collagen composite scaffolds for application in nerve tissue regeneration

Polymer Degradation and Stability 166 (2019) 73e85

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Polymer Degradation and Stability journal homepage: www.elsevier.com/locate/polydegstab

Controlled degradable chitosan/collagen composite scaffolds for application in nerve tissue regeneration Junzeng Si a, b, Yanhong Yang b, Xiaoling Xing b, Feng Yang b, Peiyan Shan a, * a b

Department of Neurology, Qilu Hospital of Shandong University, Jinan, 250012, China Department of Neurology, Jinan City People's Hospital, 271199, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 25 February 2019 Received in revised form 28 April 2019 Accepted 18 May 2019 Available online 22 May 2019

The biodegradable materials have been widely used for fabricating various scaffolds in the application of peripheral nerve tissue engineering. The porous chitosan/collagen composite scaffolds with different tailored degradation ratio may be a candidate. In the present study, the porous chitosan/collagen composite scaffolds were prepared by controlled lyophylization and phase separation of corresponding composite solutions with different proportion of chitosan and collagen. The scaffolds were investigated via morphology, porosity, liquid uptake, swelling behavior, component, mechanical and In Vitro degradation testing. The cytotoxicity and cytocompatibility of the prepared composite scaffolds were evaluated using L929 fibroblasts, RSC96 cell lines and primary Schwann cells, respectively. The related molecular mechanism was further penetrated by RT-PCR and Western Blot. Finally, the In Vivo degradation behavior and inflammatory reaction were examined by subcutaneous implantation of rabbit. The results showed that the chitosan/collagen composite scaffolds possessed a surface fiber-like structure while inner porosity. Compared to a pure collagen scaffold, the addition of chitosan decreased the mean pore size, liquid uptake and degradation rate, while increased the mechanical property of the composite scaffolds. Physicochemical properties of scaffolds including porosity, swelling behavior and component were found satisfactory for intended application. The chitosan/collagen composite scaffolds showed good cytocompatibility without cytotoxicity. And our results also demonstrated that the chitosan/collagen composite scaffolds could promote the attachment, migration and proliferation of Schwann cells. The In Vivo implantation results indicated that the composites scaffolds showed obviously modulated degradation behavior without causing any inflammatory reaction. Taken together, the developed chitosan/collagen composite scaffolds here may have good potential promising application as scaffolds materials for peripheral nerve regeneration. © 2019 Elsevier Ltd. All rights reserved.

Keywords: Peripheral nerve regeneration Chitosan Collagen Tissue engineering Composite scaffolds

1. Introduction Various biomaterials scaffolds including synthetic and natural biomaterials have been widely investigated and used in tissue engineering fields, and thus opening up the world of regeneration of different organs and tissues [1]. Peripheral nerve injury, a nervous system disease, is always associated with dysfunction, movement and sensory disorder, and pain, which has largely influenced the normal health and life of the patients [2]. The autologous grafts are the priority method for effectively repairing peripheral nerve injury at the early three decades [3], however, the size mismatch,

* Corresponding author. E-mail address: [email protected] (P. Shan). https://doi.org/10.1016/j.polymdegradstab.2019.05.023 0141-3910/© 2019 Elsevier Ltd. All rights reserved.

insufficient donor source, permanent damage at the donate site and unsatisfication of large area of transplantation extremely limited the application of autologous grafts. Fortunately, the tissue engineered biomaterials scaffolds have shown potential application for promoting peripheral nerve regeneration in the last two decades [4]. As one of the three basic elements for tissue engineering, the biomaterials scaffolds play an important role in regulating the cell attachment, tissue ingrowth, neovascularization and accelerating the formation of newborn tissue structure both in vivo and in vitro by providing a three-dimensional porous spatial structure [5]. An idea biomaterials scaffold for peripheral nerve regeneration should closely mimic the natural environment of the peripheral nerve matrix and have also excellent biodegradability without cytotoxicity or inflammatory reaction at the accumulation site of biodegradation products [6,7]. Nevertheless, until now, no biodegradable

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biomaterials scaffolds matching well with the growth of newborn nerve tissue have been developed and reported. Chitosan as a kind of glycosaminoglycans could be extracted from chitin by partial deacetylation. It is reported that chitosan with good properties including antibacterial action, hemostasis, mechanics, cytocompatibility, etc., has been widely used in a variety of biomedical fields such as wound healing, drug delivery carriers, surgical thread, and tissue engineering [8]. In the last decades, chitosan has also been enormously investigated for peripheral nerve regeneration [9]. The filament and membrane made from pure chitosan was shown to promote the attachment and proliferation of Schwann cells, indicating a potential application for peripheral nerve regeneration [10]. However, the effect of pure chitosan on promoting cell proliferation and nerve regeneration is limited due to the lack of cues for inducing cell migration and differentiation on the scaffolds surface. Thus, the composite scaffolds consisting of chitosan and other synthetic or natural biomaterials was subsequently developed, such as chitosan/silk fibroin [11], chitosan/gelatin [12], chitosan/PCL [13], etc., which were all proven to be beneficial for accelerating Schwann cells growth and nerve regeneration. In addition, the chitosan/polylysine hydrogel with enhanced bulk cohesive and interfacial adhesive force was found to largely improve the axon cross ratio of the regenerated nerves, thus offering a promising approach to the repair of severed peripheral nerves [14]. In addition, the chitooligosaccharides, a kind of the degradation products of chitosan, was also shown to be beneficial for constructing microenvironment for peripheral nerve regeneration without causing inflammatory reaction from host [15]. Thus, chitosan is very suitable for using as constructs of peripheral nerve implants. However, the biodegradation of chitosan is inadequate, as its biodegradation property is largely dependent on the deacetylation degree [16]. Generally, the higher the deacetylation degree, the slower the degradation. Therefore, the modulation of the biodegradtion rate of chitosan for better matching the velocity of the newborn tissue is necessary with high significance for nerve tissue regeneration. As a kind of ECM proteins, collagen with excellent biocompatibility, biodegradability and low immunogenicity plays an important role in supporting cell attachment, migration and proliferation [17]. Collagen combined with fibronectin, laminin and some other glycosaminoglycans could form the biological microenvironment necessary for cell growth and tissue regeneration [18]. Thus, collagen displays potential promising application in scaffolds preparation and has been used widely in tissue engineering and regenerative medicine [19]. Currently, various collagen related scaffolds have been also developed for promoting nerve regeneration [20]. However, the single collagen scaffolds are easy to be out of shape and deform due to rapid degradation when in contact with cell-culture medium or body fluid [21]. Therefore, the collagen related composite scaffolds were developed. The electrospun P (LLA-CL)/collagen scaffolds and PLGA/silk fiborin/collagen scaffolds were both fabricated and both showed great potential as a substrate for accelerated regeneration of the nerve [22,23]. Chen et al. [24] found that the 3D printed collagen/heparin sulfate scaffold could effectively enhance the mechanical properties of collagen and provide continuous guidance channels for axons growth, which may improve the neurological function after spinal cord injury. Besides, the crosslinking of collagen scaffolds using glutaraldehyde (GA), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, (EDC), genipin (GP), etc is also a good method for improving the mechanical property of collagen [25]. Recently, a 3D collagen sponge scaffold was firstly prepared and then crosslinked with dialdehyde cellulose (DAC). The mechanical properties of collagen sponge scaffolds were found to be largely enhanced and may prove relevant to neural tissue engineering [26]. In addition,

various growth factors including NGF, CNTF, bFGF, etc could be incorporated into collagen scaffolds for improving nerve regeneration [27,28]. Based on the above mentioned, the chitosan scaffolds showed good mechanical properties while low degradation properties, however, the collagen scaffolds have the opposite properties. Thus, by adjusting the ratio of different components, the combination of chitosan and collagen is anticipated to result in a kind of composite scaffolds with controlled degradation velocity. Though chitosan/ collagen scaffolds have been previously reported for application in various tissue engineering fields [29e31], few has related to the degradation modulation of chitosan scaffolds by adding collagen components for potential application in peripheral nerve regeneration [32]. Therefore, in the present study, the porous chitosan/ collagen composite scaffolds were fabricated by freezing and lyophylization of blending solution with various proportions. The physicochemical properties including microstructure, porosity, swelling capacity, mechanical property, degradation behavior as well as components variation of the chitosan/collagen scaffold were investigated. Further on, the cytotoxicity and cytocompatibility of the prepared composite scaffolds for potential nerve regeneration were separately evaluated by in vitro culture of L929 fibroblasts, RSC96 cells and primary Schwann cells. The inflammatory reaction and degradation behavior In Vivo was also further determined by subcutaneous implantation experiment of rabbit. 2. Materials and methods 2.1. Materials and reagents Chitosan (average Mw ~85,000, >90% deacetylated; Marine Chemicals) from crab shell was purchased from Nitta Gelatin Inc (Osaka, Japan), collagen film from bovine tendon (Type I), Anti-Thy 1.1 antibody, forskolin, b-darabinofuranoside, 3-(4,5dimethylthiazol-2-yl)
2,5-diphenyltetrazolium bromide (MTT), polyvinylidene fluoride (PVDF) membrane were all bought from Sigma-Aldrich (St. Louis, MO). Toluidine Blue and Type I collagenase was bought from Hyclone (4A Biotech, CN). Dulbecco's modified Eagle's medium (DMEM), penicillin, streptomycin, hematoxylineosin, 1  phosphate-buffered saline (PBS) and 0.25% trypsinEDTA were all purchased from Invitrogen (Carlsbad, CA). Heatinactivated fetal bovine serum (FBS) was purchased from Hyclone (4A Biotech, CN). The distilled water (dH2O) was used for all processes. All the other chemicals and reagents with analytical grade were used as obtained without any further purification. 2.2. Fabrication of porous chitosan/collagen composite scaffolds Both chitosan and collagen solutions with concentration of 1% (w/v) were prepared by dissolving high molecular weight chitosan and collagen sheet in 1% (v/v) acetic acid under gentle shaking. The solutions were permitted to store statically overnight at room temperature for removing the air bubbles. The fabrication of porous chitosan/collagen composite scaffolds was listed as follows: Firstly, the chitosan and collagen solution was mixed thoroughly with the volume proportions of 10%, 50%, 70% and 90%, respectively. And the pure chitosan and collagen solutions were separately used as control. Then, all the sample solutions with separate volume of 1 mL were added into a 24-well cell culture plate. The sample solution was then frozen overnight in a
20  C freezer followed by lyophilization at
50  C for 24 h. After that, the dry samples were removed from the mold and treated in a 50:50 (v/v) ethanol:sodium hydroxide (1 M) solution for 2 h to neutralize the residual acetic acid. Subsequently, the ethanol/sodium hydroxide solution was trashed and the samples were incubated in PBS for 4 h until a

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neutral status reached. Finally, all the neutralized pure or composite scaffolds were lyophilized again at
50  C for 24 h and stored before further use. The scaffolds were named as follows: M1, M2, M3, M4 with the volume proportions 10%, 50%, 70% and 90% of chitosan and collagen solution, respectively. Additionally, the pure chitosan and collagen scaffolds were separately named PCS and PCO. 2.3. Characterization of chitosan/collagen composite scaffolds 2.3.1. Morphology observation The morphology and microstructure of chitosan/collagen composite scaffolds were observed using an optical microscope (OM, Zeiss) and a scanning electron microscope (SEM, JSM6010, Hitachi), respectively. Briefly, for OM observation, the scaffolds were cut into 10 mm  10 mm size and placed onto the observation platform, then the surface topography was photographed at seven random selected sites under the same magnification. The transverse section of the scaffolds was also observed using OM for thickness analysis. For SEM observation, the dry composite scaffolds with the same size were attached to the sample stage by conductive adhesive. Then, the surface of the samples was sputter-coated with a layer of gold and observed using SEM with vacuum of 1.3  10
4 Pa. Five sites on each sample surface were randomly chosen and photographed under different magnifications. The pore size of the scaffolds was measured according to SEM images with higher magnification. 2.3.2. Porosity determination The porosity of fully dried chitosan/collagen composite scaffolds was determined using the following method. In brief, all the scaffolds were cut into rectangular pieces with 10 mm  20 mm size. The mass and volume of each scaffold before immersing into anhydrous ethanol in pycnometer were recorded as W1 and V1, respectively. After immersion in anhydrous ethanol for 2 h, the mass of each saturated sample was recorded as W2. Finally, the porosity of the composite scaffolds was determined in terms of the following equation: Porosity¼(W2eW1)/(rV1)  100% Where r ¼ 789 kg/m3 is the density of ethanol at room temperature. Five parallel samples were used for the measurement. 2.3.3. Equilibrium swelling measurement and mass variation The equilibrium swelling measurement (liquid uptake) and absolute mass variation were characterized using PBS and DMEM, respectively. The fully dried scaffolds with 10 mm  10 mm size were firstly weighed and the mass was recorded as M0. Then, the scaffolds were immersed into PBS or DMEM separately in a sealed container and placed in a 37  C oven. After predetermined times (2 h), the scaffolds were carefully taken out and the excessive liquid was removed using filter paper immediately. The weight of the wet scaffolds was recorded as M1. The equilibrium swelling measurement (ESM) was determined in terms of the following equation: ESM ¼ M1/M0  100% The absolute mass variation (MV) of the scaffolds was calculated using the following equation: MV ¼ M1-M0 The sample number for each measurement was three.

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2.3.4. FTIR spectroscopy The attenuated total reflectance/fourier transform infrared (ATR-FTIR) spectra of various scaffolds were recorded using a Thermo Nicolet spectrophotometer (Nicolet 5700, Thermo Nicolet Corporation, Madison, WI) affiliated with ZnSe crystal ATR accessory. The detection of all scaffolds was performed using KBr tablet mode, the scaffolds were mixed with KBr thouroughly and pressed to be a round tablet with a diameter of 10 mm. Then, FTIR spectra were recorded in transmission mode. All spectra were recorded from 500 cm
1 to 4000 cm
1. 2.3.5. Mechanical property The mechanical properties of the single CS, CO and various chitosan/collagen composite scaffolds under wet conditions (after immersion in PBS for 2 h) were determined using a universal mechanical testing apparatus (Tianyuan Co., Ltd.). The scaffolds were stretched with a 100 N load cell using a constant deformation rate of 10 mm/min at room temperature. The size of scaffolds was 10 mm  50 mm length and the measurement was performed along the length. The elastic modulus, ultimate tensile strength and strain at failure were calculated according to the slope of the linear region from the stress-strain curve corresponding to 0e0.2 strain. 2.3.6. Degradation kinetics In vitro biodegradation behavior of the single CS, CO and various chitosan/collagen composite scaffolds was determined by weighing the samples at different times after PBS immersion. In brief, the scaffolds with known weight (Wb) and size of 10 mm  10 mm were placed in 6-well cell culture plate and incubated in PBS at 37  C for 3, 5, 7, 10, 12 and 15 days, respectively. At each time point, the scaffolds were collected, frozen at
20  C for 24 h and lyophilized at
20  C for 48 h. The weight of each scaffold (Wd) was recorded. The fresh prepared PBS was added to the wells after each weighing. The degradation ratio (Dr) was calculated by the following equation: Dr¼ (Wb-Wd)/Wb  100%

2.4. Cytotoxicity test The cytotoxicity of the single CS, CO and various chitosan/ collagen composite scaffolds was tested using MTT assay by culturing L929 fibroblast as reported by Ozdemir et al. [33], and the distribution of fibroblasts was observed by TBO staining. The scaffolds with 10 mm  10 mm size were placed into the 24-well culture plate and sterilized with 75% ethanol for 30 min. Then the scaffolds were thoroughly washed with PBS for three times, and the cell suspension (1 mL) with cell density of 5  104 cells/mL was inoculated onto each scaffold and cultivated for 24 h. After that, the medium was removed and scaffolds were rinsed repeatedly with PBS followed by incubation in 1 mL MTT solution (5 mg/mL) at 37  C for 4 h. Thus, the insoluble formazan crystals were formed due to MTT reduction by the mitochondria of living cells. Subsequently, 1 mL of acidic isopropanol were added to each well and shaken for 30 min to dissolve the intracellularly formed formazan crystals. Finally, 200 mL of the supernatant from each well was transferred to a 96-well culture plate and the absorbance at 570 nm was read using a microplate reader (Bio-Tech Instruments, USA). A standard curve was prepared using cell suspensions with known cell numbers, thus the relative growth ratio (RGR) of L929 in each scaffold could be calculated by a linear correlation between cell concentration and absorbance. The distribution of L929 in each scaffold was detected using TBO staining. In short, after 24 h of

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culture, the medium was trashed, samples were washed with PBS for three times and fixed with formalin (4%) for 6 h. Then, 1 mL TBO solution (1%) in 75% ethanol was added to each sample after PBS rinse and incubated at room temperature for 30 min. Afterwards, the samples were rinsed with PBS for five times and observed under an optical microscope. Ten randomly chosen sites of each sample were photographed for obtaining a statistical analysis. All the tests were performed in triplicate.

scaffolds were all sterilized with 75% ethanol for 30 min and equilibrated using PBS for 1 h, and the greatest diameter of the final scaffolds were measured. The scaffolds were then implanted into a subcutaneous pocket on the dorsum of the rabbit. The skin was sealed with three simple mattress sutures. After that, all animals were housed and the implants were monitored by external inspection weekly until harvest at the 2nd, 6th and 12th post surgery. 2.8. Histological analysis

2.5. RSC96 culture The culture of RSC96 cells was performed to initially evaluate the effect of the single CS, CO and various chitosan/collagen composite scaffolds on nerve regeneration. Cell viability was measured using the CCK-8 assay and TBO staining. CCK-8 solution was prepared by dissolving CCK-8 reagent in DMEM with the volume ration of 1:10. TBO staining solution was prepared by dissolving 1 g of TBO powder into 100 mL 75% ethanol. Briefly, the scaffolds with 10 mm  10 mm size were put into a 24-well culture plate and sterilized with 75% ethanol. RSC96 cells were then seeded onto the scaffolds at a density of 5  104 cells/mL. After culture for 1, 3 and 5 days, respectively, the original medium in each well was changed to CCK-8 solution and incubated for 4 h at 37  C. Then, the supernatant with volume of 200 mL was transferred to a 96-well culture plate, and the absorbance was read at a wavelength of 490 nm using an ELISA plate reader (Bio-Tech Instruments, USA). The TBO staining of RSC96 cells was performed according to the above method used for L929 distribution. All the tests were performed in triplicate. 2.6. Primary schwann cells evaluation In peripheral nervous system, Schwann cells are the main supportive cells for the myelination of axon growth and nerve regeneration. Thus, the effect of the single CS, CO and various chitosan/ collagen composite scaffolds on peripheral nerve regeneration was further evaluated using primary Schwann cells culture. The isolation, purification and culture of Schwann cell from the sciatic nerve of 5-day-old Sprague-Dawley (SD) rats was performed as described previously [14]. In brief, the sciatic nerve of SD rat was dissected into 0.5-mm segments and enzymatically dissociated in collagenase (0.2%) for 30 min. Then, the mixture was stirred, centrifuged, and incubated in DMEM containing 10% fetal bovine serum for 24 h in a culture dish. After that, the medium was replaced by fresh prepared DMEM containing 10 mM cytosine arabinoside (Ara-C) for another 24 h to remove fibroblasts. Schwann cells were then incubated in DMEM supplemented with 2 ng/mL heregulin and 2 mM forskolin for promoting cell proliferation. When a 90% confluence of cell coverage reached in culture dish, the cells were treated with trypsin for 3 min and seeded on various scaffolds at a density of 1  106 cells/mL, and incubated at 37  C for 1, 3, and 5 days, respectively. At each time point, the TBO staining and CCK-8 test as introduced above were performed to evaluate the influence of various composite scaffolds on Schwann cells growth. All the tests were performed in triplicate. 2.7. Subcutaneous implantation experiment To further evaluate the degradation and possible inflammatory reaction of the various prepared scaffolds, a subcutaneous implantation experiment in the back of rabbit was performed in accordance with Guides for the Care and Use of Laboratory Animals from the Chinese Ministry of Public Health. The implantation was divided into four groups: the pure CS group (N ¼ 6), the pure CO group (N ¼ 6), the chitosan/collagen group (10% CS, N ¼ 6), and the chitosan/collagen group (50% CS, N ¼ 6). Before implantation, the

At each time point, the rabbit in each group were sacrificed by anesthetic overdose and the scaffolds were collected. The gross appearance, greatest diameter, and weight were recorded immediately. The scaffolds were then fixed in 4% phosphate-buffered formalin over 24 h. After being dehydrated and transparentized, the scaffolds were embedded in paraffin, and cross-sectioned longitudinally into 5 mm sections using a Leica RM2245 microtome for further HE staining and analysis. Briefly, the longitudinal sections of the scaffolds were immersed into a 2% (w/v) Harris solution for 5 min and rinsed with dH2O for 1 min. Then, the sections were back to blue in basic solution. Thereafter, the sections were immersed into a 0.5% (w/v) eosin solution for 1 min and separately dehydrated with 95% and 100% ethanol, each for 1 min. After that, the sections were made transparent with xylene and sealed with neutral balsam. Finally, the images were taken using an optical microscope. 2.9. Statistical analysis All of the numerical data were statistically analyzed using SPSS 17.0 software. The data were expressed as means ± standard deviation. The statistical analysis was performed with a Student's t-test program and a two-tailed analysis of variance (ANOVA). The differences were considered statistically significant when p < 0.05. 3. Results and discussion 3.1. Morphological index analysis The cross-sectional morphology of various prepared scaffolds was examined by optical microscope (Fig. 1A up) and scanning electron microscopy (Fig. 1A down), respectively. It was observed in optical microscope image that a fiber-like structure mainly appeared in pure CO scaffolds and the chitosan/collagen composite scaffolds with lower chitosan concentration. Notably, the fiber-like structure gradually disappeared when CS concentration increased to 90% and 100%, while a cobblestone-like structure was observed. SEM was further used to examine the micromorphology of the scaffolds. Like the results in optical microscope, the scaffolds with lower chitosan concentration mainly displayed fiber-like structure. And an interconnecting network was obtained. Interestingly, the density of pore structure on scaffolds surface was decreased with the increasing chitosan concentration in all scaffolds. Obviously, almost no pores on scaffolds surface were observed when the chitosan concentration increased to 90% and 100%. However, the pore structure actually existed in the inner scaffolds when transsectional observed as shown in Fig. 1E and F. The reason of morphology variation was mainly ascribed to the introduction of chitosan. Chitosan with good mechanical property and positive charges [34] could tightly bind to collagen via electrostatic interaction, thus the density of surface pores decreased. But the detailed mechanism should be further revealed in future. To further examine the structure of the prepared scaffolds, the thickness, pore size distribution and porosity of each scaffold were measured and shown in Fig. 1 B-D. The results clearly showed that the thickness of

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Fig. 1. Morphology observation of the pure collagen scaffolds (PCO), pure chitosan (PCS) scaffolds and various chitosan/collagen composite scaffolds (M1, M2, M3, M4). (A). Upper: optical microscope, lower: SEM. (B) Thickness **P < 0.05 versus other samples (C) pore size, *P < 0.05, **P < 0.05, ***P < 0.05 versus other samples, and (D) porosity of the various prepared scaffolds. N ¼ 3.

the scaffolds increased with the improved chitosan concentration though the same volume of solution was used for scaffolds preparation. The pure chitosan scaffolds displayed a little lower thickness than that with 90% chitosan. However, the result of surface pore size possessed an opposite trend compared to that of the thickness. The pure CO scaffolds showed the largest surface pore size with about 130 mm compared to that of all other scaffolds (P < 0.05), while the composite scaffolds with chitosan concentration larger than 50% only exhibited a distribution of pore size in the range 30e60 mm. In fact, a suitable pore size could regulate cell ingrowth, proliferation and angiogenes processes [35], an average pore diameter from 20 mm to 125 mm was found to be optimal morphological active for tissue regeneration [36]. Thus, the pore size here may be believed to be helpful for cell penetration during tissue regeneration. Besides, porosity is the percentage of void space in a solid [37]. Fig. 1D showed that the porosity of all scaffolds was found to vary from 82.5% to 92.9%. In particular, no significant differences of porosity were found among all scaffolds, indicating that the addition of chitosan did not affect significantly the porosity of the composite scaffolds. The porosity is very important for cell growth and migration, transport of waste products as well as access of nutrients. Generally, the higher the porosity, the more space and nutrition for the cells and tissue. Based on this hypothesis and experimental results above, the chitosan/collagen scaffolds with higher porosity in the present study may be beneficial for peripheral nerve regeneration.

3.2. Equilibrium swelling and mass variation The equilibrium swelling ratio of various prepared scaffolds was measured via the uptake of PBS and DMEM, respectively. And the mass variation of the scaffolds after PBS and DMEM uptake was also detected. The liquid uptake of the scaffold could provide necessary nutrition for cell growth and tissue regeneration. More, the liquid in the scaffolds could be beneficial for signal transformation and substance exchange. The results of liquid uptake of the prepared scaffolds are shown in Fig. 2A and Fig. 2B. It could be seen clearly that both the pure CO scaffolds and chitosan/collagen composite

scaffolds with chitosan concentration of 10% displayed significantly higher uptake of DMEM (Fig. 2A) and PBS (Fig. 2B) (P < 0.05). The chitosan/collagen composite scaffolds with chitosan concentration of 10% showed the largest uptake of DMEM and PBS, with the ratio of 208.6% and 182.7%, respectively. However, all the other composite scaffolds and pure CS scaffolds exhibited much less uptake of DMEM and PBS with the ratio of less than 50%. In addition, the mass variation of various scaffolds in Fig. 2C showed that after uptake of DMEM or PBS, both the pure CS scaffolds and the chitosan/collagen composite scaffolds with chitosan concentration of 90% possessed significant higher mass variation than other scaffolds. Nevertheless, the pure CO showed the least mass variation compared to other scaffolds. Moreover, the mass variation of the composite scaffolds increased with the enhanced concentration of chitosan, indicating the increasing amounts of chitosan in the composite scaffolds proportionally resulted in larger mass measurements due to the increased water content. It should be mentioned here that all the scaffolds displayed an opposite trend of the uptake of DMEM and PBS compared to that of the mass variation. The higher uptake of DMEM and PBS corresponded to a lower mass variation of the scaffolds. The reason may be that the molecular weight of CO is smaller than that of CS, thus the pure CO scaffolds and the composite scaffolds with lower chitosan concentration have low density and mass when fabricated with the same solution volume. In addition, the hydroxyl groups on chitosan could adsorb a large amount of water molecules [38], thus further causing huge mass variation as a function of chitosan concentration increasing. The results above demonstrated that the chitosan/collagen composite scaffolds with higher chitosan concentration could retain higher amount of liquids, thus may be helpful for tissue regeneration.

3.3. FTIR and mechanical properties To examine the chemical components and mechanical property of the scaffolds, the ATR/FTIR spectra and tensile stress of the pure CO scaffolds, pure CS scaffolds and chitosan/collagen composite scaffolds with various chitosan concentrations were measured using an ATR/FTIR apparatus and a universal mechanical machine.

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Fig. 2. Equilibrium swelling properties and mass variation of various prepared scaffolds. Uptake of DMEM (A) and PBS (B) of scaffold decreases with the enhanced chitosan concentration and equilibrates approximately within 2 h. In addition, the variation in mass (C) is dependent on the concentration of chitosan within scaffold. N ¼ 3, *P < 0.05, **P < 0.05.

Fig. 3. The chemical component detection (A) and representative stress-strain curve (B) of pure CS, CO and composite scaffolds with different chitosan concentrations.

Fig. 3A displays the FTIR spectra of various scaffolds. It was seen clearly that the pure collagen scaffolds mainly showed peaks of amide I band, C]O stretching at 1642 cm
1, amide II band, NeH deformation at 1536 cm
1 and amide A band, NeH stretching at 3320 cm
1. The pure chitosan scaffolds mainly showed characteristic peaks of saccharide unit at 893 cm
1, amide I band at 1642 cm
1, amide II band at 1536 cm
1 and amide III band at 1386 cm
1. Both the FTIR spectra of pure chitosan and collagen were consistent well with the previous studies [39,40]. After complexation, the spectra of all chitosan/collagen composite scaffolds displayed both chitosan and collagen characteristic peaks of amide band and saccharide unit. The peak intensities at 1642 cm
1 (amide I) for all composite scaffolds were noted to decrease with the increasing chitosan concentration. In addition, a blue shift of the peak at 1642 cm
1 was observed for the composite scaffolds when the chitosan concentration increased from 10% to 90%. Interestingly, a new peak at 1125 cm
1 appeared for all composite scaffolds, which signified successful complexation through inter/ intra molecular amide linkage in chitosan/collagen composite scaffolds. The results of FTIR indicated that the chitosan/collagen

composite scaffolds were successfully constructed. The mechanical properties of the biomaterials scaffolds are very important for cell growth and tissue regeneration [41]. A suitable mechanical property matching well with the surrounding tissues could avoid the collapse of the scaffolds and may promote cell or tissue growth via mechanical transduction [42]. Moreover, it may be beneficial for supporting cell penetration, migration and proliferation and providing space of substance exchange. Thus, the tensile stress of the prepared scaffolds was tested in the present study. Fig. 3B displays the stress-strain curves of various scaffolds obtained by applying uni-axial strain in the long axis of sheets. The test could assess differences in the elastic modulus, ultimate tensile strength, and strain at failure due to directionality. It was seen that the pure CO scaffolds showed the lowest stress (~1  10
3MPa) and smallest strain (<100%) compared to all other scaffolds, while the pure chitosan scaffolds had the highest stress (~1  10
3MPa), but the chitosan/collagen composite scaffolds with chitosan concentration of 90% showed the largest strain (>200%). In addition, with increasing content of chitosan, it is observed that both the stress and strain of the composite scaffolds increased in the order of: M1

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(10% chitosan)70%). Almost no morphology variation of the scaffolds occurred in the pure CS scaffolds. SEM results in Fig. 4B exhibited the detailed morphology of various scaffolds after degradation of 15 days. Compared with the SEM morphology of the scaffolds before PBS immersion, the surface pore structure collapsed and disappeared in the pure CO scaffolds and composite scaffolds with lower chitosan content (10%, 50% and 70%). In contrast, a fiber-like structure was mainly observed on the surface of the scaffolds. However, there was no any change of the surface morphology in the composite scaffolds with higher chitosan content (90%, 100%). Otherwise, the degradation kinetic of various scaffolds was measured and shown in Fig. 4C. Obviously, the pure CO scaffolds showed about 40% biodegradation in 7 days and more

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than 80% degradation by 15-day study period. Similarly, in agreement well with the observation by optical microscope, the composite scaffolds with lower chitosan content of 10% and 50% also underwent a rapid degradation behavior, but both was less than 50% of degradation within 15 days, which was significantly lower than that of pure CO scaffolds (P < 0.05). In addition, the pure chitosan scaffolds and composite scaffolds with higher chitosan content of 70% and 90% displayed almost no degradation ratio with less than 10%, which was consistent well with that observed by optical microscope. The results above indicated that the biodegradation behavior of the scaffolds could be well modulated and controlled by varying the chitosan content in the composite scaffolds. 3.5. Cytotoxicity evaluation Generally, the cytocompatibility is very important for the application of artificial developed scaffolds [45], thus the cytotoxicity of the prepared scaffolds should be well evaluated before further investigation for future implantation. L929 fibroblasts are usually used to examine the ctytocompatibility of the new developed artificial scaffolds [46]. Thus, in the present study, to evaluate the cytotoxicity of the various prepared scaffolds, the viability of L929 fibroblasts in different scaffolds was assessed quantitatively and qualitatively after 24 h using MTT and TBO staining assay, respectively (Fig. 5). It was seen clearly in Fig. 5A that a RGR of L929 fibroblasts with higher than 80% was obtained for all the scaffolds, and there was no obvious difference of RGR among all the scaffolds (P > 0.05). Fig. 5B further showed the distribution and morphology of L929 fibroblasts in various prepared scaffolds. All the scaffolds exhibited superior cellular response, in terms of good initial attachment by 24 h. The L929 fibroblasts distributed evenly in the scaffolds, but a little of more cells could be seen in the pure CO scaffolds compared to other scaffolds. Nevertheless, there was no obvious morphological difference of cells in various scaffolds. The results indicated that prepared chitosan/collagen composite scaffolds showed no cytotoxicity, which may have potential application for tissue regeneration. 3.6. RSC96 evaluation RSC96 cell has been used as an important supplement of primary SCs for peripheral nerve regeneration, as it is derived from the long term culture of rat primary SCs [47]. The RSC96 cell lines as a kind of Schwann cells were firstly used to evaluate the effect of the prepared scaffolds on possible nerve regeneration, because the cell lines are free of genetic variations, readily available, easy proliferation and could be expanded without any limitations, while the primary Schwann cells are difficult to isolate and purify with worse tissue heterogeneity. Thus, RSC96 cell lines are widely used as indispensable tools in the fields of biomedical research and tissue engineering [48]. Fig. 6A displayed the RSC96 cell distribution on various scaffolds stained by TBO. It was seen clear that at the 1st day of culture, only few cells attached on the surface of different scaffolds. At the 3rd day of culture, more cells proliferated in

Table 1 Calculated indexes describing the mechanical properties of the pure CS, CO scaffolds and composite scaffolds with different chitosan concentrations. Sample ID

Maximum loading (N)

Maximum displacement (mm)

Maximum Load displacement (%)

Young's modulus (MPa)

Yield elongation (%)

PCO M1 M2 M3 M4 PCS

0.203 0.257 0.348 0.416 0.496 0.567

10.157 10.312 10.624 14.073 19.412 12.223

101.572 103.124 106.242 140.735 194.117 122.234

0.002 0.002 0.002 0.003 0.003 0.004

3.159 3.624 2.979 3.159 3.453 3.776

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Fig. 4. Degradation behavior of pure chitosan scaffolds, pure collagen scaffolds and composite scaffolds with various chitosan concentrations after incubation in PBS for different periods. (A) Observation by optical microscope. (B) SEM observation, (C) degradation kinetics, N ¼ 4, *P < 0.05, **P < 0.05 compared with other samples.

Fig. 5. Cytotoxicity evaluation of pure CS scaffolds, CO scaffolds and chitosan/collagen composite scaffolds with various chitosan concentrations by L929 fibroblasts culture for 24 h. (a) MTT assay, *P > 0.05, (b) optical microscopic images of L929 fibroblasts after TBO staining.

various scaffolds with some cell aggregation on the surface of pure CO scaffolds and composite scaffolds with lower chitosan content (10%, 50%). Conversely, no cell aggregation was observed on the surface of pure CS scaffolds and composite scaffolds with higher chitosan content (70%, 90%). After further culture for 5 days, it was obviously seen that a much more aggregated cell clusters appeared

on the surface of pure CO scaffolds compared with other scaffolds, though there was still enhanced cell aggregation in the scaffolds with chitosan content of 10% and 50%. Notably, cells on other three scaffolds surface displayed homogenous distribution and a confluent coverage after 5 days of culture. Fig. 6BeD showed the density of RSC96 cells on various prepared scaffolds, there was no

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Fig. 6. Culture of RSC96 cells on pure CS scaffolds, CO scaffolds and chitosan/collagen composite scaffolds with various chitosan concentrations for 1, 3 and 5 days, respectively. (A) TBO staining assay for morphology and distribution observation, (BeD) density of RSC96 cells on various prepared scaffolds. N ¼ 4.

obvious difference of cell number among all scaffolds at each same time point (P > 0.05), however, a significant increase of cell density was obtained with elongated culture periods for the same scaffolds (P < 0.05), indicating the RSC96 cells could grow and proliferate well in our prepared chitosan/collagen scaffolds. In addition, it should be mentioned from the TBO staining results that the complexation of chitosan to collagen could regulate the proliferation and distribution of RSC 96 cells in the scaffolds. The pure CO scaffolds may be easy to cause cell aggregation due to the cell binding site on the backbone of collagen. However, the adding of chitosan to CO scaffolds may block or inhibit the binding site of cells, and thus reduce the aggregation behavior. 3.7. Schwann cells evaluation Though the RSC96 cells have been successfully used for initial evaluation of scaffolds biocompatibility, unfortunately, the original phenotype of cell lines are not stable and easy to be lost. Consequently, it may influence the experiment results and subsequent interpretation of the experimental data. Additionally, some unique feature of RSC96 cells including the overexpressed platelet derived growth factor receptors (PDGFRs) and mature Schwann cells markers are found to be different from that of primary Schwann

cells [49]. Therefore, the primary Schwann cells here were further cultured for evaluating the effect of various prepared scaffolds on peripheral nerve regeneration. Fig. 7A displays the morphology and distribution of primary Schwann cells on various scaffolds after culture for 1, 3 and 5 days by TBO staining, respectively. Obviously, the growth of cells on different scaffolds was uniform. At the 1st day of culture, Schwann cells on pure CS scaffolds and composite scaffolds with chitosan content of 90% mainly showed rounded shape without spreading behavior, while cells on other scaffolds displayed a certain spreading. After further culture for 3 and 5 days, Schwann cells on all scaffolds exhibited spreading with obvious neurites elongation except the pure CS scaffolds and composite scaffolds with chitosan content of 90%. Fig. 7B shows that the number of Schwann cells on pure CO scaffolds at the 1st day of culture was the largest compared to other scaffolds. After culture for 3 and 5 days, respectively, Schwann cells on composite scaffolds with chitosan content of 50% displayed significantly larger number than all other scaffolds (P < 0.05). In addition, an obvious proliferation of Schwann cells on all scaffolds was observed from the 1st day to the 5th day of culture. For better evaluation of cell behavior on various scaffolds, the morphological indexes including the length and number of neurites were further analyzed after 1, 3 and 5 days of culture, and shown in Fig. 7C and D. The length of neurites

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Fig. 7. Culture of primary Schwnan cells on pure CS scaffolds, CO scaffolds and chitosan/collagen composite scaffolds with various chitosan concentrations for 1, 3 and 5 days, respectively. (A) TBO staining assay for morphology and distribution observation. (B) Number of Schwann cells on various prepared scaffolds, *P < 0.05, (C, D) length and number of neurits. N ¼ 4. (E) SEM observation of primary Schwann cells on different scaffolds after culture for 5 days.

increased obviously with the incubation time of Schwann cells. At the 1st day of culture, most cells mainly displayed the neurites length in the range from 0 to 5 mm and neurites number of less than one, indicating a more rounded shape. However, after further culture for 3 and 5 days, the neurites length located in the range from 5 to 20 mm and neurites number of one and two. The spreading of cell was reported to have close relationships with the biological function of cell, including cell-cell communication, metabolism, DNA synthesis and signal transduction [50], which may further affect the migration, proliferation and differentiation of cells and tissue

regeneration [51]. Thus, a better cell spreading may be beneficial for accelerating tissue regeneration process. After culture for 5 days, the SEM images in Fig. 7E displayed that Schwann cells number was decreased with enhanced content of CS in the scaffolds, consistent well with the results by OM. The results also proved that Schwann cells on composite scaffolds with lower chitosan content could better induce the spreading of cells, which may be suitable for future application in peripheral nerve regeneration.

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3.8. Gross analysis after subcutaneous implantation To evaluate the degradation behavior of the various prepared scaffolds, the pure CO scaffolds, pure CS scaffolds and composite scaffolds with chitosan content of 10% and 50% were selected for subcutaneous implantation on the rabbit back for 2w, 6w and 12w, respectively. At each time point, no animal died in the control group and the experimental group. But infection was seen in the experimental group of pure CS scaffolds at 6w post implantation. No extrusion of the samples was seen in all experimental groups. Moreover, all the surgical sites healed completely after 8e10 days post implantation. After implantation for 2w, 6w and 12w, respectively, the rabbits were executed and the implanted scaffolds were exposed (Fig. 8A). Obviously, compared to the scaffolds before implantation (BI), the pure CO scaffolds showed significantly larger degradation after implantation for 2w, and almost no residual scaffolds were seen after 6w and 12w. However, the scaffolds in other groups were still observed even after 12w of implantation though the size decreased heavily compared to the original size (BI). Both the composite scaffolds exhibited obvious degradation behavior with the implantation period, while at the same time point, it was seen that the composite scaffold with higher chitosan content showed smaller degradation than that with lower chitosan content, indicating the degradation of composite scaffolds was well regulated and largely dependent on the chitosan concentration. Notably, the size variation of the pure CS scaffold during the whole implantation periods did not change too much though there was contraction occurring. Otherwise, it should be mentioned that all the visible implanted scaffolds at each time point were wrapped by a thin, soft fibrotic capsule with clear vascular network and minimal inflammatory reaction. From implant to explant, the residual volume percentage (RVP) of various scaffolds in the control group and experimental group was further analyzed and shown in Fig. 8B. The mean RVP of scaffolds preimplantation was considered as 100%. The mean RVP of pure scaffolds significantly decreased to less than 20% at 2w and further to less than 6% at 6w, while almost no data (ND) were measured at 12 w, indicating a much faster degradation rate. After complexation with chitosan, the mean RVP of the composite scaffolds still showed obvious decrease at different time points compared with that before implantation. However, the mean RVP

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of the composite scaffolds was still higher than 20% at 12w post implantation, thus the degradation rate of the composite scaffolds was well regulated by adding chitosan to collagen scaffolds. In contrast, the mean RVP of the pure chitosan scaffolds was still higher than 80% even at 12w post implantation, indicating a much slower degradation rate compared to other scaffolds. The results indicated that the addition of chitosan to collagen scaffolds could well regulate the in vivo degradation behavior of the chitosan/ collagen composite scaffolds, which was consistent well with the results of in vitro degradation study of the composite scaffolds. Thus, the composite scaffolds may have potential for application in various tissue regeneration with different degradation requests. 3.9. H&E staining and analysis For further examining the degradation behavior and possible inflammatory reaction of the various prepared scaffolds in vivo, the histological examination of the control group and experimental group stained with hematoxylin and eosin was performed at each time point. The results are shown in Fig. 9. Fig. 9A shows that at the 2nd week after implantation, the scaffolds mainly displayed a fiberlike structure, and there were obvious cell and tissue permeability into the fiber-like structure in all groups. After implantation for 6 w, the fiber-like structure in pure CO group significantly decreased compared with the pure CS group and composite group, while more tissue ingrowth was observed, indicating a much faster degradation of the pure CO scaffolds in vivo. However, no obvious difference of tissue ingrowth was observed between the pure CS group and composite scaffolds group, though all showed more tissue ingrowth than the 2nd week post implantation. Then, after further implantation for 12w, almost no fiber-like structure was seen in the pure CO group. Insteadly, an obvious fibrous tissue ingrowth was observed with more than 80% coverage of the vision. Nevertheless, a large amount of the fiber-like structure with higher coverage area was still seen clearly in other groups. In addition, it was noted that all scaffolds showed no obvious inflammatory reaction during the implantation periods. Fig. 9B further quantitatively characterized the degradation behavior of various scaffolds by analysis of the normalized percentage of scaffolds area (NPSA). In agreement well with the H&E staining results, the pure CO scaffolds displayed significant degradation with the implantation

Fig. 8. Gross observation and volume variation analysis of pure CS scaffolds, CO scaffolds and chitosan/collagen composite scaffolds with various chitosan concentrations after in vivo implantation for 2, 6 and 12 weeks, respectively. (A) Gross observation of the scaffolds, circle represents the pure CO scaffolds after 2w of implantation, white arrows represent the suture thread. (B) Residual volume percentage (RVP) of various scaffolds at each time point, *P < 0.05, **P < 0.05, ***P < 0.05 versus other scaffolds, N ¼ 6.

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Fig. 9. Histological staining and normalized percentage of scaffolds area analysis of pure CS scaffolds, CO scaffolds and chitosan/collagen composite scaffolds with various chitosan concentrations after in vivo implantation for 2, 6 and 12 weeks, respectively. (A) H&E staining of the scaffolds, (B) normalized percentage of scaffolds area (NPSA) of various scaffolds at each time point, *P < 0.05 versus other scaffolds, N ¼ 6.

time (P < 0.05). After 6w of implantation, the residual NPSA was only less than 20%. However, in other scaffolds groups, the residual NPSA was still higher than 45% even after implantation for 12w. The results above further indicated that the degradation behavior of chitosan/collagen composite scaffolds could be well modulated by varying the content of chitosan. 4. Conclusion Through lyophilization and phase separation composites, the present study demonstrates the successful development of chitosan/collagen composite scaffolds with controlled degradation behavior as a support matrix for peripheral nerve regeneration application. The physicochemical properties of the prepared composite scaffolds could be well regulated to be suitable for intended purpose in tissue engineering. The addition of chitosan was found to increase the mechanical property and decrease the degradation rate of the composite scaffolds. Further on, the chitosan/collagen composite scaffolds showed good cytocompatibility without cytotoxicity. And our results also demonstrate that the chitosan/ collagen composite scaffolds could promote the attachment, migration and proliferation of Schwann cells. The in vivo implantation results indicated that the composites scaffolds showed obviously modulated degradation behavior without causing any inflammatory reaction. Therefore, the developed chitosan/collagen composite scaffolds here may be promising candidates for future application in peripheral nerve regeneration. The application may be also extended to other tissue engineering fields, including bone, tendon and muscle.

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Acknowledgements [15]

We greatly thank other members in our lab for valuable suggestions and writing, and the Scientific Development Plan of Shandong Province (2012GGE27081).

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