Study on structure, mechanical property and cell cytocompatibility of electrospun collagen nanofibers crosslinked by common agents

International Journal of Biological Macromolecules 113 (2018) 476–486

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International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac

Study on structure, mechanical property and cell cytocompatibility of electrospun collagen nanofibers crosslinked by common agents Xueshi Luo, Zhenzhao Guo, Ping He, Tian Chen, Lihua Li, Shan Ding, Hong Li ⁎ Department of Material Science and Engineering, Engineering Research Center of Artificial, Organs and Materials, Ministry of Education, Jinan University, Guangzhou, 510632, PR China

a r t i c l e

i n f o

Article history: Received 23 June 2017 Received in revised form 26 January 2018 Accepted 27 January 2018 Available online 31 January 2018 Keywords: Collagen Electrospun Crosslink

a b s t r a c t Collagen electrospun scaffolds properly reproduce the framework of the extracellular matrix (ECM) of tissues that are natural with the fibrous morphology of the protein by coupling large biomimetism of the biological material. However, traditional solvents employed for collagen electrospinning lead to poor mechanical attributes and bad hydro-stability. In this work, by N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride with N-hydroxysulfosuccinimide (EDC-NHS), glutaraldehyde (GTA) and genipin (GP) respectively, electrospun collagen fibers cross-linked, effectively stabilized the fiber morphology over 2 months and improved the mechanical properties in both dry and wet state, especially EDC-NHS with large ultimate tensile stress and εb. The secondary structure of collagen structure still remained and had no obvious difference among various crosslinked samples according to FTIR. On the cell assessment, electrospun collagen fibers crosslinked by EDC-NHS, GTA and GP, were found to support cell adhesion, spreading and proliferation of MC3T3-E1. By contrast, GTA was more effective in preserving explicit fibrous morphology with a relatively lower cell viability both in FBS and BSA soaked mats. Interestingly, GP also had the similar cytocompatibility of MC3T3-E1 as EDC-NHS did. The study proved the feasibility of chemical crosslinker to electrospun collagen for biomedical application. © 2018 Published by Elsevier B.V.

1. Introduction Collagen is the most abundant component in human and animal body. In native tissue, collagen molecules are assembled extracellularly to form microfibril and fibril via inter- and intra-molecular interaction. Due to its excellent biocompatibility, collagen, especially collagen fibrous mats via electrospinning has been widely applied to bone graft [1], artificial skin [2–4], vascular grafts [5,6], nerve [7] and scaffolds for tissue engineering [8,9]. Collagen fibers with diameters ranging from a few nanometers to micrometers can be produced via electrospinning technique [8], thus resembling the fibrous morphology of native natural ECM is more convenient than any other technology. Therefore, electrospun collagen mats have drawn more attention in biomedical fields for its fibrous structure. Generally, collagen electrospinning is carried out by dissolving it in fluoroalcohols as well as in acid solutions [10]. In native tissue, due to the highly ordered triple-helix, collagen of macromolecular fibrillar arranged structure and microfibrillar networks possesses strong mechanical property. Unfortunately, electrospinning of collagen often leads to protein denaturation due to the highly volatile of organic solvents. It was reported that dissociation of type I collagen in 1,1,1,3,3,3⁎ Corresponding author at: Department of Material Science and Engineering, Jinan University, Huangpu Avenue West 601, Guangzhou 510632, PR China. E-mail address: [email protected] (H. Li).

https://doi.org/10.1016/j.ijbiomac.2018.01.179 0141-8130/© 2018 Published by Elsevier B.V.

hexafluoro-2-propanol (HFIP) resulted in the denaturation of 93% of collagen [11]. After electrospinning, collagen fiber mats showed poor mechanical property. At the same time, a peculiar aspect is that the resulting electrospun collagens, fabricated starting from a non-water soluble collagen, are always readily soluble in water if they are not crosslinked. Zeugolis et al. [11] explained this finding by demonstrating that collagen dissolved in fluoroalcohols and then electrospun into fibers was completely denatured and contained no trace of insoluble triple helices. Therefore, it is crucial to improve the stability of collagen and mechanical property for electrospun collagen mats. Two methods are used to relieve these limitations. Firstly, electrospinning of collagen has been carried out in blend with other biopolymers such as chitosan [12], polylactic acid (PLA) [13], polycaprolactone (PCL) [14,15] to improve mechanical properties. However, the biological activity of collagen was reduced in blend with other biopolymers. Then, crosslinking of electrospun collagen was used to stabilize the fiber scaffolds for further applications. Chemical crosslinkers can stabilize electrospun collagen and improve mechanical properties [16,17], especially in attempt to preserve scaffold fibrous architecture. The most commonly used crosslinking reagents for collagenbased biomaterials are glutaraldehyde (GTA) [18,19], N-(3dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride with Nhydroxysulfosuccinimide (EDC-NHS) [17,20], genipin (GP) [21,22] and so on. GTA effectively crosslinks collagen molecular by forming covalent bonds of two amine groups of (hydroxyl)-lysine residues with its

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aldehyde groups, which is the most extensively-used method. Unfortunately, induction of cytotoxicity [19] and calcification [23] in host tissues are among the most commonly associated problems. Typical protocols for crosslinking electrospun collagen with EDC frequently suffer from poor results with respect to the structural aspect of the scaffold. It has been reported that the collagen scaffold shrinked during crosslinking and the open structure which was desirable for the cell repopulation on the scaffold was almost obstructed by film-like structure on the surface of the electrospun mat [16,24]. GP-cross-linking was effective in maintaining collagen fiber integrity in aqueous and cell culture media environments for up to 7 days [21], but others have reported the decrease of cell viability on GP-crosslinked scaffolds [25,26] and even the cytotoxic effect of GP in culture medium at certain levels [27]. Anyway, the choice of the crosslinking agent must take into account its effectiveness in stabilizing the fibrous morphology; as well as costs and cytotoxicity. Up to now, there is still lack of systematic analysis on the crosslinking agents for electrospun collagen. Structural properties of collagen electrospun fibers are still a debated subject and there are conflicting reports in the literature addressing the presence of different crosslinkers of collagen in electrospun fibers. Here in, we successfully stabilized the fibrous morphology of electrospun collagen crosslinked by three common crosslinkers (GTA, EDC-NHS and GP). We highlighted differences in terms of morphology and properties of electrospun collagen when different crosslinkers were applied. The results of the study allowed optimizing the most suitable crosslinker for electrospun collagen mats among EDC-NHS, GTA and GP. The study also would provide suggestion of the feasibility of chemical crosslinker to electrospun collagen. 2. Materials and methods 2.1. Materials Type I collagen from rat tail was obtained from Sichuan Mingrang Bio-Tech Co., Ltd. (Sichuan, China). Genipin was purchased from Sichuan Victory Bio-Tech Co., Ltd. (Sichuan, China). Acetic acid was purchased from Guangzhou Chemical Reagent Factory (Guangzhou, China). Eagle's alpha minimum essential medium (α-MEM), fetal bovine serum (FBS) and bovine serum albumin (BSA) were purchased from Hyclone Inc. (Logan, Utah, U.S.), and 0.25% trypsin-EDTA was from Gibco-BRL (Grand Island, U.S.). HFIP, EDC, NHS, and GTA were purchased from Sigma-Aldrich (Saint louis, MO, U.S). 2.2. Collagen mats through electrospinning Solvent was prepared by mixing HFIP with acetic acid at ratios of 50:50 (v/v). The electrospinning solution was fabricated by dissolving collagen from rat tail in the solvent systems at 120 mg/mL concentration under magnetic stirring at 4 °C for 2 h. The prepared electrospinning solutions were loaded into a 10 mL syringe with a blunt end nozzle, controlled by a syringe pump. The solution was pushed through a capillary blunt steel needle (21 gauge, 0.7 mm i.d. × 50 mm length) at a constant speed (1 mL/h). The steel needle was coupled to a high voltage source and electrospun at 20 kV with the air gap distance of 12 cm. The electrospinning collagen samples for AFM were collected by a 20 × 20 mm cover slip for 30 min and other electrospinning collagen samples were collected on aluminum foil for 4 h. All mats were removed from the substrate for further experiments. The samples were subsequently put into the chemical hood at RT for 24 h to remove any residual HFIP. Electrospinning collagen mats were cut into 10 × 10 × 0.25 mm dimensions for all crosslinking experiments. Casting mat of collagen was obtained by casting the electrospun solution into the petri dish and put into the chemical hood at RT for 24 h to remove any residual HFIP. At last, casting mats were cut into 10 × 10 × 0.25 mm dimensions for subsequent experiments.

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2.3. Crosslinking of electrospun collagen Mats were crosslinked with vapor and liquid crosslinking agents. GTA was used for vapor crosslinking, while EDC, NHS, and GP were used in liquid crosslinking. 2.3.1. GTA cross-linking mats GTA cross-linking was achieved by placing the electrospun samples on top of a 25% (v/v) GTA solution for 24 h. The samples were then washed thoroughly in PBS and placed in a 0.1 M glycine solution overnight, to remove any un-reacted GTA. Washing 3 times in PBS was carried out to remove excess glycine. 2.3.2. GP cross-linking mats Electrospun samples were in incubated 30 mM 90% ethanol GP solutions for 24 h in centrifuge tubes at 37 °C. After crosslinking, the scaffolds were rinsed five times with ethanol for 15 min to quench unreacted GP. 2.3.3. EDC-NHS cross-linking mats Electrospun samples were soaked in 20 mM EDC and 10 mM NHS in 90% ethanol for 24 h. After crosslinking, the samples were placed in 0.1 M PBS (pH = 7.4) for 2 h and followed by deionized water five times. Electrospinning collagen mat was labeled Es-mat and different crosslinked mats were labeled: Es-GTA, Es-GP, Es-EDC-NHS. 2.4. Immersion study The cross-linking mats were sterilized in 75% ethanol and immersed in PBS in 15 mL centrifuge tube at 37 °C for 2 months. Samples were harvested at 1 week, 2 months. Mats were then air dried in chemical hood and stored in desiccator until further analysis as described in the following section. 2.5. Characterization 2.5.1. Attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy ATR-FTIR spectroscopic analysis of electrospun collagen mats were performed on spectrometer (Nicolet iS10, Thermo Scientific, waltham, America) over the range 4000–500 cm−1 at a resolution of 4 cm−1. 2.5.2. Morphological investigation The morphologies of mats were evaluated using a scanning electron microscope (SEM, TM3030, HITACHI, Tokyo, Japan). The samples were sputter-coated with gold prior to examination. The data of distribution of fiber diameters were collected on N3 different mats with 50 measurements from each mat using acquisition and image analysis software (Nano measurer 1.2). AFM measurements were conducted by Bioscope Catalyst system mounted on an inverted optical microscope (BioScope Catalyst Bruker Instruments, Mannheim, Germany). The images of collagen fibers were obtained on collagen mats with Tap 150-Al-G silicon AFM probes in ScanAsyst mode at 256 × 256 pixels, which is an imaging mode with automatic image optimization technology. The scan areas were 5 × 5 μm and 20 × 20 μm. The scan rates were from 0.1 to 1 Hz. 2.5.3. Mechanical testing Tensile stress-strain measurements were carried out on Es-mats and crosslinked electrospun mats using a DMA Q800 Dynamic Mechanical Analyzer (TA Instruments, America), with a cross-head speed of 5 mm/min. Five rectangular specimens cut from each mat (wide = 5 mm, gauge length = 10 mm) were analyzed. The average specimen thickness was measured by using a digital micrometer and was used to construct the stress-strain curves from the raw load-displacement data. The elongation at break (εb), tensile elastic modulus (E) and stress at break (σb) were given as the average value ± standard deviation.

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2.6. Cell growth and morphology The casting collagen mats and Es-GTA, Es-GP, Es-EDC-NHS collagen mats were washed in 75% ethanol for 30 min, followed by five washes in PBS for 15 min each, in a 24 multi-well plate. The samples were air dried and sterilized by UV irradiation for 30 min under laminar flow hood. After washing three times with sterile PBS, the sterile PBS was replaced by culture medium containing 10% FBS, and the cultures were kept overnight at 37 °C. MC3T3-E1 were cultured in α-MEM medium plus 10% Fetal Bovine Serum (FBS) and 1% Penicillin–Streptomycin 10000 units/mL–10 mg/mL). Sample was placed one per well in a 24well plate and a drop of 50 μL containing 1.00 × 104 cells was seeded on the center of the upper sample surface allowing cell attachment for 30 min in the incubator, before addition into each well of 1.0 mL of cell culture medium. Cells cultured directly on tissue culture plastic were used as control (cells only group). All cell-handling procedures were performed in a sterile laminar flow hood. All cell-culture incubation steps were performed at 37 °C with 5% CO2. Cell proliferation was evaluated by methyl thiazole tetrazolium (MTT) assay and cell morphology with a Zeiss LSM 700 confocal microscope (Carl Zeiss, Thornhood, NY) using phalloidin for F-actin and DAPI for the nucleus as per the manufacturer's instruction. All reagents, assay kits and fluorescent dyes for MC3T3-E1 culture and analysis were purchased from Sigma Ltd. (Saint Louis, MO. U.S.), unless otherwise noted. The morphologies of MC3T3-E1 cells that adhered on collagen membranes were observed by SEM. The collagen membranes were cut into 10 mm × 10 mm samples, and then sterilized by UV irradiation followed by PBS washing 3 times. The sample was placed in a 24-well culture plate, and the MC3T3-E1 cells were seeded at a density of 1 × 104 cells/well. After 1, 3, 5, and 7 days of cell seeding, samples were fixed with 2.5% glutaraldehyde at pH 7.4 1 h at 4 °C. After rinsing in PBS, the samples were washed with distilled water and dehydrated with graded concentrations of ethanol (50, 70, 80, 90, 100%, v/v). After freeze-drying, the samples were sputter-coated with gold for observation of their cell morphology by SEM.

Besides, the cell culture experiments have been supplemented with mats soaked overnight in 10% BSA. 2.7. Statistical analysis All statistical analyses were performed using analysis of variance (ANOVA) and Tukey's post hoc test for statistical differences at p b 0.05. 3. Results and discussion 3.1. Fourier transform infrared spectroscopy (FTIR) FTIR spectra of the samples are shown in Fig. 1. All spectra were similar and displayed the typical absorption bands of collagen as shown in Table 1. The bands typical for native collagen were observed, including N\\H stretching at 3312 cm−1 for the amide A, C\\H stretching at 3086 cm−1 for the amide B, C_O stretching at 1652 cm−1 for the amide I, and N\\H deformation at 1550 cm−1 for the amide II. Compared with native collagen, the positions of amide A of the other samples are shifted to a lower frequency of 3278 cm−1, which can be due to the effect of strong-polar solvent (HIFP) on hydrogen bonds related to peptide and\\NH group. The phenomenon suggests that the interaction between collagen microfibrillar was extensively destroyed due to both the polar solvent and the electrostatic force during electrospinning. The amide B normally held together by intramolecular hydrogen bonds, was gradually weakened after electrospun collagen fibers were crosslinked. Anyway, the positions of the other typical bands are less changed, and this fact may suggest that the secondary structure of collagen is not destroyed. Generally, GTA effectively crosslinks the collagen molecule by forming covalent binding of two amine groups of (hydroxy)-lysine residues with its aldehyde groups, which further react to form pyridinium compounds. EDC are functional coupling reagents that induce carboxylic acid groups of glutamic or aspartic acid residues to form stable amide bonds with amines of lysine and hydroxylysine

Fig. 1. FTIR spectra of the samples: the shifts of the typical positions of amide I and amide II of the samples crosslinked by EDC-NHS, GTA and GP had occurred, which indicated the interactions between the crosslinkers and collagen.

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3.2. Morphology

Table 1 The amide bands position in FTIR spectra of different collagen mats. Samples

Native collagen Casting mat Es-mat Es-EDC-NHS Es-GTA Es-GP

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Band position [cm−1] Amide A

Amide B

_CH2

Amide I

Amide II

Amide III

3312 3278 3278 3278 3278 3278

3086 3078 3078 3080 3080 3086

2926 2928 2918 2936 2936 2934

1652 1632 1628 1640 1638 1630

1550 1532 1532 1536 1540 1540

1240 1240 1240 1240 1240 1240

residues in the presence of NHS. Butler et al. proposed that cross-linking reactions take place between GP molecules and primary amine groups [28]. Because the reaction positions of crosslinking mainly were the amino resides, which had no effect on the secondary structure of collagen molecule, the electrospun fibers kept similar in the secondary structure of collagen no matter which crosslinkers were applied in the study. However, the shifts of the typical positions of amide I and amide II of the crosslinked samples also were observed as shown in Fig. 1 and Table 1. It indicated that the existing reaction involved in the stabilization process.

The morphologies of all mats were characterized by SEM (Fig. 2) and AFM (Fig. 4). Besides, the fiber diameters distribution of the mats was measured on the basis of each SEM image as shown in Fig. 3. All collagen fibers with smooth surface had no obvious bead as shown in Figs. 2 and 4, no matter which crosslinker was used. Before crosslinking, Es-mat showed uniform fiber morphologies with a diameter of 363.38 ± 148.43 nm, which stretch very well as shown in Figs. 2a and 4b and g. After crosslinked with EDC-NHS and GP, the fibers retained the no-woven fibrous morphology, but the fibers presented an obvious increment of the fiber diameters compared to uncrosslinked mats (Fig. 3). The results are in agreement with the data of Gloria et al. [25]. Meanwhile, the fibers of Es-EDC-NHS and Es-GP mats show relaxation and crimp (Fig. 2b and d), which had been termed as “crimp accentuation” [25]. It was attributed to the swelling of collagen molecule in aqueous. Anyway, fiber Fig. 3 shown that there was no statistical difference between the fiber diameters of Es-GTA sample and Es-mat, indicating that crosslinking via GTA atmosphere did lead to no obvious change in fiber diameter. Besides, Es-mat crosslinked in GTA vapor atmosphere might lead to dehydration of collagen and caused a little decrease in fiber diameter.

Fig. 2. SEM micrographs of the electrospun collagen mats before/after immersion in PBS at RT at different times: (a, e, i) Es-mat without crosslinking; (b, f, j) Es-EDC-NHS; (c, g, k) Es-GTA; (d, h, l) Es-GP; (m) shown the photographs of different mats. (a–l) scale bar = 20 μm; (m) scale bar = 1 cm. The samples for photograph in (m) are mats without substrate and the collagen mats were laid on a blue paper for taking photographs.

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Fig. 3. Fiber diameters of the uncrosslinked and crosslinked electrospun collagen mats. The data were collected on N3 different mats with 50 measurements from each mat. *p b 0.01, significantly different from Es-mat. There was no statistical difference (p N 0.05) between the fiber diameters of Es-GTA and Es-mat.

Cross-linking treatments brought about color changes on the electrospun collagen mats shown in Fig. 2. Similar discoloration results to theirs have been reported for bovine, porcine and canine pericardium and dermal sheep collagen fixed with GTA, epoxy, and GP [19,29]. The observed discoloration was attributed to the reaction of the crosslinking agent with the amino acid residues [30]. The color of the crosslinked collagen by GP changed seriously, it is because GP react with an amino acid (glycine, alanine, leucine, phenylalanine and tyrosine) to form the blue pigment (Gardenia blue dye) [30,31]. Further, AFM images showed details of the sample's microstructure in Fig. 4. Crosslinking merged the fibers and remove the identity of individual fibers at the contact points, thus forming welded or soldered junctions in Es-EDC-NHS as shown in Fig. 4c and h. Distortion could still be observed in Es-GP as shown in Fig. 4e and j, which was in agreement with the SEM observation. However, the fibers of Es-GTA still keep stretched state with a little decrease in the fiber diameter (324.56 ±

134.79 nm); no obvious welded conjunction or distorted was observed in Es-GTA in Fig. 4d and i. Since the electrospinning was performed in a poorly hydrogen bonded solvent which promotes intramolecular hydrogen bonding, the presence of higher concentration of crosslinker at the contact points might promote the inter-fiber adhesion. Thus welded junctions were only observed in Es-EDC-NHS mats and Es-GP mats. GTA crosslinking reaction was carried out at solid-vapor interface, so the morphological features of ES-GTA fiber remained intact. It was reported that the collagen or gelatin fibrous structure was immediately changed into a dense membrane structure by cross-linking in an aqueous system [32]. Therefore, we chose vapor cross-linking by GTA to avoid the collapse of the fibrous structure during cross-linking in the aqueous system. Also, ethanol worked as a solvent for GP and EDC-NHS, then fibrous structure kept less change due to the fast evaporation of ethanol. Anyway, in our study, the cross-linked collagen matrixes kept biomimetic fibrous structure of natural ECM. After immersion in PBS, uncrosslinked samples had no hydrolytic stability and immediately disintegrated (Fig. 2e and i). The fiber morphologies of crosslinked mats immersing in PBS for different time were investigated by SEM as shown in Fig. 2. The mats treated with EDC-NHS, GTA, and GP retained fibrous morphology (as shown in Fig. 2f–h, j–l) as long as 2 months. After immersion, the fiber diameters of Es-EDC-NHS, Es-GTA, Es-GP mats changed. Es-GTA mats and Es-EDCNHS mats occurred some swelling leading to an increase of fiber diameters evidently by 2 months, whereas Es-GP mats had a litter increase at 7 days and followed an obvious decrease in diameter by 2 months, which may be due to the degradation of crosslinked collagen fiber by GP. The results also show that rehydrated ES-GP may be less stability than any of the others. Crosslinking drgree also affected the stability of collagen mats. The crosslinking degree had been analyzed and the related information was described in Support Information (SI). According to the present data, the effect of crosslining degree on fiber configuration was not obvious and all crosslinked samples maintained fiber morphology as long as 2 months, which might be due to the fact that the crosslinking degree difference among Es-EDC-NHS-mat, Es-GTA mat and Es-GP mat was little. 3.3. Mechanical properties Mechanical properties of electrospun collagen mats are usually reported in the dry state, where values range from 0.02 to 30 MPa for ultimate tensile stress (UTS) [4,16,17,25,33–35] and the mechanical

Fig. 4. AFM images of different collagen mats:(a, f) Casting mat; (b, g) Es-mat; (c, h) Es-EDC-NHS; (d, i) Es-GTA; (e, j) Es-GP. Samples for AFM were collected by a 20 × 20 mm cover slip for 30 min. h

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Fig. 5. Mechanical properties: typical stress-strain curves of different collagen mats measured under various conditions: (a) Dry state: samples were measured after air drying at RT. (b) Wet state: samples were measured after immersing in PBS at RT for 12 h. The tensile strength (c), elastic modules (d) and elongation at break (d) for the mats under dry/wet state. n = 5 for all groups. *p b 0.001, all crosslinked groups are significantly greater than Es-mats; #p b 0.001, all wet crosslinked collagen mats are significantly than the dry samples of same mats.

properties under the wet state also have been reported in a few studies, where the values for tensile strength range from 0.05 to 0.75 MPa [17,25,35]. The properties under wet state are more representative of the physiological conditions. Hence, the crosslinked mats in this study were tested both in dry and wet states. Typical tensile stress-strain curves of Es-mat, casting mat, Es-EDCNHS, Es-GTA and Es-GP mats under dry and wet conditions are shown

in Fig. 5. Uncrosslinked collagen dissolved in the PBS so that they could not be tested under the wet conditions, so Es-mat and casting collagen were tested only under the dry conditions. Under dry condition, casting mats and as-electrospun collagen mat were observed in typical S-shape of the stress-strain curves. After crosslinked, Es-EDC-NHS, EsGTA and Es-GP mats were observed in brittle-like behaviors with higher peak stress (σ), higher Young's modulus (E) and lower elongation at

Fig. 6. Absorbance values for MTT assay measuring the cell proliferation of MC3T3-E1 on different mats soaked overnight in 10% FBS (a) and 10% BSA (b) before cell seeding at days 1, 3, 5, and 7. n = 5 for all groups. *p b 0.05, **p b 0.01, ***p b 0.0001. All groups of collagen mats at all time points are significantly different from the control samples (*, **, ***). #p b 0.05, ##p b 0.01, ### p b 0.0001, significantly different from Casting mats. Es-mats immediately disintegrated when immersing in culture medium so that casting mat replaced the Es-mat as positive control.

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Fig. 7. DAPI-phalloidin fluorescence assay of MC3T3-E1 on mats crosslinked with different agents, which soaked overnight in 10% FBS before cell seeding, for three days incubation. Cells nuclei were stained blue by DAPI (blue) and F-actin filaments (red) in red by phalloidin, scale bar, 50 μm.

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break (εb) compared with Es-mats and casting mats, as shown in Fig. 5a. In brief, Es-EDC-NHS mats had the highest σ (31.033 ± 6.05 MPa), E (138.03 ± 34.73 MPa), εb (5.67 ± 0.47%) among all the crosslinked groups according to Fig. 5. Furthermore, various crosslinkers have significantly different effects on electrospun collagen mats under wet condition. Typical curves for rehydrated mats consisted of a long region of constant gradient (EDCNHS and GTA) or increasing gradient (GP) which persisted until failure as shown in Fig. 5b. The hydrated collagen scaffolds demonstrated a uniaxial J-shaped stress–strain curve, which is roughly similar to that of many native tissues. The σ and E value of all crosslinked mats under wet state decreased obviously and εb increased when compared to those under dry condition. The σ values of Es-EDC-NHS, Es-GTA, Es-GP mats under wet state decreased to 1.42 ± 0.37, 0.389 ± 0.08 and 0.24 ± 0.06 MPa respectively. Besides, the E values of Es-EDC-NHS, Es-GTA, Es-GP mats under wet state decreased to 1.40 ± 0.38, 0.86 ± 0.21, 0.52 ± 0.12 MPa, respectively. On the contrary, obvious increments are shown in εb values of Es-EDC-NHS, Es-GTA, Es-GP mats (123.40 ± 28.31%, 49.60 ± 7.4%, 69.67 ± 14.12% respectively) under wet state, especially for that of Es-EDC-NHS, which also showed a pronounced UTS. Uniaxial tensile tests of dry and partially rehydrated electrospun collagen fibers produced stress-strain curves similar to those reported for semicrystalline polymers that yield and undergo plastic flow [36]. The yielding mechanism involves some form of flow that occurs within the fiber, possibly interfibrillar slippage, which plays an important role in the tensile deformation of aligned connective tissue such as tendon. Typical S-shape curves (dry fibers) have been reported for tendon [37], ligaments [38], extruded collagen fibers [39,40] and nano-meshes [41]; whilst analogous J-shape stress-curves (wet fibers/high swelling) have been re-hydrated reformed collagen fibers pericardium tissue [42] and rat tail tendon [39]. In hydrated collagenous tissue the occurrence of a J-shaped curve is often associated with collagen fibers becoming more aligned along the strain axis during stretching [29]. In the study, the strength and modulus values were reduced after rehydration while the elongation values at break increased significantly. These large decreases in the mechanical properties in the hydrated state suggests that the hydrogen and the electrostatic bonds or other chemical bonding that hold collagen fibrils together were break down by H2O molecules [29]. Furthermore, the value of UTS of rehydrated Es-EDC-NHS is significantly (p b 0.05) higher than the other mats. That may be explained by that the better hydrophilic property of EsEDC-NHS mats absorbed more water acts as a plasticizer for collagen. Many reports have shown that water content plays an important role in determining the mechanical properties of collagen fibers. It is likely that, in the absence of water molecules, these water-binding sites are available to bond inter-molecularly to stiffen the collagen triple helix and prevent slippage and translation to occur between neighboring molecules. Chemical crosslinker, such as GTA, EDC and GP, can self-polymerize or chemically bond and consequently create a three-dimensional network with long-range cross-links spanning larger gaps, which can affect the collagen fibers properties by stiffening and strengthening the fibers. This stiffening made the stress-strain curve of the crosslinked mats present a brittle behavior. Most important of all, the fibers produced in this study were characterized by mechanical properties that closely match native tissues. For example, human anterior cruciate ligament, rat tail tendon, bovine, and rabbit Achilles tendons, that can withstand mechanical loads from 15 to 53 MPa and exhibit strain at break from 7 to 40% [29,40,43–46]. Good mechanical strength and large elongation of electrospun collagen mats crosslinked by EDC-NHS provide a good candidate for tissue restorative, altogether with fibrous morphology. Additionally, as a scaffolding candidate in tissue engineering, the crosslinked fibers possesses the biomimetic structure and a defined dimensional organization of the tissue-engineered space over 2 months, which can support and maintain the normal state of differentiation within the cellular compartment.

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3.4. Cell growth and morphology Many studies on the cytocompatility and cytotoxicity about EDCNHS, GP and GTA have been reported over years. Although GTA is a less expensive, fast reacting crosslinker, it causes local toxicity contributing to the release of unreacted aldehydes [47–49]. EDC-NHS is commonly used for collagen crosslinking, which has been reported to have good biocompatibility and no cytotoxicity [25,49]. GP also drove great attention because of its biocompatibility [47,50]. Es-mats, casting mats, Es-EDC-NHS mats, Es-GTA and Es-GP mats were cultured with MC3T3-E1 cells in the study. Fig. 6a showed the cell proliferation of the samples soaked overnight in 10% FBS examined by MTT assay. Cell proliferation increased with time on all samples and the proliferation on the casting mats was the lowest at Days 1, 3, 5 and 7. On the other hand, the proliferation of Es-GTA mats was the lowest among all crosslinked mats at Days 3, 5, and 7, while the highest cell proliferation was observed in Es-EDC-NHS mats, which is consistent with other published data [26,49]. However, all three crosslinkers were favorable in supporting cell viability over time. The morphologies of MC3T3-E1 cells growing on different mats soaked overnight in 10% FBS also were observed DAPI-phalloidin fluorescence assay (Fig. 7) at Day 3 and SEM morphological analysis (Fig. 9a–c) at Day 3. Since no crosslinked collagen (Es-mat and casting collagen) dissolved in the medium, the cells on the samples show similar polygonal morphology with clear and stretched filopodia as shown in Fig. 9a-9c. Cells attached well on the crosslinked fibers whose specified fiber texture remained explicitly, and adopted spreading morphologies with cellular extensions representative of MC3T3-E1 outgrowth. The cells' filopodias stretched along the collagen fibers of Es-EDC-NHS, Es-GTA and Es-GP mats according to Fig. 7. Cellular morphology on the EsGTA are stellate, more flatter than any of the others, and most of the cells on the Es-GP and Es-EDC-NHS samples others were fusiform, which spread toward the fiber's alignment. SEM showed that polygonal cells almost formed a layer on the surface of Es-GP and Es-EDC-NHS at Day 3 while the cell's number on the Es-GTA obviously obvious is less, which keep accord with our MTT assay and the other's result [43,47,48]. For further research, the data (Figs. 6b, 8, 9d–f) about cytocompatility of the mats soaked in 10% BSA overnight which made them all more equal at the starting point were acquired. Fig. 6 indicated that there was little difference in cell proliferation among the BSA soaked groups when compared to that of the FBS soaked groups. The cell proliferation of most of the mats (except Es-EDC-NHS, Es-GP at Day 3 and Es-GP at Day 7)soaked in 10% BSA overnight showed no statistical difference, because BSA is an effective scavenger sequestering toxic substances, including pyrogens, oxygen free radicals, from the medium [51]. Cell proliferation increased with time on all the samples and the proliferation on all samples at Day 1 was nearly equal while the casting mats presented the lowest proliferation at Days 3, 5 and 7. On other hand, the highest cell proliferation was observed in Es-GP mats, which was different from the FBS soaked mats. The morphologies of MC3T3-E1 cells growing on the mats soaked in 10% BSA also were observed DAPI-phalloidin fluorescence assay (Fig. 8) at Day 3 and SEM morphological analysis (Fig. 9d–f) at Day 3. The cells assumed a more rounded and elongated phenotype and the cells' filopodias stretched more orderly along the collagen fibers of Es-EDCNHS, Es-GTA and Es-GP mats according to Fig. 8, which compared to their FBS soaked counterparts. BSA intensified cell spreading on the surfaces of the control group and the casting mat when compared to that of FBS soaked counterparts. Besides, the cells spreading area of Es-EDCNHS, Es-GTA and Es-GP mats became smaller on the surface of the BSA soaked mats. BSA soaked mats represented less cell spread, which could be explained by the non-specific nature of BSA and BSA had blocking effect on cell adhesion, deriving from its less binding domains compared to collagen I [52]. Others reported the decrease of cell viability on GP-crosslinked collagen scaffolds [25,26] and even the cytotoxic effect of GP in culture

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Fig. 9. SEM images of MC3T3-E1 grown on the crosslinked mats soaked overnight in 10% FBS (a–c) and 10% BSA (d–f) before cell seeding for three days incubation, scale bar, 20 μm.

medium at certain levels [27]. It has been also speculated that GPcrosslinked gelatin is 10,000 times less cytotoxic than GTA-crosslinked gelatin or biological tissues without any sign of calcification [47,53]. Anyway, according to our results, GP-crosslinked collagen exhibited better cell proliferation than GTA-crosslinked collagen, but there are no obvious negative effect on cell proliferation and growth when GP was applied for crosslinking electrospun collagen fiber. This the difference would be owing to the amount of GP used and additional washing steps for complete removal. It was also reported the cell repopulation on the scaffold crosslinked by EDC was almost obstructed by film-like structure on the surface of the electrospun mats [24]. In the study, both Es-EDC-NHS and Es-GP retained fiber structure in long term and were favorable in supporting cell viability over time, which is inconsistent with some published data [21,54]. Overall, MC3T3-E1 proliferation on Es-EDC-NHS mats was significantly higher when compared to any of the others among the FBS soaked mats while Es-GP mats had highest proliferation among the BSA soaked mats, and cell spreading almost provided a cell covered surface on both ES-EDC-NHS and Es-GP mats.

4. Conclusion Herein, we investigated EDC-NHS, GTA and GP cross-linking approaches to the same electrospun collagen fiber. The results of FTIR showed that the secondary structure of collagen nevertheless remained and no apparent variation in the collagen mats occurred when various cross-linking agents were used. Significantly, all three crosslinkers stabilized morphology of electrospun collagen more than two months as well as an increase of mechanical qualities in both wet and dry state, particularly EDC-NHS with large UTS and εb. All of the electrospun collagen fibers crosslinked by EDC-NHS, GTA and GP supported adhesion, proliferation and spreading of MC3T3-E1. By contrast, GTA were more effective in preserving fibrous morphology with a relatively lower cell viability, and electrospun collagen crosslinked by EDC-NHS and GP mats confirmed better cytocompatibility in both FBS and BSA soaked mats in vitro. The results indicate that the crosslinked collagen fibrous matrix can mimetic to the natural ECM proteins' biological and

structural properties in some degree, and provided a suitable level of hydro-stability of collagen. Acknowledgements The work was supported by National Natural Science Foundation of China (3157040298 and 31400824). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.ijbiomac.2018.01.179. References [1] A.M. Ferreira, P. Gentile, V. Chiono, G. Ciardelli, Collagen for bone tissue regeneration, Acta Biomater. 8 (9) (2012) 3191–3200. [2] F. Wang, M. Wang, Z. She, K. Fan, C. Xu, B. Chu, C. Chen, S. Shi, R. Tan, Collagen/chitosan based two-compartment and bi-functional dermal scaffolds for skin regeneration, Mater. Sci. Eng. C Mater. Biol. Appl. 52 (2015) 155–162. [3] G. Ramanathan, S. Singaravelu, T. Muthukumar, S. Thyagarajan, P.T. Perumal, U.T. Sivagnanam, Design and characterization of 3D hybrid collagen matrixes as a dermal substitute in skin tissue engineering, Mater. Sci. Eng. C Mater. Biol. Appl. 72 (2017) 359–370. [4] T. Zhou, N. Wang, Y. Xue, T. Ding, X. Liu, X. Mo, J. Sun, Electrospun tilapia collagen nanofibers accelerating wound healing via inducing keratinocytes proliferation and differentiation, Colloids Surf. B: Biointerfaces 143 (2016) 415–422. [5] S. Ghazanfari, A. Driessen-Mol, C.V. Bouten, F.P. Baaijens, Modulation of collagen fiber orientation by strain-controlled enzymatic degradation, Acta Biomater. 35 (2016) 118–126. [6] P. Joanne, M. Kitsara, S.E. Boitard, H. Naemetalla, V. Vanneaux, M. Pernot, J. Larghero, P. Forest, Y. Chen, P. Menasche, O. Agbulut, Nanofibrous clinical-grade collagen scaffolds seeded with human cardiomyocytes induces cardiac remodeling in dilated cardiomyopathy, Biomaterials 80 (2016) 157–168. [7] A. Timnak, F.Y. Gharebaghi, R.P. Shariati, S.H. Bahrami, S. Javadian, H. Emami Sh, M.A. Shokrgozar, Fabrication of nano-structured electrospun collagen scaffold intended for nerve tissue engineering, J. Mater. Sci. Mater. Med. 22 (6) (2011) 1555–1567. [8] T. Jiang, E.J. Carbone, K.W.H. Lo, C.T. Laurencin, Electrospinning of polymer nanofibers for tissue regeneration, Prog. Polym. Sci. 46 (2015) 1–24. [9] C. Dong, Y. Lv, Application of collagen scaffold in tissue engineering: recent advances and new perspectives, Polymer 8 (2) (2016) 42. [10] B.D. Walters, J.P. Stegemann, Strategies for directing the structure and function of three-dimensional collagen biomaterials across length scales, Acta Biomater. 10 (4) (2014) 1488–1501.

Fig. 8. DAPI-phalloidin fluorescence assay of MC3T3-E1 on mats crosslinked with different agents, which soaked overnight in 10% BSA before cell seeding, for three days incubation. Cells nuclei were stained blue by DAPI (blue) and F-actin filaments (red) in red by phalloidin, scale bar, 50 μm.

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