Article pubs.acs.org/Biomac
Synthesis and Fabrication of Nanocomposite Fibers of Collagen-Cellulose Nanocrystals by Coelectrocompaction Elvis Cudjoe,†,⊥ Mousa Younesi,‡,⊥ Edward Cudjoe,‡,∥ Ozan Akkus,*,‡ and Stuart J. Rowan*,†,§ †
Department of Macromolecular Science and Engineering, Case Western Reserve University, Cleveland, Ohio 44106, United States Department of Mechanical and Aerospace Engineering, Case Western Reserve University, Cleveland, Ohio 44106, United States § Institute for Molecular Engineering and Department of Chemistry, University of Chicago, 5640 S. Ellis Avenue, Chicago, Illinois 60637, United States ‡
S Supporting Information *
ABSTRACT: An electrochemical process has been used to compact cellulose nanocrystals (CNC) and access aligned micron-sized CNC fibers. Placing a current across aqueous solutions of carboxylic acid functionalized CNCs (t-CNC−COOH) or carboxylic acid/primary amine functionalized CNCs (t-CNC−COOH-NH2) creates a pH gradient between the electrodes, which results in the migration and concentration of the CNC fibers at their isoelectric point. By matching the carboxylic acid/amine ratio of CNCs and collagen (ca. 30:70 carboxylic acid:amine ratio), it is possible to coelectrocompact both nanofibers and access aligned nanocomposite fibers. t-CNC−COOH-NH2/collagen fibers showed a maximum increase in mechanical properties at 5 wt % of t-CNC−COOH-NH2. Compared to collagen/CNC films which have no alignment in the plane of the films, the tensile properties of the aligned fibers show a significant enhancement in the wet mechanical properties (40 MPa vs 230 MPa) for the 5 wt % of t-CNC−COOH-NH2/collagen films and fiber, respectively.
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INTRODUCTION Collagen fibers are made up of collagen molecules that are formed by the association of the triple helix polypeptide chains.1 Type-I collagen is the most abundant natural protein in the human body and is the key element that imparts structural integrity and tensile strength to many tissues in the body.2 It is recognized by cells through its cell attachment sites3 and as such, collagen is used as a key ingredient in a variety of tissue engineering applications such as drug delivery agents,4,5 wound healing biomaterials,6 and scaffolds,7 to name a few. There has been interest in recent years in developing methods to compact collagen molecules with high orientational order to broaden the application range of collagen.8−12 Controlling the assembly process in order to compact the collagen molecules into aligned structures could greatly impact the properties of the highly oriented tissue-like biomaterials. As such, researchers have investigated the use of magnetic particles8 and electrospinning13 to induce alignment of collagen molecules. In spite of extensive studies on collagen and its assembled nanofibers, the common problem of biodegradation and low © XXXX American Chemical Society
mechanical properties still exists which limits their promising application in tissue engineering. Thus, a number of studies have targeted the improvement of collagen-based biomaterials by incorporating fillers in order to enhance the mechanical properties of these materials. Incorporation of inorganic nanosized hydroxyapatite in a highly porous collagen sponge resulted in 4-fold increase in stiffness (ca. 230 to 900 Pa).14 Niu et al.15 reported collagen/silica-based composite sponges which were made by infiltrating choline-stabilized silicic acid precursors into polyallylamine-enriched collagen. At 50 wt % of silica, there was an increase in the modulus from 0.1 kPa to more than 150 kPa. An alternative method for improving the mechanical properties of collagen based-biomaterials is the incorporation of synthetic polymers.16−18 Yoo and co-workers18 developed a collagen/polycaprolactone (PCL) composite fibers with the goal of withstanding physiologic vascular conditions Received: January 2, 2017 Revised: March 3, 2017
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DOI: 10.1021/acs.biomac.7b00005 Biomacromolecules XXXX, XXX, XXX−XXX
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Biomacromolecules such as high pressure and flow. The composites demonstrated good biomechanical properties with a tensile strength of ca. 8.3 MPa compared to the control sample with a tensile strength of ca. 5.1 MPa. Cellulose nanocrystals (CNCs) have been extensively studied in recent years as a reinforcing material for polymer matrices on account of its interesting properties such as biosustainability, biorenewability, relatively low production cost, and low cytotoxicity.19 Depending on the source and method of isolation, the CNC diameter ranges from ca. 5 to 30 nm, while length can range from hundreds of nanometers to several micrometers. CNCs have been used to reinforce a variety of polymer matrices on account of a combination of properties such as high aspect ratio, high crystallinity, low density, and high elastic moduli.20−23 Relevant to this work is that CNCs have been used to reinforce collagen composite films.24−27 Zhang and co-workers24 made collagen/CNCs nanocomposites using CNCs isolated from microcrystalline cellulose. Composites were made by solution casting, and the CNC content was varied from 0 to 10 wt %. The mechanical properties of the composite films showed an increase (2.4 times) in tensile strength with up to 7 wt % filler incorporated. Above this threshold, the tensile strength of the composite decreased, and this effect was attributed to the higher content of the CNCs disrupting rather than reinforcing the collagen matrix. Interestingly, results from cell viability studies showed no apparent cytotoxicity. Pooyan et al.25 also made collagen/CNC composites from type I collagen and CNCs isolated from microcrystalline cellulose. With varying content of CNCs (1−9 wt %) in the collagen matrix, they saw an increase in shear modulus with up to about 3 wt % of CNCs and a systematic decrease with higher CNC content. The decrease in the mechanical properties was attributed to the CNC aggregation/phase separation in the composite. However, in both systems, the CNCs were dispersed in a randomly oriented collagen matrix prompting the question of how the CNCs might impact the properties of a more oriented collagen matrix. Akkus and co-workers12,28 reported an electrochemical alignment technique that controls the assembly of type I collagen and allow access to highly oriented and densely packed collagen bundles and fibers. This process involves the application of an electric current to an aqueous collagen solution that results in a compacted bundle of collagen nanofibers.12,28,29 This electrochemical compaction process is dependent on the net charge of collagen, which, on account of the fact that it contains pH-sensitive groups (carboxylic acid and amines), is known to vary depending on the pH. For example, collagen molecules have −0.8 coulombs of charge at pH 3 and +0.8 coulombs of charge at pH 11.29 The application of an electric field across an aqueous collagen solution results in the migration of the charged molecules toward the opposite charged electrode. The electric current also results in a pH gradient between the two electrodes; thus, the collagen migrates until it reaches the isoelectric point at which its net charge is zero, and the electrokinetic mobility is arrested. Aligned collagen bundles after cross-linking demonstrated a 30-fold increase in mechanical strength in the alignment direction compared to that of cross-linked randomly oriented collagen fibers. A scanning electron microscopy (SEM) image of the cross-linked aligned collagen bundle showed dense, close packed collagen fibers with a relatively uniform orientation. As expected, cell studies proved biocompatibility and good cell proliferation, which opens the door for these aligned collagen materials to be used in tissue engineering applications.12
The goal of this research is to investigate the potential of this electrochemical technique to access aligned collagen/t-CNC (t represents tunicate CNCs) fiber nanocomposites and investigate the effect that addition of t-CNCs has on the mechanical properties of the aligned collagen bundles (Figure 1). It is
Figure 1. Schematic of the electrochemical alignment process of collagen/CNC nanocomposite fibers (small (red) and large (green) fibers correspond to collagen and t-CNCs, respectively).
worth noting that polymer matrices with aligned CNCs have been prepared using electric fields,30−32 shear forces,33−35 or magnetic fields36−40 in recent years, and in all cases tested, an increase in mechanical strength is observed. For example, Eichhorn and co-workers39 demonstrated a 45% reinforcement (ca. 8.3 GPa) when CNCs were magnetically aligned in a cellulose matrix compared to that when the CNCs had no alignment (ca. 4.5 GPa). We are currently not aware of a composite system where the matrix (native collagen in this case) has also been coaligned and cocompacted with the CNCs.
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EXPERIMENTAL SECTION
Materials. 2,2,6,6-Tetramethylpiperidinyloxy (TEMPO), 4-dimethylaminopyridine (DMAP), and ethyl-3- (3-(dimethylamino)propyl) carbodiimide (EDC) were purchased from Sigma-Aldrich. tert-Butyl (2-aminoethyl)carbamate was synthesized according to previously published work.41 Acid extracted type-I bovine telocollagen solution (6 mg/mL) was purchased from Advanced BioMatrix, San Diego, CA. All other chemicals were purchased from Fisher Scientific and used without further purification. Genipin was purchased from Wako Pure Chemicals. Ultrathin carbon film on holey carbon support copper TEM grids and uranyl acetate were purchased from Ted Pella, Inc. Sea tunicates (Styela Clava) were collected from floating docks in Warwick Cove Marina (Warwick, RI) and were cleaned according to previously published procedures.42 Instrumentation. All sonication of t-CNCs was performed using a Q500 QSonica Ultrasonic processor at an amplitude of 40% Titration of carboxylic acid functional groups were performed using an Accumet AR50 dual channel pH/ion/conductivity meter. Functional group densities were determined following previously published procedures.20 t-CNC samples were freeze-dried and lyophilized on a VirTis benchtop K lyophilizer. FT-IR spectroscopy was performed on an Agilent Technologies Cary 600 series instrument. Transmission Electron Microscopy (TEM) of the t-CNCs was performed on FEI Technai F30 at 300 kV. t-CNCs were dispersed in water (0.1 wt %) and solution cast on a TEM grid which was subsequently stained with 2 wt % uranyl acetate solution. SEM was performed on FEI Helios Nanolab 650 after samples were sputter coated with ca. 7.5 nm Pd. Raman spectroscopy was performed on a Horiba Jobin Yvon 785 nm Raman system. TEMPO Oxidation of t-CNCs (t-CNC−COOH). Hydrolyzed t-CNCs isolated from mantles of sea tunicates were oxidized using TEMPO, NaBr, and NaOCl in 87% yield (by mass) following previously published procedures.20,43 The functional group density of B
DOI: 10.1021/acs.biomac.7b00005 Biomacromolecules XXXX, XXX, XXX−XXX
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Biomacromolecules Scheme 1. Synthesis of t-CNC−COOH and t-CNC−COOHNH2
t-CNC−COOH was 1021 ± 24 mmol/kg as determined by the conductometric titration described by Way et al.20 Synthesis of Amine functionalized t-CNC−COOH (t-CNC− COOH-NH2). t-CNC−COOH-NH2 was synthesized in 84% yield following procedures outlined in a prior publication20 with minor modifications. Briefly, t-CNC−COOH (0.5 mg, 0.6 mmol) was homogeneously dispersed in DMF (100 mL) using a sonicator. The solution was transferred to a 200 mL round-bottomed flask followed by the addition of DMAP (0.73 mg, 6 mmol) and EDC (1.15 g, 6 mmol). After the reactants were fully dissolved, tert-butyl (2-aminoethyl) carbamate (0.96 mg, 6 mmol) was added to the solution and allowed to stir at room temperature for 18 h. The resulting material was precipitated in excess ethyl acetate and filtered using a fine fritted filter. The filtrate was washed extensively with ethyl acetate (×5) and water (×5). The resulting functionalized t-CNC were dispersed in water (150 mL) and refluxed for 3 h to remove the Boc protecting group. The reaction was allowed to cool down after 3 h followed by extensive washing with water (×5). The solids were freeze-dried and lyophilized to yield a white fluffy t-CNC−COOH-NH2. The amine functional group density was determined to be 730 ± 39 mmol/kg. Electrical Alignment of Collagen/t-CNC Fiber Composite. Acid extracted type-I collagen solution was diluted 2-fold using RNase/DNase free water (Invitrogen, Carlsbad, CA), and the pH was adjusted to 8−10 using 1 N NaOH. The collagen solution was then dialyzed against ultrapure water at room temperature for 18 h to remove salts. t-CNC (5 mg/mL) was added to dialyzed collagen solution (5 mg/mL) at different weight percentages (ca. 0%, 2.5%, 5%, 7.5%, and 10%) to yield a total of 5 mL of aqueous solution with 0.5 wt % total solids content. The mixtures were aligned via an electrochemical compaction process as described in the previous study.44 Briefly, t-CNC/collagen mixture was loaded between two stainless steel wires (electrodes), and 20 V was applied for 60 s. In the presence of electric current, a pH gradient developed between the electrodes and the t-CNC/collagen aligned along the isoelectric point. Aligned t-CNC/ collagen was then treated with PBS buffer for 6 h at 37 °C to facilitate
Figure 2. POM image of electro-compacted 0.5 wt % (total solids) dispersions of (a) t-CNC−COOH, (b) t-CNC−COOH55−NH245, and (c) t-CNC−COOH27−NH273 at pH 7. fibril formation.45 Following PBS treatment, t-CNC/collagen threads were recovered from the electrochemical cells, immersed in isopropyl alcohol overnight, washed once with RNase/DNase free water (Invitrogen, Carlsbad, CA), and dried for 1 h. The t-CNC/collagen threads were then cross-linked by immersing into 0.625 wt % genipin (Wako Pure Chemical, Osaka, Japan) in 95 vol % ethanol solution for 3 days, followed by ultrapure water rinse and drying overnight. Electrocompaction of Nonoriented Collagen/t-CNC Film Composite. Collagen/t-CNC film composites were made following the previously published procedure.28 In short, a total of 5 mL of aqueous solution was made by mixing t-CNCs (5 mg/mL) at different weight percentages (ca. 0%, 5%, and 10%) with dialyzed collagen solution (5 mg/mL). The mixture was loaded between two carbon block electrodes which were separated from each other with a silicon rubber spacer (thickness of 2 mm). Twenty volts of electricity was applied to the electrodes, and as a result, the collagen/t-CNC solution was compacted into a disc-shaped composite film with no alignment of the components. The disc-shaped film was immersed in PBS buffer for 6 h at 37 °C, immersed in isopropyl alcohol overnight, and washed with RNase/DNase free water. The resulting composite material was dried for 1 h followed by cross-linking in 0.625 wt % genipin in 95 vol % ethanol solution for 3 days. The cross-linked film was rinsed 3 times with ultrapure water and dried overnight at room temperature. Tensile Tests. The mechanical properties of individual threads (n = 10/group) were measured using ARES (TA Instruments, New Castle, DA) as described previously.36 Briefly, 2 cm long samples C
DOI: 10.1021/acs.biomac.7b00005 Biomacromolecules XXXX, XXX, XXX−XXX
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Biomacromolecules
Figure 3. SEM image of dry electro-compacted fibers of (a) t-CNC−COOH, (b) t-CNC−COOH55−NH245, and (c) t-CNC−COOH23−NH273. were cut using a scalpel blade, and the samples were hydrated in 1× PBS for 1 h prior to testing. Samples were fixed onto the fixtures of the instrument at a gauge length of 10 mm and subjected to tensile loading until failure at a strain rate of 10 mm/min. Samples were tested in the wet state by rehydrating them briefly using a wet Q-tip prior to testing. The fractured samples were used for average crosssectional area measurement using a multiphoton confocal microscope (Leica TCS SP2) by measuring the wet area at three different locations along the length of the sample. Quantification of cross-sectional area was done using ImageJ (ImageJ 1.47v, U.S. National Institute of Health, Bethesda, MD) (ca. 0.015−0.065 mm2). Young’s modulus was determined by calculating the slope of the linear region of the stress− strain curve. Raman Spectroscopy. Raman spectroscopy of the electrochemically aligned fiber composite was collected using a 785 nm Raman system (Horiba Jobin Yvon, Edison, NJ, USA). The spectrum was obtained from 4 different locations on the fiber composite, and each spectrum was obtained as the average of 30 consecutive spectra collected for 10 s each. Polarization sensitivities of the Raman bands to orientation were collected with the aligned fibers parallel (ZZ) or perpendicular (XX) to the direction of polarization of the laser. Background subtraction from the spectra were performed using Labspec software (Horiba Jobin Yvon).
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RESULTS AND DISCUSSION Prior to this study, the only material that was shown to form aligned fibers using the electrochemical compaction process is collagen.12,28 However, conceptually the process should work for any ampholytic nanofiber. As such, the first step in this work was to determine if charged CNCs could be electrocompacted. Nonsurface modified CNCs were isolated from sea tunicates via HCl hydrolysis using standard procedures.20 To access charged water dispersible CNCs, the surface of the t-CNCs (t represents tunicates) was modified (Scheme 1) at the C6-position using well-known oxidation conditions (TEMPO,
Figure 4. POM images of the electro-compaction experiments with 0.5 wt % (total solids) dispersions of (a) 95:5 wt % collagen/t-CNC− COOH55−NH245 alignment and (b) 95:5 wt % collagen/t-CNC− COOH27−NH273.
NaOCl, and NaBr)20 that created carboxylic acid surface functional groups (t-CNC−COOH) with a functional group content of ca. 1000 mmol/kg (Figure S1). These t-CNC−COOH were then dispersed in DI water (5 mg/mL, 0.5 wt %) at a pH of 7, and 1 mL of the dispersion was exposed to 20 V of current between 2 stainless steel wires. D
DOI: 10.1021/acs.biomac.7b00005 Biomacromolecules XXXX, XXX, XXX−XXX
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Biomacromolecules
Figure 5. SEM images of dry cross-linked fiber composites with (a) 0, (b) 2.5, (c) 5, (d) 7.5, and (e) 10 wt % t-CNC−COOH27−NH273.
surface (t-CNC−COOH-NH2). It was decided therefore to prepare two different batches of such CNCs that varied in the ratio of amine and carboxylic acid groups. The first batch was targeted to have ca. 50:50 mix of the two functionalities, while the second batch was targeted to mimic the ratio of −COOH:−NH2 in collagen (ca. 30:70).46 To access these CNCs, different mole equivalents of the mono-Boc protected ethylene diamine with respect to the carboxylic acid groups were reacted with t-CNC−COOH (ca. 1000 mmol/kg). Five mole equivalents resulted in ca. 45% conversion of the carboxylic acid to the amide, while 10 mol equivalents resulted in ca. 70% conversion. After deprotection of the Boc-groups, the resulting t-CNC−COOH-NH2 have a primary amine group content of ca. 455 ± 28 mmol/kg (t-CNC−COOH55−NH245) and ca. 730 ± 39 mmol/kg (t-CNC−COOH27−NH273), and this was confirmed by conductometric titrations (Figures S3 and S4). In addition, FT-IR studies showed a lower intensity of the N−H bending for t-CNC−COOH55−NH245 at 1640 cm−1 relative to t-CNC−COOH27−NH273, consistent with a lower amine content (Figure S5). TEM images (Figure S6) showed
As postulated, on account of the surface negative charges on t-CNC−COOHs it migrates to the anode, as can be seen by the formation of a birefringent band in the polarized optical image (POM) shown in Figure 2a. Unfortunately, the extraction/ isolation of the aligned macroscopic fiber of the t-CNC−COOHs was difficult on account of the t-CNCs partly coating/adhering to the stainless steel anode (Figure S2), presumably a result of the relative high concentration of carboxylate/carboxylic acid moieties. One advantage that the CNCs have over collagen is the relative ease with which it is possible to alter the chemistry of their surface functional groups. This imparts the ability to tailor the nature of the CNCs surface charge and thus where they will concentrate between the two electrodes. Some of us (Rowan and co-workers) have previously shown, for example, that amine functionalized CNCs20 can be accessed (Scheme 1) using standard amide coupling conditions (EDC/DMAP) with a mono-Boc protected ethylene diamine, followed by the removal of the protecting Boc-group. The resulting CNCs contain a mixture of carboxylic acid and amine functionalities on the E
DOI: 10.1021/acs.biomac.7b00005 Biomacromolecules XXXX, XXX, XXX−XXX
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Biomacromolecules no significant degradation of the CNCs after this surface functionalization. Applying a voltage across an aqueous dispersion (1 mL of a 5 mg/mL dispersion) of these t-CNC−COOH-NH2 (at pH 7) results in the alignment of the fibers at their isoelectric point. Figures 2b and c show the POM images of these experiments indicating the formation of birefringent bands at different points between the electrodes, with t-CNC−COOH55−NH245 being aligned closer to the midpoint between the two electrodes and t-CNC−COOH27−NH273 aligning closer to the cathode owing to the higher content of positively charged species. Attempts to extract/isolate the aligned fibers with a pair of tweezers after alignment was extremely difficult on account of the narrow size (fiber diameter ca. 20 μm; see Figure S7 for lower magnification SEM) and brittle nature of the t-CNC nanofibers which caused them to break. However, SEM images on the recovered pieces showed similar features in all samples (Figure 3a−c) with densely packed nanofibers suggesting an effective compaction of the aqueous dispersion of t-CNC nanofibers. Having successfully demonstrated the ability to electrochemically compact the charged t-CNCs into fibers, the next step was to see if aligned collagen/t-CNC fiber nanocomposites could be obtained. In order to access larger fibers, these experiments used 5 mL of a 0.5 wt % (total solids collagen + t-CNC) aqueous dispersion, instead of 1 mL of a 0.5 wt % t-CNC aqueous solution used in the prior experiments. Unfortunately, attempts to align collagen/t-CNC−COOH55−NH245 (with a solid content of 95:5 wt % collagen/t-CNC−COOH55−NH245) resulted in no formation of a birefringent phase (Figure 4a) and no fiber formation. One possible reason for this is the difference in carboxylic acid/amine ratio between the two bioderived nanofibers which would result in the concentration of the two components at different points between the electrodes. If this is true, then it should be possible to access nanocomposite fibers by matching the carboxylic acid/amine ratio of the collagen and t-CNCs. Collagen itself is known to have a carboxylic acid/ amine ratio of 30:70.46 Gratifyingly, applying a voltage across an aqueous solution of 95:5 wt % collagen/t-CNC−COOH27− NH273 did result in the formation of the fiber nanocomposite (Figure 4b). Collagen nanocomposite fibers incorporating t-CNC− COOH27−NH273 at 0, 2.5, 5, 7.5, and 10 wt % were then prepared, and the nanocomposite fibers were treated with PBS at neutral pH and incubated at 37 °C for 6 h. The resulting macroscopic fiber was cross-linked with genipin in a 95% ethanol solution for 3 days to allow more facile handling and characterization of the aligned fiber. SEM images (Figure 5) of the dried cross-linked composite fibers appear relatively densely packed suggesting a relatively good compaction. As expected, the use of 5 mL solutions (vs the 1 mL solutions used in the CNC only experiments) resulted in much thicker fibers (c.f. Figures S7 and S8) with the average dimeter of the composite fibers being >100 nm versus
