2. X-ray Diffraction (XRD) of SiO2 /DAC The X-ray diffraction patterns of the SiO2 /DAC hybrid materials were recorded using Cu Kα radiation (λ = 0.154056 nm) on a diffractometer (Panalytical X’pert Pro MPD, PANalytical B.V. Co., Almelo, The Netherlands) at a scanning rate of 1◦ /min in the 2θ range from 5 to 70◦ . 2.3.3. Thermogravimetric Analysis (TGA) of SiO2 /DAC Thermogravimetric analysis of the SiO2 /DAC hybrid materials was performed with a thermal analyzer (Netzsch TG 209, NETZSCH Co., Selb, Germany). Samples (~2.0 mg) were heated from 30 to 800 ◦ C (ramp of 10 ◦ C/min) in a high-purity nitrogen atmosphere flowing at 80 cm3 /min.
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2.4. Cross-Linking of Collagen with SiO2 /DAC The freeze-dried collagen was dissolved in water acidified to pH 4.0 using dilute HCl to obtain a concentration of 6 mg/mL, and then the pH of the solution was adjusted to 11.0 by dropwise addition of NaOH (1.0 mol/L). DAC, SiO2 /DAC hybrid materials and SiO2 –NH2 in powder format were slowly added to the collagen solutions so that the final mass ratio of cross-linker/collagen reached 1/50 for all the samples. These solutions were gently stirred for 12 h at 20 ◦ C, during which time all of the cross-linkers were dissolved, due to the effect of NaOH, and the resultant solutions were clear. The mixtures were further kept in the refrigerator for 24 h at 10 ◦ C. The cross-linking reaction between cross-linkers and collagen were then terminated by adding excessive glycine, since the amino group in glycine is able to react with the active aldehyde groups in DAC or SiO2 /DAC. After this step, these solutions were dialyzed against 0.05 mol/L acetic acid solution, and thus the residual cross-linkers were removed. Finally, these resultants were lyophilized in the freeze dryer. 2.5. Characterization of Cross-Linked Collagen 2.5.1. Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) of Collagens Electrophoretic patterns were measured according to the method of Laemmli [26] with a slight modification, using 10% resolving gel and 4% stacking gel. Collagens were mixed with a sample buffer containing 10% β-Mercaptoethanol, to reach a final collagen concentration of 1 mg/mL, and the mixed solution was boiled for 5 min. After electrophoresis, the gel was stained for 45 min using 0.25% Coomassie brilliant blue R250 solution and destained using 7.5% acetic acid and 5% methanol. 2.5.2. FTIR of Collagens FTIR spectra of the collagen samples were recorded with a FTIR spectrophotometer (Thermo Scientific Nicolet IS10, Thermo Fisher Scientific Co., Waltham, MA, USA) using potassium bromide (KBr) pellets. The measurements were performed at a data acquisition rate of 2 cm−1 per point and in the range from 450 to 4000 cm−1 . 48 scans were performed for each sample. 2.5.3. Differential Scanning Calorimetry (DSC) of Collagens The cross-linking effect on collagen imposed by the SiO2 /DAC hybrid materials was evaluated by DSC (Netzsch DSC 200PC, NETZSCH Co., Selb, Germany). The freeze-dried collagens (~2 mg) were weighted accurately into aluminum pans and sealed; they were scanned over the range from 30 to 60 ◦ C at a heating rate of 3 ◦ C/min in a nitrogen atmosphere. Liquid nitrogen was used as a cooling medium, and empty pans were used as the reference. 2.5.4. Oscillatory Rheological Measurements of Collagens The rheological measurements of solutions were performed on a Rheometer System (MARS III, HAAKE Co., Karlsruhe, Germany) equipped with a cone-and-plate geometry (diameter 30 mm; angle 2◦ ). For the precise control of sample temperature, temperature was controlled to within an accuracy of ±0.10 ◦ C. The samples for oscillatory rheological tests were measured after each equilibration time for 3 min. Rheological properties were measured by dynamic frequency sweeps at a constant strain of 5% within the linear range. Dynamic frequency sweep tests for all the samples were performed from 0.1 to 10 Hz at constant temperature of 20 ◦ C. The storage modulus (G’), loss modulus (G”) and tan δ were recorded. 2.5.5. Field Emission Scanning Electron Microscopy (FESEM) of Collagens The morphologies of lyophilized DAC, SiO2 –NH2 and the modified collagen were examined with a field-emission scanning electron microscope (Nova FEI NanoSEM 230, FEI Co., Hillsboro, OR, USA), operated at an accelerating voltage of 10 or 5 kV.
The morphologies of lyophilized DAC, SiO2–NH2 and the modified collagen were examined with a field-emission scanning electron microscope (Nova FEI NanoSEM 230, FEI Co., Hillsboro, OR, USA), operated at an accelerating voltage of 10 or 5 kV. Polymers 2018, 10, 550
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3. Results and Discussion 3.1. Preparation and Characterization of SiO2/DAC Hybrid Materials Scheme 1 displays the synthetic of SiO and the SiO2/DAC hybrid materials. After 2, DAC 3.1. Preparation and Characterization of routes SiO2 /DAC Hybrid Materials the Schiff base reaction between the aldehyde groups of DAC and the amino group of SiO2–NH2, Scheme 1 displays the synthetic routes of SiO2 , DAC and the SiO2 /DAC hybrid materials. SiO2 was immobilized onto the chains of DAC. Therefore, the prepared hybrid materials can be After the Schiff base reaction between the aldehyde groups of DAC and the amino group of SiO2 –NH2 , described as SiO2 immobilized DAC, possessing both of the aldehyde groups and SiO2 as the SiO2 was immobilized onto the chains of DAC. Therefore, the prepared hybrid materials can be functional constituents reacting with collagen. described as SiO2 immobilized DAC, possessing both of the aldehyde groups and SiO2 as the functional constituents reacting with collagen.
Scheme 1. Schematic illustration of the preparation of SiO /DAC hybrid materials. Scheme 1. Schematic illustration of the preparation of SiO22/DAC hybrid materials.
As shown in Figure 1, the FTIR spectra of DAC showed two characteristic peaks at 1726 cm−1 −1 As shown in Figure thewith FTIRresults spectrareported of DAC previously showed two[27,28]. characteristic peaks 1726 and 880 cm−1, which is in1,line A diffuse bandatat 880cm cm−1 − 1 − 1 and 880 cm , which is in line with results reported previously [27,28]. A diffuse band at 880 cm can be assigned to the hemiacetal and hydrated form [29]. The sharp peak at 1726 cm−1 is a characteristic band of carbonyl groups [28]. The absorption band at 3452 cm−1 of the SiO2 indicated that there were plenty of hydroxyl groups on its surface. While the absorptions at 1081 cm−1 , 798 cm−1 and
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can be be assigned assigned to the hemiacetal and hydrated form [29]. The sharp peak at at 1726 1726 cm cm−1−1 is is aa can Polymers 2018, 10, 550 to the hemiacetal and hydrated form [29]. The sharp peak 6 of 15 −1 characteristic band band of of carbonyl carbonyl groups groups [28]. [28]. The The absorption absorption band band at at 3452 3452 cm cm−1 of of the the SiO SiO2 indicated indicated characteristic 2 that there were plenty of hydroxyl groups on its surface. While the absorptions at 1081 cm−1−1,, 798 798 that there were plenty of hydroxyl groups on its surface. While the absorptions at 1081 cm −1 −1 − 1 −1 −1 cm and 469 cm were attributed to the antisymmetric stretching vibration, symmetric stretching 469 cm werecm attributed to the antisymmetric stretchingstretching vibration, vibration, symmetricsymmetric stretching stretching vibration cm and 469 were attributed to the antisymmetric vibration and flexural vibration of Si–O–Si, respectively [30]. After reaction with SiO hybrid 2, the and flexural vibration of Si–O–Si, respectively [30]. After reaction with SiO , the hybrid materials vibration and flexural vibration of Si–O–Si, respectively [30]. After reaction2 with SiO2, the hybrid −1 − 1 −1 materials SiO /DAC exhibited new characteristic peaks located at 1067, 795 and 468 cm , as well as 2 SiO2 /DAC exhibited new characteristic peaks located 1067, 795 as well 1060, materials SiO new characteristic peaksatlocated at and 1067,468 795cm and, 468 cm as , as well801 as 2/DAC exhibited −1 − 1 −1 1060, 801 and 469 cm , for samples with SiO /DAC of 1/10 and 1/2, respectively, which were 2 and 469 with SiO2 /DAC 1/10 and of 1/2, respectively, which were derived 1060, 801cmand, for 469samples cm , for samples with of SiO 1/10 and 1/2, respectively, which from were 2/DAC derived from the absorption of Si–O–Si. It is therefore suggested that SiO 2 was probably the absorption of Si–O–Si. It is therefore suggested that SiO was probably immobilized onto the derived from the absorption of Si–O–Si. It is therefore 2 suggested that SiO2 was probably immobilized onto the DAC molecules. DAC molecules. immobilized onto the DAC molecules.
Figure 1. 1. FTIR FTIR spectra spectra of of DAC (a), SiO (d) and the SiO /DAC hybrid materials with ratios of of 1/10 (b) Figure 1. FTIR spectra of DAC DAC (a), (a), SiO SiO222(d) (d)and andthe theSiO SiO22/DAC hybrid materials with ratios 1/10 2 /DAC Figure hybrid materials with ratios of 1/10 (b) andand 1/2 1/2 (c). (c). (b) and 1/2 (c).
The TG thermographs thermographs and the the DTG curves curves (the inserts) inserts) for for samples samples are are shown shown in in Figure Figure 2. 2. As The The TG TG thermographsand and theDTG DTG curves(the (the inserts) for samples are shown in FigureAs 2. indicated by the high residual masses after the decomposition step of the TG curves, it was found indicated by the high residual masses after the decomposition step of the TG curves, it was found As indicated by the high residual masses after the decomposition step of the TG curves, it was found that DAC DAC had had a low low char char yield yield on on pyrolysis, pyrolysis, while while the the char char yields yields were were increased increased with with the increase increase that that DAC had aa low char yield on pyrolysis, while the char yields were increased with thethe increase of of SiO /DAC ratios. This could be attributed to the hybridization of DAC with SiO –NH , as as the the 2 2 2 of ratios. This could be attributed the hybridization ofwith DACSiO with SiO –NH 2/DAC SiOSiO ratios. This could be attributed to thetohybridization of DAC the2,latter is 2 /DAC 2 –NH 2 , 2as latter is an inorganic matter and exhibited a high char yield, as shown in Figure 2. latter is an inorganic matter and exhibited a high char yield, as shown in Figure 2. an inorganic matter and exhibited a high char yield, as shown in Figure 2.
Figure 2. 2. Thermogravimetric Thermogravimetric curves curves of of the the SiO SiO2/DAC /DAC hybrid materials. materials. Figure Figure 2. Thermogravimetric curves of the SiO hybrid materials. 22/DAC hybrid
From the the DTG DTG curves, curves, the the corresponding corresponding temperature temperature of of the the maximum maximum decomposition decomposition rate rate From From thebeDTG curves,We thehave corresponding temperature of the maximum decomposition rateof(T359.6 max ) (T ) could obtained. previously observed that the native cellulose had a T max max (T max) could be obtained. We have previously observed that the native cellulose had a Tmax of ◦ C359.6 could be After obtained. We have previously observed that the native cellulose hadthe a Tresultant [31]. max of 359.6 °C [31]. treatment with NaIO , the T was reduced to 328.2 °C for DAC. The 4 max °C [31]. After treatment with NaIO4, the Tmax was reduced 328.2 °C for the resultant DAC. The ◦ Cto After treatment with NaIO , the T was reduced to 328.2 for the resultant DAC. The reduction in max 4 reduction in decomposition temperature for DAC compared to that of cellulose has been reported reduction in decomposition temperature for DAC compared to that of cellulose has been reported decomposition temperature for DAC compared to that of cellulose has been reported by Kim et al. [25]. by Kim Kim et et al. al. [25]. [25]. However, However, the the SiO SiO2/DAC /DAC hybrid hybrid materials materials had had TTmax of of 355.4 355.4 and and 341.8 341.8 °C °C for for by 2 max◦ However, the SiO /DAC hybrid materials had T of 355.4 and 341.8 C for SiO /DAC ratios of max 2 2 SiO2/DAC /DAC ratios ratios of of 1/10 1/10 and and 1/2, 1/2, respectively, respectively, indicating indicating that that the the thermal thermal stability stability of of DAC DAC could could be be SiO 2 1/10 and 1/2, respectively, indicating that the thermal stability of DAC could be improved by the
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improved introduction matter. It should be noted that there was no introduction inorganic matter.of It inorganic should be noted that was distinct for the improved by byofthe the introduction of inorganic matter. It there should beno noted thatweight there loss waspeak no distinct distinct weight loss peak for the pure SiO that the sample was inorganic. 2, suggesting pure SiO , suggesting that the sample was inorganic. weight loss 2 peak for the pure SiO2, suggesting that the sample was inorganic. Figure peaks observed at Figure 33 shows the XRD XRDpatterns patternsof ofall allthe thesamples. samples.For ForDAC, DAC,the thediffraction diffraction peaks observed shows the the XRD patterns of all the samples. For DAC, the diffraction peaks observed at ◦ ◦ ◦ 14.8°, 16.3° and 22.4° were attributed to the cellulose I pattern [32], but it was also reported that the at 14.8 , 16.3 and 22.4 were attributed to the cellulose I pattern [32], but it was also reported that 14.8°, 16.3° and 22.4° were attributed to the cellulose I pattern [32], but it was also reported that the ◦ crystallinity would be than that [33]. aa broad peak ~23.5°, which the crystallinity would be lower that of cellulose [33]. SiO2 exhibited a broad peak at ~23.5 crystallinity would be lower lower thanthan that of of cellulose cellulose [33]. SiO SiO22 exhibited exhibited broad peak at at ~23.5°, which, was in accordance with the reported result and suggested an amorphous form for SiO [34]. In the 2 which in accordance with the reported and suggested an amorphous form for SiOIn was inwas accordance with the reported result result and suggested an amorphous form for SiO the 2 [34]. 2[34]. case of hybrid material with SiO mass ratios of 1/10 and 1/2, the diffraction peaks 2/DAC In the case of hybrid material with SiO /DAC mass ratios of 1/10 and 1/2, the diffraction peaks case of hybrid material with SiO2/DAC mass ratios of 1/10 and 1/2, the diffraction peaks 2 disappeared significantly and and disproportionately in terms of the SiO /DAC mass ratios, indicating disappeared significantly and disproportionately disproportionately in in terms termsof ofthe theSiO SiO2 2/DAC mass ratios, ratios, indicating indicating 2/DAC mass the obvious decline in the degree of crystallinity of DAC. This observation suggests that there the obvious obvious decline declineininthe thedegree degree crystallinity of DAC. observation suggests that might there of of crystallinity of DAC. ThisThis observation suggests that there might be an interaction between DAC and SiO particles, restricting the capacity of DAC to form 2SiO might be an interaction between DAC and particles, restricting the capacity of DAC to form aaa be an interaction between DAC and SiO2 particles, restricting the capacity of DAC to 2 well-defined order. well-defined well-defined order. order.
Figure3. 3. XRD patterns patterns of the the SiO22/DAC /DAC hybrid Figure hybrid materials. materials. Figure 3.XRD XRD patternsof of theSiO SiO2/DAC hybrid materials.
3.2. 3.2. FESEM of SiO SiO22-DAC -DAC FESEM of -DAC Figure shows the FESEM images for DAC and SiO (1/2). ItIt can that DAC 2/DAC (1/2). Figure can be be seen seen Figure 444 shows shows the the FESEM FESEM images imagesfor forDAC DACand andSiO SiO can be seen that that DAC DAC 2 /DAC 2/DAC (1/2). It exhibited long and short rods (Figure 4a). For SiO /DAC, there were many particles of SiO -NH 2 2 2 exhibited long and there were many particles of SiO -NH and short short rods rods(Figure (Figure4a). 4a).For ForSiO SiO2 /DAC, /DAC, there were many particles of SiO -NH 2 22 2 2 bonded to surface of DAC, which further demonstrates that interaction occurred between the bonded of DAC, which further demonstrates that interaction occurred occurred between the molecules bonded totosurface surface of DAC, which further demonstrates that interaction between the molecules of SiO -NH and DAC. In addition, it was obviously found that the size of SiO -NH 2 2 2 2 was of SiO2 -NH and2-NH DAC. In addition, it was obviously found that the size tens to molecules of2 SiO DAC. In addition, it was obviously found that of theSiO size of SiO 2 -NH 2 was 2 and 2-NH 2 was tens of For in SiO to hundreds of nanometers. For simplicity, in the following, SiO refers SiO -NH tens to to hundreds hundreds of nanometers. nanometers. For simplicity, simplicity, in the the following, following, SiO2torefers refers to SiO SiO. 2-NH -NH2.. 2
2
2
2
2
2
Figure 4. FESEM images (scale bar = 3 μm) of DAC (a), and SiO2/DAC (1/2) (b) (Small figure at Figure 4. 4. FESEM FESEM images images (scale (scale bar bar == 33 µm) μm)ofofDAC DAC(a), (a),and andSiO SiO/DAC (1/2) (b) (b) (Small (Small figure figure at at 2/DAC (1/2) Figure bottom left has a magnification of 200,000 (the blue dashed outline).2 Bars, 500 nm.). bottom left has a magnification of 200,000 (the blue dashed outline). Bars, 500 nm.). bottom left has a magnification of 200,000 (the blue dashed outline). Bars, 500 nm.).
3.3. 3.3. The The Cross-Linking Cross-Linking of of Collagen Collagen Using Using SiO SiO22/DAC /DAC Hybrid Hybrid Materials Materials as as Modifiers Modifiers
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The preparedSiO SiO22/DAC /DAC hybrid hybrid materials, materials,asaswell wellasasthe the pure DAC applied in The prepared pure DAC andand SiOSiO applied in the 2, were 2 , were the modification of collagen solution. the modifiers were into added into collagen the modification of collagen solution. As theAs modifiers were added collagen solution, solution, the powders powders were dispersed quickly withand stirring and then dissolved to of thebasic effectpH. of were dispersed quickly with stirring then dissolved gradually gradually due to thedue effect basic pH. be It should out thatthe although wasDAC just could 11.0, DAC could bebecause dissolved It should pointedbe outpointed that although pH wasthe justpH 11.0, be dissolved its because has been destroyed during processes, the previous processes, which included structureits hasstructure been destroyed during the previous which included hydrolysis andhydrolysis oxidation. and oxidation. Aftermixtures reaction, could clear mixtures could be obtained. Scheme 2 presents themechanism cross-linking After reaction, clear be obtained. Scheme 2 presents the cross-linking of mechanism of SiO collagen with SiO /DAC, which could possibly be ascribed to two types of reaction in 2 collagen with /DAC, which could possibly be ascribed to two types of reaction in the mixtures. 2 the mixtures. Firstly,the there exists thereaction Schiff base reaction the amino groups and of collagen and Firstly, there exists Schiff base between the between amino groups of collagen the paired the pairedgroups aldehyde of the cross-linking [19]; secondly, the hydroxyl groups aldehyde of groups the cross-linking reagents reagents [19]; secondly, the hydroxyl groups of SiO2 of inSiO the2 in the hybrid materials form hydrogen bonds with thegroups hydroxyl groups or amino groups of hybrid materials can formcan hydrogen bonds with the hydroxyl or amino groups of collagen [21]. collagen [21]. In fact, the cross-linking mechanism might be more complex. The cross-linking In fact, the cross-linking mechanism might be more complex. The cross-linking products were then products weretothen characterized to evaluate the influences of SiO2/DAC materials on the characterized evaluate the influences of SiO2 /DAC hybrid materials on thehybrid structural properties of structural properties of collagen solutions. collagen solutions.
Scheme collagen with SiO (a) Scheme 2. 2. Schematic Schematicillustration illustrationofofthe thecross-linking cross-linkingmechanism mechanismof of collagen with SiO 2/DAC. 2 /DAC. Interaction between the the amino groups of collagen andand the the paired aldehyde groups of DAC; (b) (a) Interaction between amino groups of collagen paired aldehyde groups of DAC; hydrogen bonds formed between thethehydroxyl SiO22/DAC the hydroxyl hydroxyl (b) hydrogen bonds formed between hydroxylgroups groupsof of SiO SiO22 in in SiO /DAC and and the groups oramino aminogroups groups of collagen; (c) more SiO SiO2/DAC replaced aldehyde groups and groups or of collagen; (c) more SiO2 in SiO replaced aldehyde groups and bonded 2 in 2 /DAC bonded to the molecules of collagen). to the molecules of collagen).
SDS-PAGE was performed performed to to identify identify the the molecular molecular weight weight distribution distribution of of collagens. collagens. As As can can SDS-PAGE was be seen from from Figure Figure 5, 5, the the pattern pattern for for all all the the collagens collagens consists consists of of two two α-chains α-chains (α1 (α1 and and α2) α2) as as the the be seen major constituents. Components with higher molecular weight (MW), including βand major constituents. Components with higher molecular weight (MW), including β- and γ-components, γ-components, were This also suggests observed.that This that the native triple-helix of destroyed collagen was not were also observed. thesuggests native triple-helix of collagen was not by the destroyed the introduction of confirmed cross-links, which confirmed by the quite between similar introductionby of cross-links, which was by the quitewas similar characteristics observed characteristics observed between the FTIR spectra of all of the collagens (as shown in Figure 6).by In the FTIR spectra of all of the collagens (as shown in Figure 6). In the case of collagen modified the case of collagen modified by DAC, the band intensity for α-chains and β-components was significantly reduced, which could be due to the formation of cross-linked aggregates with higher
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99 of 15 of 15 and β-components was significantly reduced, which could be due to the formation of cross-linked aggregates with higher MW that could not get through the gel [35]. MW not through gel as in gel patterns, the MW that that could could not get getin through the gel [35]. [35]. However, as displayed displayedeffect in the theon gel patterns, the chemical chemical However, as displayed the gel the patterns, theHowever, chemical cross-linking collagen became weaker cross-linking effect on collagen became weaker when SiO /DAC or pure SiO were employed. cross-linking effect on collagen became weaker when SiO /DAC or pure SiO were employed. 2 2 2 2 when SiO2 /DAC or pure SiO2 were employed. Polymers 2018, 10, Polymersthe 2018, 10, xx FOR FOR PEER PEER REVIEW REVIEW DAC, band intensity for α-chains
Figure 5. patterns of native collagen and collagens modified by the SiO hybrid Figure 5. 5. SDS-PAGE SDS-PAGE patterns patterns of of native native collagen collagenand andcollagens collagensmodified modifiedby bythe theSiO SiO /DAC hybrid hybrid Figure SDS-PAGE 22/DAC 2 /DAC materials: DAC; materials: (lane 1) 1) protein protein markers; markers; (lane 2) 2) native native collagen; (lane 3) collagen modified by materials: (lane (lane 1) protein markers; (lane (lane 2) native collagen; collagen; (lane (lane 3) 3) collagen collagen modified modified by by DAC; DAC; (lane 4) collagen collagenmodified modifiedby bySiO SiO /DAC (1/10); (lane 5) collagen modified by SiO /DAC (1/2); (lane /DAC (1/10); (lane 5) collagen modified by SiO /DAC (1/2); (lane 6) (lane 4) 4) collagen modified by SiO /DAC (1/10); (lane 5) collagen modified by SiO /DAC (1/2); (lane 6) 22 2 22 2 (lane 6) collagen modified collagen modified by collagen modified by SiO SiO22..by SiO2 .
Figure 6. of native collagen (a), and collagen cross-linked by the SiO hybrid Figure 6. 6. FTIR FTIR spectra spectra of of native native collagen collagen (a), (a), and andcollagen collagencross-linked cross-linkedby bythe theSiO SiO /DAC hybrid hybrid Figure FTIR spectra 22/DAC 2 /DAC materials: by DAC (b); collagen cross-linked by SiO (1/10) (c); collagen materials: collagen collagen cross-linked cross-linked by by DAC DAC(b); (b);collagen collagencross-linked cross-linkedby bySiO SiO /DAC(1/10) (1/10) (c); (c); collagen collagen materials: collagen cross-linked 22/DAC 2 /DAC cross-linked cross-linked by SiO /DAC(1/2) (1/2)(d); (d);and andcollagen collagencross-linked cross-linkedby bySiO SiO (e). cross-linked by by SiO SiO222/DAC /DAC (1/2) (d); and collagen cross-linked by SiO 22 (e). 2(e).
3.4. 3.4. DSC DSC Thermograms of of Cross-Linked Cross-Linked Collagens Collagens 3.4. DSC Thermograms Thermograms of Cross-Linked Collagens DSC native collagen and collagen modified by the SiO hybrid materials DSC thermograms thermograms of of native native collagen collagen and andcollagen collagenmodified modifiedby bythe theSiO SiO /DAC hybrid hybrid materials materials 2/DAC DSC thermograms of 22/DAC are shown in Figure 7. Endothermal peaks associated with the helix-coil transition due to the are shown in Figure 7. Endothermal peaks associated with the helix-coil transition due to the the are shown in Figure 7. Endothermal peaks associated with the helix-coil transition due to thermal collagen, thermal disruption disruption of of hydrogen hydrogen bonds, bonds, which which was was thought thought to to stabilize stabilize the the triple triple helix helix of of collagen, collagen, thermal disruption of hydrogen bonds, which was thought to stabilize the triple helix of were detectable from the heat flow curves as a function of temperature. The un-modified collagen were detectable from the heat flow curves as a function of temperature. The un-modified collagen were detectable from the heat flow curves as a function of temperature. The un-modified collagen ◦ had of had aaa denaturation denaturation temperature (T of 38.9 38.9 °C, °C, which which was was in in line line with with previous previous reports reports [36,37]. [36,37]. As As dd)) had denaturationtemperature temperature(T (T d ) of 38.9 C, which was in line with previous reports [36,37]. ◦ C. for collagen cross-linked by DAC, the TTdd T was significantly increased by 14.4 °C. However, the T for for collagen cross-linked by by DAC, thethe was significantly increased by 14.4 °C. However, the T of ddTof As collagen cross-linked DAC, was significantly increased by 14.4 However, the d d cross-linked collagen by SiO /DAC decreased with increasing SiO /DAC ratios, but was still cross-linked collagen by SiO /DAC decreased with increasing SiO /DAC ratios, but was still 22 2 /DAC decreased with increasing SiO 222 /DAC ratios, but was still of cross-linked collagen by SiO obviously obviously higher higher than than that that of of the the un-modified un-modified collagen. collagen. Note Note that that the the cross-linking cross-linking of of pure pure SiO SiO22 had had aa slight slight effect effect on on the the thermal thermal stability stability of of collagen. collagen.
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obviously higher than that of the un-modified collagen. Note that the cross-linking of pure SiO2 had a 10 of 15 Polymerseffect 2018, 10, FORthermal PEER REVIEW slight onxthe stability of collagen.
Figure 7.7.DSC DSCthermograms thermograms native collagen (a), collagen and collagen cross-linked by 2the SiOhybrid 2/DAC Figure of of native collagen (a), and cross-linked by the SiO /DAC hybrid materials: collagen cross-linked by DAC (b); collagen cross-linked by SiO /DAC (1/10) (c); 2 materials: collagen cross-linked by DAC (b); collagen cross-linked by SiO2 /DAC (1/10) (c); collagen collagen cross-linked by SiO /DAC (1/2) (d); and collagen cross-linked by SiO (e). 2 cross-linked by SiO2 /DAC (1/2) (d); and collagen cross-linked by SiO2 (e). 2
3.5. Dynamic of Cross-Linked Cross-Linked Collagens Collagens 3.5. Dynamic Rheology Rheology Tests Tests of It was was reported reported that that cross-linked cross-linked collagen collagen was was more more resistant resistant to to deformation deformation and and to to flow flow than than It native collagen collagen[38], [38],which whichwas wasreflected reflectedinin viscoelastic properties. The frequency dependence of native itsits viscoelastic properties. The frequency dependence of G’, G’, tan δ and η* for all the samples over the frequency range of 0.01–10 Hz at 25 °C is depicted in ◦ tan δ and η* for all the samples over the frequency range of 0.01–10 Hz at 25 C is depicted in Figure 8. Figure can be seen from Figure 8, of theG’values of G’ wereexponentially increased exponentially with the As can 8. be As seen from Figure 8, the values were increased with the addition of addition of DAC. However, the values of G’ decreased remarkably for the collagen modified by DAC. However, the values of G’ decreased remarkably for the collagen modified by SiO2 /DAC (1/10), SiO2/DACthey (1/10), although theythan were stillofgreater than thosecollagen. of the un-modified collagen. With although were still greater those the un-modified With increasing SiO2 /DAC increasing SiO2/DAC the values of G’ decreased continuously. It was to note that ratios, the values of G’ratios, decreased continuously. It was interesting to note thatinteresting collagen modified by collagen modified by SiO displayed a higher G’ than that of native collagen when the shearing 2 SiO2 displayed a higher G’ than that of native collagen when the shearing frequency was no more than frequency wasthe novalues more were than 0.2 Hz, thenative valuescollagen were close to the those of native collagenfurther when 0.2 Hz, while close to while those of when shearing frequency the shearing further to 10 Hz. It seemed that the network of increased to 10frequency Hz. It seemed that increased the entanglement network of collagen was entanglement not stable enough because collagen was not stable enough because of the weak interactions between collagen and SiO 2, and of the weak interactions between collagen and SiO2 , and thus the network was easily destroyed when thussamples the network wasa strong easily mechanical destroyed treatment. when the samples underwent a strong mechanical the underwent treatment. The technique of dynamic oscillation is useful in resolving the structural properties of materials technique dynamicresponse oscillation in resolving theGenerally, structural of into aThe solid-like and aofliquid-like (G’ is anduseful G”, respectively) [39]. theproperties ratio G”/G’, materials into a solid-like a liquid-like response (G’ and G”, respectively) [39]. Generally, the which is defined as the lossand tangent (tan δ), was used to describe the flowing behavior of molecules, ratio G”/G’, which is defined as the loss tangent (tan δ), was used to describe the flowing behavior and it crossed the threshold (tan δ = 1) from solid-like to liquid-like behavior [40]. That is, the smaller of molecules, and it crossed the threshold (tan δ =the 1) behavior from solid-like to liquid-like behavior [40]. the value of tan δ, the more rubbery or elastomeric of the material was [41]. The tan δ That is, the smaller the value of tan δ, the more rubbery or elastomeric the behavior of the material value of all samples was smaller than 1 under our experimental conditions, indicating a solid-like was [41].ofThe tan δ solution. value of all samplesaswas smaller than 1the under our experimental conditions, network collagen However, DAC was added, modified collagen exhibited tan δ indicating a solid-like network of collagen solution. However, as DAC was added, the modified values below 0.17, indicating the absolute solid-like behavior of this sample. When the SiO2 /DAC collagenmaterials exhibitedwere tan δused, values indicating the absolute solid-like behavior of thisthat sample. hybrid thebelow tan δ 0.17, of collagen increased to different extents, suggesting the When the SiO /DAC hybrid materials were used, the tan δ of collagen increased to different extents, 2 solid-like network tended to become a liquid-like network, due to the moderate cross-linking effect of suggesting that the solid-like network become abyliquid-like network, due to the moderate the hybrid modifier. With respect to thetended sampletomodified SiO2 , the tanδ curves displayed a trend cross-linking effect of the hybrid modifier. With respect to the sample modified by SiO2,the theweak tanδ similar to that of G’ when the shearing frequency was increased, which again confirmed curves displayed a trend similar to that of G’ when the shearing frequency was increased, which interaction between collagen and SiO2 . againFrom confirmed the weak interaction between(η*) collagen and 2. the changes in the complex viscosity (Figure 8c),SiO it could be seen that all samples showed From the changes in the complex viscosity (η*) (Figure 8c), it could be samples a shear thinning behavior. Note that the η* values of collagen cross-linked by seen DACthat wereallfar larger showed a shear thinning behavior. Note that the η* values of collagen cross-linked by DAC were far than those for the other samples, indicating that a rapid cross-linking reaction had occurred between larger than those for the other samples, indicating that a rapid cross-linking reaction had occurred between collagen and DAC, while the η* values of collagen cross-linked by SiO2/DAC or SiO2 decreased as the ratio of SiO2 in SiO2/DAC increased. The decrease in viscosity due to the introduction of SiO2 was in favor of processing for collagen solution.
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collagen2018, and10,DAC, the η* values Polymers x FORwhile PEER REVIEW
of collagen cross-linked by SiO2 /DAC or SiO2 decreased11 asofthe 15 ratio of SiO2 in SiO2 /DAC increased. The decrease in viscosity due to the introduction of SiO2 was in favor of processing for collagen solution.
Figure 8. G’ (a), tan tan δδ (b) (b) and and η* η*(c) (c)ofofnative nativecollagen collagenand andcollagen collagencross-linked cross-linkedby bythe theSiO SiO Figure 8. G’ (a), 2/DAC 2 /DAC hybrid hybrid materials. materials.
3.6. 3.6. FESEM FESEM of of Cross-Linked Cross-Linked Collagens Collagens The 9, displayed displayed aa well-ordered well-ordered porous porous structure structure for for native native The FESEM FESEM images, images, as as shown shown in in Figure Figure 9, collagen (Figure 9a). As for collagen modified by DAC (Figure 9b), the sponge had a heterogeneous collagen (Figure 9a). As for collagen modified by DAC (Figure 9b), the sponge had a heterogeneous structure, result of of the the excessively excessively fast fast cross-linking cross-linking effect. effect. It be observed observed from from structure, which which was was aa result It could could be Figure that the the permeability permeability of of pores pores was was decreased, decreased, and and the the aggregation aggregation of of collagen collagen was was Figure 8b 8b that enhanced due to the cross-linking effect of DAC. In contrast, with the increased ratio of SiO /DAC, enhanced due to the cross-linking effect of DAC. In contrast, with the increased ratio of SiO2 /DAC, 2 the morphological characteristics characteristics of of porosity porosity and and fibrillar fibrillar network network of of modified modified collagen collagen became became the morphological evident, for the the sample sample modified modified by bySiO SiO2 /DAC(1/2) that aa evident, particularly particularly for (Figure 9d), 9d), suggesting suggesting that 2/DAC(1/2) (Figure moderate cross-linkingeffect effectononcollagen collagen could be imposed by hybrid the hybrid modifier. It should be moderate cross-linking could be imposed by the modifier. It should be noted noted that collagen modified by the pure SiO2 had a distinct morphology compared to the other samples; that is, a large number of fibers were visible. This morphology was quite similar to that reported previously [34], indicating that the aggregation behavior of collagen was influenced by the addition of nano-SiO2.
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that collagen modified by the pure SiO2 had a distinct morphology compared to the other samples; that is, a large number of fibers were visible. This morphology was quite similar to that reported previously [34], indicating that the aggregation behavior of collagen was influenced by the addition of 12 of 15 Polymers 2018, nano-SiO 2 . 10, x FOR PEER REVIEW
Figure 9. FESEM FESEMimages images (scale = 500 of native collagen (a),collagen and collagen cross-linked Figure 9. (scale barbar = 500 μm)µm) of native collagen (a), and cross-linked by the by SiO2 hybrid /DAC hybrid materials: collagen cross-linked DAC (b);collagen collagen cross-linked cross-linked by SiOthe materials: collagen cross-linked by by DAC (b); by 2/DAC SiO22/DAC(1/10) /DAC(1/10)(c); (c);collagen collagen cross-linked SiO /DAC(1/2) (d); and collagen cross-linked by cross-linked byby SiO /DAC(1/2) (d); and collagen cross-linked by SiO 2 2 2 SiO (e). 2 (e).
4. Conclusions A novel cross-linking agent of collagen was prepared via the hybridization of DAC and nano-SiO2. The structure of the SiO2/DAC hybrid materials was studied. Then, SiO2/DAC hybrid materials were added into collagen solutions. Multiple sources of evidence were provided that the
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4. Conclusions A novel cross-linking agent of collagen was prepared via the hybridization of DAC and nano-SiO2 . The structure of the SiO2 /DAC hybrid materials was studied. Then, SiO2 /DAC hybrid materials were added into collagen solutions. Multiple sources of evidence were provided that the triple-helix of collagen was not destroyed; rather, the thermal stability could be significantly improved due to the interactions between SiO2 /DAC and collagen. Compared to collagen modified by pure DAC, collagen modified by the SiO2 /DAC hybrid materials exhibited a better flowability, protecting collagen solutions against excessive gelation. This will be of significance for the development of new cross-linkers and for the process and design of materials based on collagen in the format of solution. Author Contributions: M.Z. and C.D. conceived and designed the experiments; Y.Z., B.Y. and X.Y. performed the experiments; M.Z. and C.D. analyzed the data; R.S. contributed part of reagents/materials/analysis tools; C.D. and M.Z. wrote the paper. Acknowledgments: This work was financially supported by the National Natural Science Foundation of China (Grant Nos. 21606046 and21306024), the Natural Science Foundation of Fujian Province (Grant No. 2016J01208), and the Special Fund for Science and Technology Innovation of FAFU (Grant Nos. KFA17217A and KFA17476A). Conflicts of Interest: The authors declare no conflict of interest.
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